Bacterium of genus corynebacterium capable of producing isopropanol, and method for producing isopropanol using microorganism

Abstract

As one aspect, the present disclosure provides a bacterium of the genus Corynebacterium capable of producing isopropanol with high productivity. As another aspect, the present disclosure provides a method which enables the production of isopropanol with high productivity while suppressing the production of by-products.　In one aspect, the present disclosure relates to a bacterium of the genus Corynebacterium capable of producing isopropanol, the bacterium being produced by introducing into a bacterium of the genus Corynebacterium a specific gene (A) that encodes an enzyme having an activity of producing acetoacetic acid from acetoacetyl-CoA and / or a specific gene (B) that encodes an enzyme having an activity of producing acetone from acetoacetic acid in a manner that enables expression thereof. In another aspect, the present disclosure relates to a method for producing isopropanol, the method comprising: allowing a genetically modified bacterium of the genus Corynebacterium capable of producing isopropanol to carry out a fermentation reaction in a fermentation tank to produce a fermentation product containing isopropanol; continuously or intermittently withdrawing a portion of a reaction solution containing the fermentation product from the fermentation tank during the fermentation reaction; separating isopropanol from the withdrawn reaction solution by pervaporation; and supplying to the fermentation tank the reaction solution from which isopropanol has been separated.

Description

Corynebacterium bacterium having the ability to produce isopropanol, and method for producing isopropanol using a microorganism

[0001] The present disclosure relates to a Corynebacterium bacterium having the ability to produce isopropanol and a method for producing isopropanol. The present disclosure relates to a method for producing isopropanol by subjecting the Corynebacterium bacterium or a genetically modified microorganism having the ability to produce isopropanol to a fermentation reaction.

[0002] Biorefinery is a general term for technologies and industries that produce biofuels and chemicals from biomass, and great expectations are placed on it as a technology for reducing CO2 emissions and realizing a carbon recycling society.

[0003] Isopropanol is a type of secondary alcohol. Propylene obtained by dehydrating isopropanol is a basic raw material for synthetic resins and petrochemical products such as acrylic acid, and attempts have been made to produce isopropanol from biomass raw materials. In recent years, as a method for producing isopropanol from biomass raw materials, attempts have been made to produce isopropanol from saccharides using recombinant microorganisms (Patent Documents 1 to 4).

[0004] U.S. Patent Application Publication No. 2023 / 272436 International Publication No. 2009 / 131040 International Publication No. 2009 / 028582 International Publication No. 2013 / 022070

[0005] However, it cannot be said that sufficient productivity of isopropanol has been obtained by conventional methods, and further improvement in productivity is required.

[0006] The present disclosure provides, as one aspect, a new Corynebacterium bacterium capable of improving the productivity of isopropanol. The present disclosure provides, as other aspects, methods capable of producing isopropanol with high productivity while suppressing the production of by-products.

[0007] This disclosure relates, in one embodiment, to a Corynebacterium bacterium having isopropanol-producing ability, obtained by introducing at least one of the following genes (A) and (B) into the bacterium so as to be expressible. Gene (A): At least one gene selected from the group consisting of (A1) to (A9) below, which encodes an enzyme that has the activity to produce acetoacetate from acetoacetyl-CoA; (A1) A gene from which the genus Pseudomonas originates; (A2) A gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 14; (A3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 14; (A4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in Sequence ID No. 14; (A5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding the polypeptide having the amino acid sequence shown in Sequence ID No. 14; (A6) A gene having the nucleotide sequence shown in Sequence ID No. 4; (A7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 4; (A8) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, with 1 to 10 nucleotides being deleted, substituted and / or added per unit; (A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 4;Gene (B): At least one gene selected from the group consisting of (B1) to (B9) below, which encodes an enzyme having the activity to produce acetone from acetoacetate; (B1) A gene derived from at least one microorganism selected from the group consisting of the genera Paenibacillus, Bradyrhizobium, Amycolatopsis, Paraburkholderia, and Burkholderia; (B2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, as shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B6) A gene having a nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65; (B7) A gene having a nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65;(B8) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, with 1 to 10 nucleotides deleted, substituted and / or added per unit, as shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65; (B9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65.

[0008] In other embodiments, the present disclosure relates to a method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified Corynebacterium bacteria having isopropanol-producing ability to produce a fermentation product containing isopropanol; continuously or intermittently withdrawing a portion of the reaction solution containing the fermentation product from the fermenter during the fermentation reaction; separating isopropanol from the withdrawn reaction solution by pervaporation; and supplying the reaction solution from which isopropanol has been separated to the fermenter, wherein the Corynebacterium bacteria have at least one of the activities of producing isopropanol from acetone and producing acetone from acetoacetic acid enhanced by the genetic modification.

[0009] In other embodiments, this disclosure relates to a method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified microorganisms capable of producing isopropanol to produce a fermentation product containing isopropanol; separating isopropanol from at least a portion of the reaction solution containing the fermentation product in the fermenter; supplying the reaction solution from which isopropanol has been separated to the fermenter; and maintaining the isopropanol concentration in the fermenter at 6 g / L or less.

[0010] In further embodiments, the present disclosure relates to a method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified microorganisms capable of producing isopropanol to produce a fermentation product containing isopropanol; separating isopropanol from the reaction solution by passing a portion of the reaction solution containing the fermentation product in the fermenter through a pervaporation module; supplying the reaction solution that has passed through the pervaporation module to the fermenter; and maintaining the isopropanol concentration in the fermenter at 6 g / L or less.

[0011] The present disclosure further relates to a method for producing isopropanol, comprising using a genetically modified Corynebacterium bacterium capable of producing isopropanol to produce isopropanol through a fermentation reaction, wherein the Corynebacterium bacterium is the Corynebacterium bacterium of the present disclosure.

[0012] According to this disclosure, in one embodiment, a novel Corynebacterium bacterium capable of improving the productivity of isopropanol can be provided. According to this disclosure, in another embodiment, a method for producing isopropanol with high productivity while suppressing the production of by-products can be provided.

[0013] Figure 1 is a schematic diagram showing the configuration of the experimental apparatus used in Reference Example 1. Figure 2 is a graph showing the change in isopropanol concentration of the isopropanol aqueous solution in the tank due to pervaporation separation in Reference Example 1. Figure 3 is a graph showing the relationship between aeration stirring conditions and permeation flux in Reference Example 1. Figure 4 is a graph showing the relationship between the separation coefficient β and the isopropanol concentration in the tank in Reference Example 1. Figure 5 is a graph showing the culture time elapsed for the value obtained by multiplying OD by the culture volume (L) in Reference Example 2. Figure 6 is a graph showing the culture time elapsed for the value obtained by multiplying OD by the culture volume (L) in Example 3 (pervaporation) and Reference Example 2 (gas stripping). Figure 7 is a schematic diagram showing the configuration of the experimental apparatus used in Reference Example 3. Figure 8 is a graph showing the change in the concentration of the isopropanol aqueous solution in the tank due to pervaporation separation in Example 3. Figure 9 is a graph showing the relationship between linear velocity and permeation flux in Example 3. Figure 10 is a graph showing the relationship between linear velocity and pervaporation membrane selectivity in Reference Example 3. Figure 11 is a graph showing the relationship between vacuum level and permeation flux in Reference Example 4. Figure 12 schematically shows one form of isopropanol production in genetically modified Corynebacterium bacteria usable in the methods of this disclosure. Figure 13 is an overall metabolic pathway diagram illustrating one form of the biosynthetic pathway for isopropanol production in genetically modified Corynebacterium bacteria usable in the methods of this disclosure.

[0014] Various methods have been proposed for producing isopropanol by microbial fermentation using recombinant microorganisms. One such method involves culturing microbial cells in a fermenter while continuously producing isopropanol from the cells, and then separating the produced isopropanol by gas stripping (for example, Patent Document 4). In gas stripping, sterilized air is introduced into the fermenter to promote the volatilization of isopropal in the tank, and the exhaust gas containing the resulting isopropanol is supplied to the capture liquid.

[0015] The inventors conducted extensive research to provide a new method capable of improving the productivity of isopropanol. In the process, the inventors discovered that in the production of isopropanol using microbial fermentation reactions, sequentially separating volatile components such as isopropanol during the fermentation reaction by pervaporation significantly improves the productivity of isopropanol and reduces the generation of by-products such as acetone and acetic acid. This disclosure is based on this discovery.

[0016] Although the detailed mechanism of the effects of this disclosure is not clear, it is presumed to be as follows: Genetically modified Corynebacterium bacteria capable of producing isopropanol are subjected to a fermentation reaction to produce isopropanol. In parallel with this fermentation reaction, isopropanol and other volatile components are separated from the reaction solution containing the produced isopropanol and other volatile components by pervaporation, and the separated reaction solution is used for further fermentation reactions. By separating by pervaporation in parallel with the fermentation reaction and reusing the separated reaction solution, the concentration of isopropanol in the reaction solution during the fermentation reaction can be maintained at a low level. As a result, the microorganisms can be kept active for the fermentation reaction, thereby improving the productivity of isopropanol and suppressing the production of by-products such as acetone and acetic acid. Furthermore, since the production ratio of isopropanol to by-products can be shifted towards the isopropanol side, it is thought that the yield of isopropanol can be improved. However, this disclosure does not have to be interpreted as being limited to these mechanisms.

[0017] Furthermore, this disclosure relates to a gene (A) encoding an enzyme having the activity to produce acetoacetate from acetoacetyl-CoA consisting of the nucleotide sequence shown in SEQ ID NO: 4, a gene (B) encoding an enzyme having the activity to produce acetone from acetoacetate consisting of the nucleotide sequence shown in SEQ ID NOs: 7, 17, 20, 23, 26, 29, or 32, and acetone consisting of the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50 This finding is based on the discovery that the productivity of isopropanol can be improved by introducing a gene (C) encoding an enzyme that has the activity to produce isopropanol (IPA) from tone, and a gene (D) encoding an enzyme that has the activity to produce acetoacetyl-CoA from acetyl-CoA and malonyl-CoA consisting of the base sequence shown in Sequence ID No. 1, into Corynebacterium glutamicum in an expressible manner, using microorganisms (Corynebacterium bacteria).

[0018] [Method for Producing Isopropanol (First Embodiment)] The method for producing isopropanol according to the first embodiment of this disclosure includes: causing a fermentation reaction in a fermenter using genetically modified Corynebacterium bacteria having isopropanol-producing ability to produce a fermentation product containing isopropanol; continuously or intermittently withdrawing a portion of the reaction solution containing the fermentation product from the fermenter during the fermentation reaction; separating isopropanol from the withdrawn reaction solution by pervaporation; and supplying the reaction solution from which isopropanol has been separated to the fermenter. The Corynebacterium bacteria used in the method for producing isopropanol according to the first embodiment of this disclosure are genetically modified Corynebacterium bacteria having isopropanol-producing ability, wherein at least one of the activities of producing isopropanol from acetone and producing acetone from acetoacetic acid is enhanced by the genetic modification. According to the first embodiment of this disclosure, in one or more embodiments, isopropanol can be produced with high productivity while suppressing the production of by-products. According to a first aspect of the present disclosure, in one or more embodiments, the production ratio of isopropanol to by-products can be improved. According to a first aspect of the present disclosure, in one or more embodiments, an aqueous isopropanol solution having an isopropanol concentration of 30 g / L or more can be obtained.

[0019] In one or more embodiments, by-products include volatile components other than isopropanol. In one or more embodiments, volatile components include acetone, etc. In one or more embodiments, other by-products include acetic acid, alanine, and valine, etc. In one or more embodiments, which are not particularly limited, the fermentation product may contain by-products other than isopropanol. In one or more embodiments, which are not particularly limited, the fermentation product may contain isopropanol and at least one of acetone and acetic acid, or it may contain isopropanol, acetone and acetic acid, and may further contain alanine and valine.

[0020] The Corynebacterium bacteria used in the methods of this disclosure are capable of producing isopropanol. These Corynebacterium bacteria have been genetically modified to enhance at least one of the activities of producing isopropanol from acetone and producing acetone from acetoacetic acid. In this disclosure, "at least one of the activities of producing isopropanol from acetone and producing acetone from acetoacetic acid is at least enhanced" means that the activity of producing isopropanol from acetone is at least enhanced, the activity of producing acetone from acetoacetic acid is at least enhanced, or both the activity of producing isopropanol from acetone and the activity of producing acetone from acetoacetic acid are at least enhanced.

[0021] In this disclosure, "enhanced activity" means, in one or more embodiments, that the expression of the gene encoding the polypeptide having the activity is enhanced, the expression level of the gene encoding the polypeptide having the activity is increased, or the transcription level of the gene encoding the polypeptide having the activity is increased, compared to a strain in which the activity is not enhanced (or before the enhancement of activity is performed).

[0022] Examples of Corynebacterium bacteria whose activity in producing isopropanol from acetone is at least enhanced by genetic recombination include, in one or more embodiments, Corynebacterium bacteria in which isopropanol dehydrogenase activity or alcohol dehydrogenase activity is enhanced. In one or more embodiments of said Corynebacterium bacteria, which are not particularly limited, examples include Corynebacterium bacteria in which at least one of the genes (C1) to (C9) described later is introduced to be expressible, Corynebacterium bacteria in which the expression of at least one of said genes (C1) to (C9) is enhanced, or Corynebacterium bacteria in which the expression and / or transcription level of at least one of said genes (C1) to (C9) is increased. Whether or not a Corynebacterium bacterium has at least enhanced activity in producing isopropanol from acetone can be confirmed in one or more embodiments by measuring the expression level or transcription level of the gene encoding the polypeptide having said activity and comparing it with that of the parent strain (Corynebacterium bacteria that have not undergone said enhancement).

[0023] Examples of Linebacterium bacteria whose activity in producing acetone from acetoacetic acid is at least enhanced by genetic recombination include, in one or more embodiments, Corynebacterium bacteria in which acetacetate decarboxylase activity is enhanced. In one or more embodiments of said Corynebacterium bacteria, which are not particularly limited, examples include Corynebacterium bacteria in which at least one of the genes (B1) to (B9) described later is introduced to be expressible, Corynebacterium bacteria in which the expression of at least one of said genes (B1) to (B9) is enhanced, or Corynebacterium bacteria in which the expression and / or transcription of at least one of said genes (B1) to (B9) is increased. Whether or not a Corynebacterium bacterium has at least enhanced activity in producing acetone from acetoacetic acid can be confirmed in one or more embodiments by measuring the expression or transcription of the gene encoding the polypeptide having said activity and comparing it with that of the parent strain (Corynebacterium bacteria without said enhancement).

[0024] Examples of Corynebacterium bacteria in which the activity of producing acetone from acetoacetic acid and the activity of producing isopropanol from acetone are enhanced by genetic recombination include, in one or more embodiments, Corynebacterium bacteria in which acetacetate decarboxylase activity and isopropanol dehydrogenase activity or alcohol dehydrogenase activity are enhanced. In one or more embodiments of the Corynebacterium species described below, at least one of the genes (B1) to (B9) described later is introduced to be expressible, the expression of at least one of the genes (B1) to (B9) is enhanced, or the expression level and / or transcription level of at least one of the genes (B1) to (B9) is increased, and the Corynebacterium species described below also includes those in which at least one of the genes (C1) to (C9) described later is introduced to be expressible, the expression of at least one of the genes (C1) to (C9) is enhanced, or the expression level and / or transcription level of at least one of the genes (C1) to (C9) is increased. Whether a Corynebacterium bacterium has enhanced activity in producing acetone from acetoacetic acid and isopropanol from acetone can be determined in one or more embodiments by measuring the expression or transcription level of the gene encoding the polypeptide having said activity and comparing it with that of the parent strain (a Corynebacterium bacterium without said enhancement).

[0025] In one or more embodiments, the Corynebacterium species exemplified in the genetically modified Corynebacterium species described later in this disclosure may be used as the Corynebacterium species used in the method of this embodiment.

[0026] <Isopropanol Production Process by Fermentation Reaction> The method according to this embodiment includes producing a fermentation product containing isopropanol by carrying out a fermentation reaction in a fermenter using genetically modified Corynebacterium bacteria having the ability to produce isopropanol.

[0027] In one or more embodiments, the fermentation reaction can be carried out by culturing the genetically modified Corynebacterium bacteria in a reaction solution containing fermentation raw materials. In one or more embodiments, the production process includes culturing the Corynebacterium bacteria in a reaction solution containing the genetically modified Corynebacterium bacteria, fermentation raw materials, and culture medium. In one or more embodiments, the production process may include adding the fermentation raw materials to the reaction solution during the culturing (fermentation reaction). Furthermore, as described later, the method of this embodiment includes supplying the reaction solution from which isopropanol has been separated by pervaporation to a fermenter continuously or intermittently during the culturing (fermentation reaction).

[0028] In one or more embodiments, the production process may include aerobic cultivation of the Corynebacterium species under the medium, temperature, and pH conditions described later, thereby allowing the Corynebacterium species to undergo a continuous fermentation reaction to produce a fermentation product containing isopropanol. Therefore, in one or more embodiments, the production process is preferably carried out by continuous cultivation using the genetically modified Corynebacterium species described above.

[0029] In one or more embodiments, carbon sources can be used as fermentation raw materials. Examples of carbon sources in one or more embodiments include carbohydrates or sugar alcohols such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, molasses, sorbitol, and glycerin; organic acids such as fumaric acid, maleic acid, and gluconic acid; and alcohols such as ethanol and propanol. One type of fermentation raw material may be used alone, or two or more types may be used in combination. In one or more embodiments, sugars such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, and molasses can be preferably used as fermentation raw materials.

[0030] In one or more embodiments, the culture medium may be a natural or synthetic medium containing a carbon source, a nitrogen source, inorganic salts, and other nutrients. In one or more embodiments, which are not particularly limited, the culture medium may be Medium A [Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)], and Medium BT [Omumasaba, CA et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)].

[0031] The carbon sources are as described above. One type of carbon source may be used alone, or two or more types may be used in mixture. The concentration of these carbon sources in the reaction solution is about 0.01 (w / v%) to about 50 (w / v%) in one or more embodiments.

[0032] Examples of nitrogen sources in one or more embodiments include inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, as well as urea, aqueous ammonia, sodium nitrate, and potassium nitrate. In one or more embodiments, nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolysates, and amino acids can also be used as nitrogen sources. One type of nitrogen source may be used alone, or two or more types may be used in mixture. The concentration of the nitrogen source in the reaction solution varies depending on the nitrogen compound used, but in one or more embodiments, it is about 0.1 (w / v%) to about 10 (w / v%).

[0033] Examples of inorganic salts in one or more embodiments include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, ammonium carbonate, and calcium carbonate. These inorganic salts may be used individually or in mixtures of two or more. The concentration of inorganic salts in the reaction solution varies depending on the inorganic salt used, but in one or more embodiments, it is approximately 0.01 (w / v%) to approximately 1 (w / v%).

[0034] Other nutrients may include, in one or more embodiments, meat extract, peptone, polypeptone, yeast extract, dried yeast, corn steep liquor, skim milk powder, skim soybean hydrochloride hydrolysate, and extracts and decomposition products of animals, plants, and microorganisms. The concentration of nutrients in the reaction solution varies depending on the nutrients used, but in one or more embodiments, it is about 0.1 (w / v%) to about 10 (w / v%). Vitamins may be added as needed. Examples of vitamins may include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

[0035] The temperature and pH of the reaction solution in the production process can be set as appropriate. In one or more embodiments, the temperature of the reaction solution in the production process is 20°C to 40°C, 25°C to 35°C, or about 30°C. In one or more embodiments, the method of this embodiment may include setting the temperature inside the fermenter to 20°C to 40°C, 25°C to 35°C, or about 30°C. In one or more embodiments, the pH of the reaction solution in the production process is pH 6 to 8. pH adjustment can be performed in one or more embodiments using an alkaline substance such as ammonia.

[0036] The method of this embodiment may, in one or more embodiments, include controlling the dissolved oxygen concentration of the reaction solution in the fermenter. To suppress the accumulation of acetic acid contained in the reaction solution, the method may include controlling the dissolved oxygen concentration of the reaction solution in the fermenter to be maintained above 0 ppm. In other words, the method of this embodiment may, in one or more embodiments, include maintaining a state in which oxygen is dissolved in the reaction solution in the fermenter.

[0037] In one or more embodiments, the method of this embodiment may include stirring the reaction solution in the fermenter during the fermentation reaction (culture). The stirring speed is 10 rpm to 1000 rpm or 40 rpm to 600 rpm in one or more embodiments. In one or more embodiments, the method of this embodiment may include passing gas through the reaction solution in the fermenter during the fermentation reaction (culture). The amount of gas to pass through is 0.02 vvm to 3.0 vvm, 0.1 vvm to 2 vvm, or 0.1 vvm to 1 vvm in one or more embodiments (vvm = gas volume (mL) / reaction solution volume (mL) / time (minutes)).

[0038] <Isopropanol Separation Step by Pervaporation> The method according to this embodiment includes continuously or intermittently withdrawing a portion of the reaction solution containing the fermentation product from the fermenter during the fermentation reaction, and separating isopropanol from the reaction solution withdrawn from the fermenter by pervaporation.

[0039] The reaction solution may be withdrawn continuously or intermittently, but continuous withdrawal is preferable in order to further improve the productivity of isopropanol. The reaction solution withdrawal rate can be appropriately set based on the linear velocity of the reaction solution passing through the pervaporation module. In one or more embodiments, the reaction solution withdrawal rate is 3 L / h to 30 L / h per L of reaction solution.

[0040] In one or more embodiments, the reaction solution withdrawn from the fermentation tank is supplied to a pervaporation module equipped with a pervaporation membrane. When the reaction solution withdrawn from the fermentation tank is passed through the pervaporation module, isopropanol and other volatile components in the reaction solution evaporate through the pervaporation membrane in the pervaporation module and are separated from the reaction solution. By bringing the separated isopropanol and other volatile components into contact with a collecting liquid for collection, isopropanol and other volatile components can be recovered. The separation step includes, in one or more embodiments, at least recovering isopropanol separated from the reaction solution by pervaporation. In one or more embodiments, isopropanol can be recovered as an aqueous solution of isopropanol. The aqueous solution of isopropanol may contain other volatile components other than the aqueous solution of isopropanol in one or more embodiments.

[0041] In one or more embodiments, examples of the collecting liquid include water, organic solvents, etc., and preferably water. From the viewpoint of improving the recovery efficiency of isopropanol, in one or more embodiments, the collecting liquid is preferably cooled, and more preferably cooled to 0°C or lower.

[0042] In one or more embodiments, the separation step includes passing the reaction solution withdrawn from the fermentation tank through the pervaporation module and bringing the isopropanol separated from the reaction solution by the passing into contact with a collecting liquid for collection.

[0043] In one or more embodiments, the pervaporation module consists of a housing and a pervaporation membrane, such as a hollow fiber membrane, a flat membrane, or a spiral membrane, placed inside the housing. One side of the space separated by the membrane and the housing is called the circulation side, and this space is provided with a connection port (inlet) at one end of the housing for supplying the reaction liquid (treated water) and a connection port (outlet) at the other end for discharging the reaction liquid (degassed water) from which the volatile components have been separated. The other side of the space separated by the membrane and the housing is called the permeation side, and the housing in this space has an outlet formed therein for discharging volatile components that have passed through the hollow fiber membrane (pervaporation membrane) to the outside. The outlet can be connected to a vacuum pump, and a collection container (trap) containing a collection liquid for recovering the separated volatile components is placed between the outlet and the vacuum pump. When the reaction solution is supplied to the inlet of the pervaporation module, volatile components in the reaction solution are separated through the hollow fiber membrane (pervaporation membrane) inside the pervaporation module, and the separated volatile components are collected in a collection container connected to the outlet. The reaction solution from which the volatile components have been separated is discharged outside the pervaporation module through the outlet and supplied to the fermentation tank through piping connected to the outlet. Examples of materials for the pervaporation membrane include silicone and zeolite in one or more embodiments.

[0044] In one or more embodiments, the vacuum level on the permeation side of the pervaporation module is 15 kPa or less, 10 kPa or less, 7.5 kPa or less, 5 kPa or less, 4 kPa or less, 3 kPa or less, 2 kPa or less, or 1 kPa or less, from the standpoint of more efficiently separating isopropanol from the reaction solution and further improving the productivity of isopropanol. In one or more embodiments, which are not limited, the vacuum level on the permeation side of the pervaporation module may be 0.1 kPa or more, 1 kPa or more, 2 kPa or more, 3 kPa or more, 4 kPa or more, or 5 kPa or more. In order to more efficiently separate isopropanol from the reaction solution and further improve the productivity of isopropanol, the vacuum level on the permeation side of the pervaporation module can be, in one or more embodiments, 0.1 kPa to 15 kPa, 0.1 kPa to 10 kPa, 0.1 kPa to 7.5 kPa, 0.1 kPa to 5 kPa, 0.1 kPa to 4 kPa, 0.1 kPa to 3 kPa, 0.1 kPa to 2 kPa, or 0.1 kPa to 1 kPa. In order to more efficiently separate isopropanol from the reaction solution and further improve the productivity of isopropanol, the vacuum level on the permeation side of the pervaporation module may be 1 kPa to 15 kPa, 2 kPa to 15 kPa, 3 kPa to 10 kPa, 4 kPa to 10 kPa, or 5 kPa to 7.5 kPa in one or more embodiments. In this disclosure, "vacuum level on the permeation side of the pervaporation module" refers to the vacuum level (pressure unit (Pa) representing the degree of vacuum) set in the vacuum pump, and the vacuum level on the permeation side can be controlled by the vacuum pump connected to the above-mentioned outlet in one or more embodiments.

[0045] The flow of the reaction solution into the pervaporation module is carried out, in one or more embodiments, such that the reaction solution passes through the pervaporation module at a linear velocity of 0.0018 m / s or more, from the viewpoint of more efficiently separating isopropanol from the reaction solution and further improving the productivity of isopropanol. The flow of the reaction solution into the pervaporation module is, from the same viewpoint, in one or more embodiments, when using a pervaporation module with a linear distance from the inlet to the outlet of 0.2 m, the reaction solution has a linear velocity of 0.0019 m / s or more, 0.002 m / s or more, 0.003 m / s or more, 0.004 m / s or more, 0.005 m / s or more, 0.006 m / s or more, or 0.007 m / s or more. The flow of the reaction solution into the pervaporation module may, in one or more embodiments, when the linear distance from the inlet to the outlet of the pervaporation module is 0.2 m, be carried out such that the reaction solution passes through at a linear velocity of 0.05 m / s or less. The flow of the reaction solution into the pervaporation module may, in one or more embodiments, when the linear distance from the inlet to the outlet of the pervaporation module is 0.2 m, be carried out such that the reaction solution passes through at a linear velocity of 0.0018 m / s to 0.05 m / s, 0.0019 m / s to 0.05 m / s, 0.002 m / s to 0.05 m / s, 0.003 m / s to 0.05 m / s, 0.004 m / s to 0.05 m / s, 0.005 m / s to 0.05 m / s, 0.006 m / s to 0.05 m / s, or 0.007 m / s to 0.05 m / s. The linear velocity at which the reaction solution passes through the pervaporation module can be appropriately determined, for example, by conducting tests as shown in Reference Example 3, according to the length of the pervaporation module (for example, the effective length of the pervaporation membrane, etc.).

[0046] In the present disclosure, the "linear velocity" refers to the distance that the fluid moves per unit time in the inner hollow cross-section of the pervaporation membrane in the pervaporation module.

[0047] In one or more embodiments, the pervaporation module is connected to a vacuum pump, thereby reducing the pressure inside the pervaporation module. A collection container (trap) for recovering isopropanol may be installed between the pervaporation module and the vacuum pump. The number of traps is not particularly limited and may be one, two or three or more in one or more embodiments. In one or more embodiments, additional traps may be installed on the exhaust side of the vacuum pump. In one or more embodiments, which are not particularly limited, one trap is installed between the pervaporation module and the vacuum pump, and at least two additional traps are installed on the exhaust side of the vacuum pump.

[0048] When multiple traps are installed, in one or more embodiments, the temperature of each trap is set to a different temperature. To recover isopropanol more efficiently and further improve the productivity of isopropanol, it is preferable that at least one trap is cooled to 0°C or below, and more preferably that the trap located closest to the pervaporation module is cooled to 0°C or below. Examples of temperatures below 0°C in one or more embodiments include -1°C or below, -2°C or below, -3°C or below, -4°C or below, -5°C or below, -6°C or below, -7°C or below, -8°C or below, and -9°C or below. In one or more embodiments, the temperature of the traps is -20°C or above, and is not particularly limited. In one or more embodiments, the cooling temperature of the trap may be -20°C to -1°C, -20°C to -2°C, -20°C to -3°C, -20°C to -4°C, -20°C to -5°C, -20°C to -6°C, -20°C to -7°C, -20°C to -8°C, and -20°C to -9°C. In one or more embodiments, when one trap is installed between the pervaporation module and the vacuum pump, and two traps are installed on the exhaust side of the vacuum pump, the trap installed between the pervaporation module and the vacuum pump may be cooled to 0°C or below, and of the two traps installed on the exhaust side of the vacuum pump, the trap on the vacuum pump side may be kept at room temperature, while the other trap may be kept below room temperature.

[0049] <Process of supplying reaction solution to fermentation tank (reuse)> The method of this embodiment includes supplying the reaction solution from which isopropanol has been separated to a fermentation tank during the fermentation reaction. This makes it possible to maintain a low concentration of isopropanol in the fermentation tank and further improve the productivity of isopropanol. In one or more embodiments of this embodiment, the method includes contacting Corynebacterium bacteria with fermentation raw materials in a fermentation tank containing the reaction solution from which isopropanol has been separated, and further includes causing the Corynebacterium bacteria to carry out a fermentation reaction in the fermentation tank containing the reaction solution.

[0050] The rate at which the reaction solution from which isopropanol has been separated is not particularly limited, and can be carried out in the same way as the withdrawal rate described above, in order to keep the amount of reaction solution in the fermenter approximately constant.

[0051] The method of this embodiment may, in one or more embodiments, include maintaining the isopropanol concentration of the reaction solution in the fermenter at 6 g / L or less, in order to further improve the productivity of isopropanol. Therefore, the method of this embodiment, in order to further improve the productivity of isopropanol, includes, in one or more embodiments, causing a fermentation reaction by Corynebacterium bacteria in a reaction solution in which the isopropanol concentration is controlled to 6 g / L or less. The isopropanol concentration in the fermenter is, in one or more embodiments, 5.9 g / L or less, 5.8 g / L or less, 5.7 g / L or less, 5.6 g / L or less, 5.5 g / L or less, 5.4 g / L or less, or 5.3 g / L or less. In one or more embodiments, the isopropanol concentration in the fermenter is preferably 0 g / L to 5.9 g / L, 0 g / L to 5.8 g / L, 0 g / L to 5.7 g / L, 0 g / L to 5.6 g / L, 0 g / L to 5.5 g / L, 0 g / L to 5.4 g / L, or 0 g / L to 5.3 g / L. In this disclosure, the isopropanol concentration of the reaction solution in the fermenter can be measured in one or more embodiments using ultra-high performance liquid chromatography or gas chromatography, for example, based on the examples.

[0052] In one or more embodiments, the method of this embodiment may include removing the reaction solution from the fermenter and / or supplying the reaction solution from which the isopropanol has been separated to the fermenter so that the isopropanol concentration in the fermenter is maintained at 6 g / L or less.

[0053] In order to further improve the productivity of isopropanol, the method of this embodiment may, in one or more embodiments, include maintaining the isopropanol concentration in the fermenter at 5 g / L or less, 4.9 g / L or less, 4.8 g / L or less, or 4.7 g / L or less 24 hours after the start of the fermentation reaction (cultivation), by sequentially separating isopropanol from the reaction solution by pervaporation. In order to further improve the productivity of isopropanol, the method of this embodiment may, in one or more embodiments, include maintaining the isopropanol concentration in the fermenter at 0 g / L to 5 g / L, 0 g / L to 4.9 g / L, 0 g / L to 4.8 g / L, or 0 g / L to 4.7 g / L 24 hours after the start of the fermentation reaction (cultivation).

[0054] The method of this embodiment may, in order to further improve the productivity and yield of isopropanol, include, in one or more embodiments, allowing Corynebacterium bacteria to undergo a fermentation reaction under conditions in which the production ratio (weight ratio) of isopropanol to by-products in the fermenter is shifted to the isopropanol side by sequential separation of isopropanol by pervaporation. The method of this embodiment may, in order to further improve the productivity and yield of isopropanol, include, in one or more embodiments, performing the separation of isopropanol by pervaporation such that the weight ratio of isopropanol to acetone in the fermenter (isopropanol / acetone) is maintained at 3 or higher. The weight ratio of isopropanol to acetone in the fermenter (isopropanol / acetone) is 3 or higher in one or more embodiments, preferably 4 or higher, 5 or higher, 7 or higher, 10 or higher, or 20 or higher, in order to further improve the productivity and yield of isopropanol. The weight ratio of isopropanol to acetone in the fermenter (isopropanol / acetone) is 100 or less in one or more embodiments, not limited to this set. The weight ratio of isopropanol to acetone in the fermenter (isopropanol / acetone) is 3 to 100 in one or more embodiments, not limited to this set, preferably 4 to 100, 5 to 100, 7 to 100, 10 to 100, or 20 to 100. The weight ratio of isopropanol to acetone in the fermenter (isopropanol / acetone) can be calculated by measuring the concentrations (g / L) of isopropanol and acetone in the reaction solution in the fermenter using ultra-high performance liquid chromatography or gas chromatography, and then dividing the concentration of isopropanol by the concentration of acetone. The concentrations of isopropanol and acetone can be measured based on the examples. The weight ratio of isopropanol to acetone in the fermentation tank can be controlled in one or more embodiments by the degree of vacuum on the permeation side of the pervaporation module and the rate at which the reaction solution flows into the pervaporation module.

[0055] In one or more embodiments, separation by pervaporation may be carried out under conditions such that the weight ratio of isopropanol production to acetic acid production at 48 hours of culture (isopropanol / acetic acid) is 15 or more. In this disclosure, "production amount" means the sum of the amount separated and recovered by pervaporation and the amount contained in the culture medium (amount produced in the fermenter). In one or more embodiments, the weight ratio of production amount (isopropanol / acetic acid) at 48 hours of culture is 20 or more, 25 or more, 30 or more, 35 or more, or 40 or more. In one or more embodiments that are not limited, the weight ratio of production amount (isopropanol / acetic acid) at 48 hours of culture is 50 or less. In one or more embodiments that are not limited, the weight ratio of production amount (isopropanol / acetic acid) at 48 hours of culture is 20 to 50, 25 to 50, 30 to 50, 35 to 50, or 40 to 50. The weight ratio of isopropanol to acetic acid produced at 48 hours of culture is the weight ratio of isopropanol to acetic acid produced by the fermentation reaction of genetically modified Corynebacterium bacteria during the 48 hours of culture from the start of culture. This can be calculated by dividing the amount of isopropanol produced at 48 hours of culture by the amount of acetic acid produced at 48 hours of culture. The amount of isopropanol produced at 48 hours of culture can be obtained by measuring the concentration of isopropanol in the permeate separated by pervaporation between the start of culture and 48 hours, and the concentration of isopropanol in the reaction solution in the fermenter after 48 hours of culture, using ultra-high performance liquid chromatography, and then adding these together. The amount of acetic acid produced at 48 hours of culture can be obtained by measuring the concentration of acetic acid in the reaction solution in the fermenter after 48 hours of culture using ultra-high performance liquid chromatography. The concentrations of isopropanol and acetic acid can be measured according to the examples. In this disclosure, "start of culture" means inoculating a pre-culture solution into a fermenter that has reached pre-set culture conditions. "Reached pre-set culture conditions" means that the temperature, pH, stirring speed, etc. of the reaction solution have reached the set conditions."Pre-culture medium" refers to the culture medium obtained during pre-culture, which is a small-scale culture performed prior to the main culture under the same or similar conditions as the normal culture conditions.

[0056] Separation by pervaporation may, in one or more embodiments, be performed under conditions such that the weight ratio of isopropanol production to acetic acid production at the end of culture (isopropanol / acetic acid) is 15 or more. In this disclosure, "end of culture" means the time when the end time set in the production plan in advance is reached, or the time when culture can no longer be continued due to unforeseen circumstances, etc. The weight ratio of production at the end of culture (isopropanol / acetic acid) is 20 or more, 25 or more, 30 or more, 35 or more, or 40 or more in one or more embodiments. The weight ratio of production at the end of culture (isopropanol / acetic acid) is 50 or less in one or more embodiments that are not limited. The weight ratio of production at the end of culture (isopropanol / acetic acid) is 20 to 50, 25 to 50, 30 to 50, 35 to 50, or 40 to 50 in one or more embodiments that are not limited. The weight ratio of isopropanol to acetic acid at the end of culture is the weight ratio of isopropanol to acetic acid produced by fermentation in genetically modified Corynebacterium bacteria from the start to the end of culture, and can be calculated by dividing the amount of isopropanol produced at the end of culture by the amount of acetic acid produced at the end of culture. The amount of isopropanol produced at the end of culture can be obtained by measuring the concentration of isopropanol in the permeate separated by pervaporation from the start to the end of culture and the concentration of isopropanol in the reaction solution in the fermenter at the end of culture using ultra-high performance liquid chromatography, and then adding these together. The amount of acetic acid produced at the end of culture can be obtained by measuring the concentration of acetic acid in the reaction solution in the fermenter at the end of culture using ultra-high performance liquid chromatography. The concentrations of isopropanol and acetic acid can be measured according to the examples.

[0057] In one or more embodiments, the method of this embodiment includes separating isopropanol from the reaction solution by pervaporation under conditions where the weight ratio of isopropanol production to acetic acid production (isopropanol / acetic acid) is 15 or more, and it is preferable to continue separating isopropanol from the reaction solution by pervaporation under conditions where the isopropanol / acetic acid ratio is 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, or 40 or more after 48 hours from the start of cultivation. In one or more embodiments, the method of this embodiment includes separating isopropanol from the reaction solution by pervaporation under conditions where the weight ratio of isopropanol production to acetic acid production (isopropanol / acetic acid) is 15 or more and 50 or less. After 48 hours from the start of cultivation, the separation of isopropanol from the reaction solution by pervaporation may be continued under conditions where the isopropanol / acetic acid ratio is 15 or more and 50 or less, 20 or more and 50 or less, 25 or more and 50 or less, 30 or more and 50 or less, 35 or more and 50 or less, or 40 or more and 50 or less.

[0058] The weight ratio of isopropanol production to acetic acid production, as described above, can be controlled in one or more embodiments by the degree of vacuum on the permeation side of the pervaporation module and the rate at which the reaction solution flows into the pervaporation module.

[0059] In the method of this embodiment, it is preferable to carry out isopropanol production by fermentation reaction (cultivation) continuously. The duration of the fermentation reaction (cultivation) is 40 hours or more, 50 hours or more, 100 hours or more, 200 hours or more, or 500 hours or more in one or more embodiments, which are not particularly limited.

[0060] The method of this embodiment may, in one or more embodiments, include discharging a portion of the reaction liquid in the fermenter to the outside of the system, in order to reduce the concentration of non-volatile components (e.g., salts and acids) that may adversely affect the growth of microorganisms in the fermenter.

[0061] The method of this embodiment may, in one or more embodiments, include, in terms of recovering microbial cells trapped in a pervaporation module (pervaporation membrane, etc.), discharging a portion of the reaction solution from the fermentation tank, removing microbial cells from the discharged reaction solution, and supplying the reaction solution from which the microbial cells have been removed to a pervaporation module, and then supplying the reaction solution that has passed through the pervaporation module to the fermentation tank. By supplying the reaction solution from which the microbial cells have been removed to a pervaporation module, it is possible to recover microbial cells trapped in the pervaporation module (pervaporation membrane, etc.). Examples of microbial cells include the Corynebacterium species described above, which have undergone a fermentation reaction in a reaction tank, in one or more embodiments.

[0062] The isopropanol obtained by the method of this disclosure may be used as is or purified in one or more embodiments. Therefore, the method of this disclosure may, in one or more embodiments, include purifying the recovered isopropanol or aqueous isopropanol solution. Examples of purification methods in one or more embodiments include membrane separation and distillation.

[0063] [Method for Producing Isopropanol (Second Embodiment)] A second embodiment of the present disclosure relates to a method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified microorganisms capable of producing isopropanol to produce a fermentation product containing isopropanol; separating isopropanol from at least a portion of the reaction solution containing the fermentation product in the fermenter; supplying the reaction solution from which the isopropanol has been separated to the fermenter; and maintaining the isopropanol concentration in the fermenter at 6 g / L or less.

[0064] A second aspect of the present disclosure is characterized by carrying out a fermentation reaction with a genetically modified microorganism capable of producing isopropanol in a fermenter in which the isopropanol concentration is maintained at 6 g / L or less, and separating isopropanol from the reaction solution in parallel with the fermentation reaction. In one or more embodiments, the isopropanol concentration in the fermenter is 5.9 g / L or less, 5.8 g / L or less, 5.7 g / L or less, 5.6 g / L or less, 5.5 g / L or less, 5.4 g / L or less, or 5.3 g / L or less. In one or more embodiments, the isopropanol concentration in the fermenter is preferably 0 g / L to 5.9 g / L, 0 g / L to 5.8 g / L, 0 g / L to 5.7 g / L, 0 g / L to 5.6 g / L, 0 g / L to 5.5 g / L, 0 g / L to 5.4 g / L, or 0 g / L to 5.3 g / L. According to a second aspect of this disclosure, in one or more embodiments, isopropanol can be produced with high productivity while suppressing the production of by-products. According to a second aspect of this disclosure, in one or more embodiments, the production ratio of isopropanol to by-products can be improved. According to a second aspect of this disclosure, in one or more embodiments, an aqueous solution of isopropanol having an isopropanol concentration of 30 g / L or more can be obtained.

[0065] Examples of genetically modified microorganisms include, in one or more embodiments, coryneform bacteria, Escherichia coli (Escherichia species, particularly Escherichia coli), solvent-resistant bacteria, and yeast.

[0066] Corynebacteria are a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology (Vol. 8, 599 (1974)), and are not particularly limited as long as they grow under normal aerobic conditions. Examples of Corynebacteria include, in one or more embodiments, bacteria of the genera Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, and Micrococcus. Examples of solvent-resistant bacteria include, in one or more embodiments, Pseudomonas putida S12, Pseudomonas aeruginosa, Pseudomonas paucimobilis, Pseudomonas alcaligenes, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas oleovorans, Pseudomonas sp., Rhodococcus erythropolis, Rhodococcus opacus, Burkholderia cepacia, and Paenibacillus illinoisensis.

[0067] In one or more embodiments, genetically modified microorganisms may have at least one of the activities of producing isopropanol from acetone and producing acetone from acetoacetic acid enhanced by genetic modification.

[0068] In the methods of this embodiment, one or more embodiments may use a Corynebacterium bacterium of the genus described herein as the genetically modified microorganism used. In the methods of this embodiment, one or more embodiments may use the transformant described in Japanese Patent No. 5395063 as the genetically modified microorganism used.

[0069] The isopropanol production step by fermentation in the present embodiment can be carried out in the same manner as the first embodiment, except that a genetically modified microorganism capable of producing isopropanol is subjected to a fermentation reaction in a fermenter in which the isopropanol concentration is maintained at 6 g / L or less.

[0070] The method for recovering isopropanol in this embodiment is not particularly limited and can be carried out using methods known in the art. Examples of isopropanol recovery methods in one or more embodiments include gas stripping, pervaporation, distillation, adsorption, ion exchange resin, and solvent extraction.

[0071] When isopropanol is separated from the reaction solution by pervaporation, the procedure can be carried out in the same manner as the isopropanol separation step by pervaporation in the first embodiment.

[0072] The supply (reuse) of the reaction solution to the fermenter in the method of this embodiment can be carried out in the same manner as the supply of the reaction solution to the fermenter in the first embodiment, except that the isopropanol concentration in the fermenter is maintained at 6 g / L or less.

[0073] [Method for Producing Isopropanol (Third Embodiment)] A third embodiment of the present disclosure relates to a method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified microorganisms capable of producing isopropanol to produce a fermentation product containing isopropanol; separating isopropanol from the reaction solution by passing a portion of the reaction solution containing the fermentation product in the fermenter through a pervaporation module; supplying the reaction solution that has passed through the pervaporation module to the fermenter; and maintaining the isopropanol concentration in the fermenter at 6 g / L or less.

[0074] A third aspect of the present disclosure is characterized by carrying out a fermentation reaction with genetically modified microorganisms capable of producing isopropanol in a fermenter in which the isopropanol concentration is maintained at 6 g / L or less, and separating the isopropanol in parallel with the fermentation reaction by pervaporation. In one or more embodiments, the isopropanol concentration in the fermenter is 5.9 g / L or less, 5.8 g / L or less, 5.7 g / L or less, 5.6 g / L or less, 5.5 g / L or less, 5.4 g / L or less, or 5.3 g / L or less. In one or more embodiments, the isopropanol concentration in the fermenter is preferably 0 g / L to 5.9 g / L, 0 g / L to 5.8 g / L, 0 g / L to 5.7 g / L, 0 g / L to 5.6 g / L, 0 g / L to 5.5 g / L, 0 g / L to 5.4 g / L, or 0 g / L to 5.3 g / L. According to a third aspect of this disclosure, in one or more embodiments, isopropanol can be produced with high productivity while suppressing the production of by-products. According to a third aspect of this disclosure, in one or more embodiments, the production ratio of isopropanol to by-products can be improved. According to a third aspect of this disclosure, in one or more embodiments, an aqueous solution of isopropanol having an isopropanol concentration of 30 g / L or more can be obtained.

[0075] As genetically modified microorganisms, the microorganisms exemplified in the second embodiment can be used.

[0076] The method of this embodiment can be carried out in one or more embodiments based on the second embodiment, except that the separation of isopropanol from the reaction solution is performed by pervaporation. The separation of isopropanol by pervaporation can be carried out in the same manner as the isopropanol separation step by pervaporation of the first embodiment.

[0077] [Method for Producing Isopropanol (Fourth Aspect)] A fourth aspect of the present disclosure relates to a method for producing isopropanol, comprising causing a fermentation reaction in a genetically modified Corynebacterium bacterium of the present disclosure, as described later, to produce isopropanol.

[0078] The production of isopropanol by fermentation in the fourth aspect of this disclosure can be carried out with reference to the first to third aspects. The method for recovering isopropanol in the fourth aspect of this disclosure is not particularly limited. Examples of isopropanol recovery methods in one or more embodiments include gas stripping, pervaporation, distillation, adsorption, ion exchange resin, and solvent extraction. When isopropanol is separated from the reaction solution by pervaporation, it can be carried out in the same manner as the isopropanol separation step by pervaporation in the first aspect.

[0079] According to the methods of this disclosure (referred to as "Methods of the First to Fourth Aspects of this Disclosure," hereinafter the same), an isopropanol productivity of 0.5 g / L / hr or more can be achieved in one or more embodiments. The isopropanol productivity in the methods of this disclosure is 0.55 g / L / hr or more, 0.6 g / L / hr or more, or 0.65 g / L / hr or more in one or more embodiments. According to the methods of this disclosure, an isopropanol-to-sugar yield of 25% (mol / mol) or more can be achieved in one or more embodiments. The isopropanol-to-sugar yield in the methods of this disclosure is 26% (mol / mol) or more, 27% (mol / mol) or more, or 28% (mol / mol) or more in one or more embodiments. According to the methods of this disclosure, since the reaction liquid from which isopropanol has been separated by pervaporation is supplied (reused) to a fermenter, the amount of waste liquid can be reduced.

[0080] The method of this disclosure can suppress the generation of by-products in one or more embodiments. The acetone-to-sugar yield in the method of this disclosure is 10% (mol / mol) or less in one or more embodiments, preferably 5% (mol / mol) or less, 4.5% (mol / mol) or less, or 4% (mol / mol) or less. The amount of acetone in the fermenter in the method of this disclosure is 1.5 g / L or less, 1 g / L or less, 0.9 g / L or less, 0.8 g / L or 0.7 g / L or less in one or more embodiments. The acetic acid-to-sugar yield in the method of this disclosure is 5% (mol / mol) or less in one or more embodiments, preferably 4% (mol / mol) or less, 3% (mol / mol) or less, 2% (mol / mol) or less, or 1% (mol / mol) or less. In the method of the present disclosure, the amount of acetic acid in the fermenter is, in one or more embodiments, 1.5 g / L or less, 1.4 g / L or less, 1.3 g / L or less, 1.2 g / L or less, or 1.1 g / L or less.

[0081] According to the method of this disclosure, in one or more embodiments, an aqueous isopropanol solution can be obtained having an isopropanol concentration of 30 g / L or more, 35 g / L or more, 40 g / L or more, 45 g / L or more, 50 g / L or more, or 55 g / L or more.

[0082] The isopropanol produced by the method of this disclosure can be used as a raw material for propylene and acrylic acid, etc., in one or more embodiments. Therefore, the method of this disclosure can also be described in one or more embodiments as a method for producing isopropanol for use in the production of propylene or acrylic acid or derivatives thereof.

[0083] [Method for producing propylene or acrylic acid or derivatives thereof] In other aspects, this disclosure relates to a method for producing propylene or acrylic acid or derivatives thereof, characterized in that isopropanol produced by the method of this disclosure is used as a raw material to produce propylene or acrylic acid or derivatives thereof.

[0084] In one or more embodiments, the production method of this embodiment includes dehydrating isopropanol produced by the method of the present disclosure to obtain propylene, and obtaining acrylic acid or a derivative thereof from the obtained propylene. In one or more embodiments, the production method of this embodiment includes obtaining acrylic acid or acrolein by partially oxidizing the propylene obtained by dehydrating isopropanol produced by the method of the present disclosure. In one or more embodiments, the dehydration of isopropanol and the partial oxidation of propylene may be carried out stepwise or simultaneously, but it is preferable to produce them continuously using a catalyst.

[0085] Acrylic acid can be reacted with alcohol to obtain acrylic acid esters, and can also be derived into various derivatives such as acrylate salts, acrylic polymers, and superabsorbent polymers. Specifically, acrylic acid, acrylic acid esters, and acrylate salts can be (co)polymerized individually or in combination to produce various hydrophilic and lipophilic acrylic polymers. Crosslinked resins such as superabsorbent polymers can also be produced by partial crosslinking. Examples of propylene derivatives in one or more embodiments include polypropylene, acrylonitrile, ABS resin, acetone, bisphenol A, phenol, and acrylic acid. Examples of acrylic acid derivatives in one or more embodiments include acrylate salts, methacrylate esters, acrylic acid esters, polyacrylic acid, acrylic polymers, and superabsorbent polymers (SAP). Examples of acrylic acid esters in one or more embodiments include methyl acrylate, ethyl acrylate, butyl acrylate, and 2-ethylhexyl acrylate. The above acrylic acids and various derivatives can be suitably used in various applications such as adhesives, paints, detergents, and sanitary materials.

[0086] [Genetically Modified Corynebacterium Bacteria] In one aspect, this disclosure relates to a genetically modified Corynebacterium bacterium having the ability to produce isopropanol. The genetically modified Corynebacterium bacterium of this disclosure can be used in the methods for producing isopropanol according to this disclosure (including the first to fourth aspects described above). The genetically modified Corynebacterium bacterium of this disclosure has at least one of gene (A) and gene (B) introduced into it so as to be expressible, wherein gene (A) is at least one gene selected from the group consisting of (A1) to (A9) below and is a gene that encodes an enzyme having the activity to produce acetoacetate from acetoacetyl-CoA, and gene (B) is at least one gene selected from the group consisting of (B1) to (B9) below and is a gene that encodes an enzyme having the activity to produce acetone from acetoacetate.

[0087] The genetically modified Corynebacterium bacteria of this disclosure have at least one of the following genes introduced by genetic recombination: gene (A) encoding an enzyme that has the activity to produce acetoacetate from acetoacetyl-CoA selected from the group consisting of (A1) to (A9) below, and gene (B) encoding an enzyme that has the activity to produce acetone from acetoacetate selected from the group consisting of (B1) to (B9) below. In one or more embodiments, the Corynebacterium bacteria of this disclosure can produce isopropanol from acetoacetyl-CoA and / or acetoacetate (for example, Figure 12). In other embodiments of this disclosure, the Corynebacterium bacteria of this disclosure can produce isopropanol from a carbon source such as glucose (for example, Figure 13).

[0088] In this disclosure, "90% or more identity" with respect to an amino acid sequence or base sequence means at least 90% identity, and in one or more embodiments, it means 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more, or 100% identity.

[0089] In this disclosure, "identity of amino acid sequence or nucleotide sequence" can be performed using readily available sequence comparison computer programs. Examples of such computer programs in one or more embodiments include the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387), the BLAST package (Ausubel et al. (1999) ibid-Ch. 18), and FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410).

[0090] In this disclosure, “stringent conditions” means conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. In one or more embodiments, stringent conditions include conditions under which highly identical base sequences hybridize, but less identical base sequences do not hybridize. In one or more embodiments, high identity between base sequences includes cases where the identity is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% or more. In one or more embodiments, stringent conditions may also be those described in Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Vol2, p11.45. Specifically, hybridization may occur at a temperature 5°C to 10°C lower than the melting temperature (Tm) of the complete hybrid.

[0091] The Corynebacterium bacteria of this disclosure are, in one or more embodiments, Corynebacterium bacteria having isopropanol production ability and possessing at least one of the above gene (A) and the above gene (B). In one or more embodiments, the Corynebacterium bacteria of this disclosure may have one or more of the above two types of genes (the above gene (A) and the above gene (B)) expressedly introduced, or the expression of one or more genes may be enhanced, or one or more genes may be expressedly introduced and the expression of one or more genes may be enhanced. Therefore, in one or more embodiments, the microorganisms of this disclosure can also be called transformants. In one or more embodiments, the microorganisms of this disclosure can also be called transformants in which isopropanol production ability is improved by introducing and / or enhancing the expression of the above genes.

[0092] In this disclosure, "transformed organisms obtained by introducing a gene" may include transformed organisms obtained by introducing the gene in a way that enables expression, and transformed organisms that can be obtained by introducing the gene in a way that enables expression.

[0093] Methods for introducing genes include, in one or more embodiments, methods using general genetic engineering techniques (for example, the method described in Michael R. Green & Joseph Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory Press). Gene introduction methods include, in one or more embodiments, gene introduction using plasmid vectors or integration into the chromosomes of a microbial host.

[0094] In this disclosure, "introducing a gene in an expressible manner" means, in one or more embodiments, introducing the introduced gene in a manner that allows it to be expressed in the transformant. Methods for introducing a gene in an expressible manner include, in one or more embodiments, introducing the gene together with an expression regulatory sequence such as a promoter capable of inducing increased gene expression. In this disclosure, the gene introduced together with a promoter capable of inducing increased expression may, in one or more embodiments, be a single gene or an operon capable of expressing multiple genes. Known promoters can be used as promoters capable of inducing increased expression.

[0095] In this disclosure, "a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: XX" means a gene encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: XX. In this disclosure, "a gene having the nucleotide sequence shown in SEQ ID NO: XX" means a gene containing the nucleotide sequence shown in SEQ ID NO: XX.

[0096] [Gene (A)] In one or more embodiments, the genetically modified Corynebacterium bacteria of this disclosure may have a gene (A) selected from the group consisting of (A1) to (A9) below, which encodes an enzyme having the activity to produce acetoacetic acid from acetoacetyl-CoA. In one or more embodiments, gene (A) encodes a polypeptide having acyl-CoA thioesterase (tes, EC number: 3.1.2.-) activity. In the genetically modified Corynebacterium bacteria of this disclosure, gene (A) may, in one or more embodiments, be an exogenous gene, an intrinsically present gene of the microorganism, or an intrinsically present gene of the microorganism with enhanced expression.

[0097] In one or more embodiments, the origin of gene (A) may be the genus Pseudomonas, etc. The names (designations) of the gene-derived organisms shown in this disclosure are examples only, and synonymous alternative names may exist.

[0098] Examples of the genus Pseudomonas include Pseudomonas putida in one or more embodiments. Examples of the tes gene derived from Pseudomonas putida in one or more embodiments include the gene consisting of the nucleotide sequence shown in SEQ ID NO: 4. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 4 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0099] In one or more embodiments, the tes gene derived from Pseudomonas putida is preferred for gene (A) because it further improves the productivity of isopropanol.

[0100] Gene (A) is selected from the following group consisting of (A1) to (A9): (A1) A gene from which the genus Pseudomonas originates; (A2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 14, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14; (A3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 14, or a gene encoding a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 14; (A4) A gene encoding a polypeptide having an amino acid sequence in which 100 amino acids constitute one unit, with 1 to 10 amino acids deleted, substituted, and / or added per unit, or a gene encoding a polypeptide consisting of an amino acid sequence in which 100 amino acids constitute one unit, with 1 to 10 amino acids deleted, substituted, and / or added per unit, in the amino acid sequence shown in SEQ ID NO: 14; (A5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 14, or a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14; (A6) A gene having the nucleotide sequence shown in SEQ ID NO: 4, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 4; (A7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 4, or a gene consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 4; (A8) A gene having a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 4, where 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted and / or added per unit; or a gene consisting of a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 4, where 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted and / or added per unit;(A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 4, or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 4.

[0101] In order to further improve the productivity of isopropanol, gene (A) is preferably, in one or more embodiments, a gene encoding a polypeptide having or comprising the amino acid sequence shown in SEQ ID NO: 14, a gene encoding a polypeptide having or comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 14, a gene having or comprising the base sequence shown in SEQ ID NO: 4, or a gene having a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 4.

[0102] [Gene (B)] In one or more embodiments, the genetically modified Corynebacterium bacteria of the present disclosure may have a gene (B) selected from the group consisting of (B1) to (B9) below, which encodes an enzyme having the activity to produce acetone from acetoacetate. In one or more embodiments, gene (B) encodes a polypeptide having acetacetate decarboxylase (adc, EC number: 4.1.1.4) activity. In the genetically modified Corynebacterium bacteria of the present disclosure, gene (B) may, in one or more embodiments, be an exogenous gene, an intrinsically present gene of the microorganism, or an intrinsically present gene of the microorganism with enhanced expression.

[0103] In one or more embodiments, the gene (B) may originate from the genera Paenibacillus, Bradyrhizobium, Amycolatopsis, Paraburkholderia, and Burkholderia. In one or more embodiments, the genus Paenibacillus may include Paenibacillus polymyxa and Paenibacillus graminis. In one or more embodiments, the adc gene derived from Paenibacillus polymyxa may consist of the nucleotide sequence shown in SEQ ID NO: 7. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 7 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15. In one or more embodiments, the adc gene from Paenibacillus graminis may consist of the nucleotide sequence shown in SEQ ID NO: 23. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 23 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 55. Examples of the genus Bradyrhizobium include, in one or more embodiments, Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, and Bradyrhizobium japonicum. An example of an adc gene derived from Bradyrhizobium diazoefficiens, in one or more embodiments, is the gene consisting of the nucleotide sequence shown in SEQ ID NO: 17. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 17 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 53. An example of an adc gene from Bradyrhizobium elkanii, in one or more embodiments, is the gene consisting of the nucleotide sequence shown in SEQ ID NO: 26. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 26 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 56. An example of an adc gene from Bradyrhizobium japonicum, in one or more embodiments, is the gene consisting of the nucleotide sequence shown in SEQ ID NO: 29.The gene consisting of the nucleotide sequence shown in SEQ ID NO: 29 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 57. Examples of the genus Amycolatopsis include, in one or more embodiments, Amycolatopsis mediterranei. Examples of the adc gene of Amycolatopsis mediterranei include, in one or more embodiments, the gene consisting of the nucleotide sequence shown in SEQ ID NO: 20. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 20 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 54. Examples of the genus Paraburkholderia include, in one or more embodiments, Paraburkholderia caribensis. Examples of the adc gene of Paraburkholderia caribensis include, in one or more embodiments, the gene consisting of the nucleotide sequence shown in SEQ ID NO: 32. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 32 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 58. Examples of the genus Burkholderia include, in one or more embodiments, Burkholderia glumae. Examples of the adc gene of Burkholderia glumae include, in one or more embodiments, the gene consisting of the nucleotide sequence shown in SEQ ID NO: 65. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 65 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 68.

[0104] In one or more embodiments, the adc gene (B) is preferred from Bradyrhizobium japonicum, Bradyrhizobium elkanii, Paenibacillus polymyxa, Paenibacillus graminis, Amycolatopsis mediterranei, Bradyrhizobium diazoefficiens, Paraburkholderia caribensis, or Burkholderia glumae, as it further improves the productivity of isopropanol.

[0105] Gene (B) is selected from the following group consisting of (B1) to (B9): (B1) A gene derived from at least one microorganism selected from the group consisting of the genera Paenibacillus, Bradyrhizobium, Amycolatopsis, Paraburkholderia, and Burkholderia; (B2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NOs. 15, 53, 54, 55, 56, 57, 58, or 68, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs. 15, 53, 54, 55, 56, 57, 58, or 68; (B3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NOs. 15, 53, 54, 55, 56, 57, 58, or 68, or a gene encoding a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NOs. 15, 53, 54, 55, 56, 57, 58, or 68; (B4) A gene encoding a polypeptide having an amino acid sequence in which 100 amino acids are considered as one unit, and 1 to 10 amino acids are deleted, substituted and / or added per unit, as shown in the amino acid sequence of SEQ ID NOs. 15, 53, 54, 55, 56, 57, 58, or 68; or a gene encoding a polypeptide consisting of an amino acid sequence in which 100 amino acids are considered as one unit, and 1 to 10 amino acids are deleted, substituted and / or added per unit, as shown in the amino acid sequence of SEQ ID NOs. 15, 53, 54, 55, 56, 57, 58, or 68; (B5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NOs. 15, 53, 54, 55, 56, 57, 58, or 68, or a gene having a nucleotide sequence complementary to a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs. 15, 53, 54, 55, 56, 57, 58, or 68, and a gene that hybridizes under stringent conditions;(B6) A gene having the nucleotide sequence shown in SEQ ID NOs: 7, 17, 20, 23, 26, 29, 32, or 65, or a gene consisting of the nucleotide sequence shown in SEQ ID NOs: 7, 17, 20, 23, 26, 29, 32, or 65; (B7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NOs: 7, 17, 20, 23, 26, 29, 32, or 65, or a gene consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NOs: 7, 17, 20, 23, 26, 29, 32, or 65; (B8) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, with 1 to 10 nucleotide deletions, substitutions, and / or additions per unit, as shown in SEQ ID NOs: 7, 17, 20, 23, 26, 29, 32, or 65, or a gene consisting of a nucleotide sequence in which 100 nucleotides constitute one unit, with 1 to 10 nucleotide deletions, substitutions, and / or additions per unit; (B9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NOs: 7, 17, 20, 23, 26, 29, 32, or 65, or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NOs: 7, 17, 20, 23, 26, 29, 32, or 65.

[0106] In terms of further improving the productivity of isopropanol, gene (B) is preferably, in one or more embodiments, a gene encoding a polypeptide having or consisting of the amino acid sequence shown in SEQ ID NOs. 57, 56, 15, 55, 54, 53, 58, or 68; a gene encoding a polypeptide having or consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NOs. 57, 56, 15, 55, 54, 53, 58, or 68; a gene having or consisting of the nucleotide sequence shown in SEQ ID NOs. 29, 26, 7, 23, 20, 17, 32, or 65; or a gene having or consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NOs. 29, 26, 7, 23, 20, 17, 32, or 65.

[0107] [Gene (C)] In one or more embodiments, the genetically modified Corynebacterium bacteria of the Disclosure may be expressibly introduced to gene (C), which encodes an enzyme having the activity to produce isopropanol from acetone, in order to further improve the productivity of isopropanol. In one or more embodiments, gene (C) encodes a polypeptide having alcohol dehydrogenase (adh, EC number: 1.1.1.2) activity. In the genetically modified Corynebacterium bacteria of the Disclosure, gene (C) may, in one or more embodiments, be an exogenous gene, be a gene inherent to the microorganism, or be a gene whose expression is enhanced from a gene inherent to the microorganism.

[0108] The gene (C) may originate from the genera Levilactobacillus, Limosilactobacillus, Blautia, and Clostridium in one or more embodiments. Examples of the Levilactobacillus gene include Levilactobacillus brevis in one or more embodiments. An example of the adh gene derived from Levilactobacillus brevis in one or more embodiments is the gene consisting of the nucleotide sequence shown in SEQ ID NO: 10. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 10 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16. Examples of the Limosilactobacillus gene in one or more embodiments include Limosilactobacillus gastricus and Limosilactobacillus fermentum. An example of the adh gene derived from Limosilactobacillus gastricus in one or more embodiments is the gene consisting of the nucleotide sequence shown in SEQ ID NO: 35. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 35 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 59. In one or more embodiments, an adh gene derived from Limosilactobacillus fermentum is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 38. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 38 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 60. In one or more embodiments, an example of the genus Blautia is Blautia producta. In one or more embodiments, an adh gene of Blautia producta is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 41. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 41 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 61.Examples of the genus Clostridium include, in one or more embodiments, Clostridium isatidis, Clostridium cochlearium, and Clostridium tyrobutyricum. In one or more embodiments, the adh gene of Clostridium isatidis is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 44. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 44 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 62. In one or more embodiments, the adh gene of Clostridium cochlearium is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 47. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 47 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 63. In one or more embodiments, the adh gene of Clostridium tyrobutyricum is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 50. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 50 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 64.

[0109] Gene (C) is preferably an adh gene derived from Limosilactobacillus gastricus, Clostridium tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, Levilactobacillus brevis, Clostridium isatidis, or Blautia producta in one or more embodiments, in terms of further improving the productivity of isopropanol, and more preferably an adh gene derived from Limosilactobacillus gastricus, Clostridium tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, Levilactobacillus brevis, or Clostridium isatidis, and more preferably an adh gene derived from Limosilactobacillus gastricus, Clostridium tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, or Levilactobacillus A brevis-derived ADH gene is even more preferable.

[0110] In one or more embodiments, gene (C) may be at least one gene selected from the group consisting of (C1) to (C9) below: (C1) A gene derived from the genera Levilactobacillus, Limosilactobacillus, Blautia, or Clostridium; (C2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NOs. 16, 59, 60, 61, 62, 63, or 64, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs. 16, 59, 60, 61, 62, 63, or 64; (C3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NOs. 16, 59, 60, 61, 62, 63, or 64, or a gene encoding a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NOs. 16, 59, 60, 61, 62, 63, or 64; (C4) A gene encoding a polypeptide having an amino acid sequence in which 100 amino acids constitute one unit, with 1 to 10 amino acids deleted, substituted, and / or added per unit, as shown in the amino acid sequence of SEQ ID NO: 16, 59, 60, 61, 62, 63, or 64; or a gene encoding a polypeptide consisting of an amino acid sequence in which 100 amino acids constitute one unit, with 1 to 10 amino acids deleted, substituted, and / or added per unit, as shown in the amino acid sequence of SEQ ID NO: 16, 59, 60, 61, 62, 63, or 64; (C5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having an amino acid sequence of SEQ ID NO: 16, 59, 60, 61, 62, 63, or 64; or a gene that hybridizes under stringent conditions with a nucleotide sequence complementary to a gene encoding a polypeptide consisting of an amino acid sequence of SEQ ID NO: 16, 59, 60, 61, 62, 63, or 64; (C6) A gene having the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50, or a gene consisting of the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50;(C7) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50, or a gene consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50; (C8) A gene having a nucleotide sequence in the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50, where 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted, and / or added per unit; or a gene consisting of a nucleotide sequence in the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50, where 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted, and / or added per unit; (C9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50, or a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NOs: 10, 35, 38, 41, 44, 47, or 50, and a gene that hybridizes under stringent conditions.

[0111] In terms of further improving the productivity of isopropanol, in one or more embodiments, gene (C) is preferably a gene encoding a polypeptide having or consisting of the amino acid sequence shown in SEQ ID NOs. 59, 64, 63, 60, 16, 62, or 61; a gene encoding a polypeptide having or consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NOs. 59, 64, 63, 60, 16, 62, or 61; a gene having or consisting of the nucleotide sequence shown in SEQ ID NOs. 35, 50, 47, 38, 10, 44, or 41; or a gene having or consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NOs. 59, 64, 63, 60, 16, or 62; or a gene encoding a polypeptide having or consisting of the amino acid sequence shown in SEQ ID NOs. 59, 64, 63, 60, 16, or 62 More preferably, a gene encoding a polypeptide having or consisting of an amino acid sequence having the above-mentioned identity, a gene having or consisting of a nucleotide sequence shown in SEQ ID NOs. 35, 50, 47, 38, 10, or 44, or a gene having or consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NOs. 35, 50, 47, 38, 10, or 44 is preferred. Even more preferably, a gene encoding a polypeptide having or consisting of an amino acid sequence shown in SEQ ID NOs. 59, 64, 63, 60, or 16, a gene encoding a polypeptide having or consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NOs. 59, 64, 63, 60, or 16, a gene having or consisting of a nucleotide sequence shown in SEQ ID NOs. 35, 50, 47, 38, or 10, or a gene having 90% or more identity with the nucleotide sequence shown in SEQ ID NOs. 35, 50, 47, 38, or 10 is preferred.

[0112] [Gene (D)] In one or more embodiments, the genetically modified Corynebacterium bacteria of the Disclosure may be expressibly introduced to gene (D), which encodes an enzyme having the activity to produce acetoacetyl-CoA from acetyl-CoA and malonyl-CoA, in order to further improve the productivity of isopropanol. In one or more embodiments, gene (D) encodes a polypeptide having acetoacetyl-CoA synthase (aacs, EC number: 2.3.1.194) activity. In the genetically modified Corynebacterium bacteria of the Disclosure, gene (D) may, in one or more embodiments, be an exogenous gene, a gene inherent to the microorganism, or an enhanced expression of a gene inherent to the microorganism.

[0113] In one or more embodiments, the gene (D) may originate from the genus Streptomyces, etc. In one or more embodiments, the genus Streptomyces may include Streptomyces tendae, etc. In one or more embodiments, the aacs gene derived from Streptomyces tendae may consist of the nucleotide sequence shown in SEQ ID NO: 1. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13.

[0114] In one or more embodiments, the aacs gene derived from Streptomyces tendae is preferred as gene (D) from the viewpoint of further improving the productivity of isopropanol.

[0115] In one or more embodiments, gene (D) may be at least one gene selected from the group consisting of (D1) to (D9) below: (D1) A gene from which the genus Streptomyces originates; (D2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 13, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13; (D3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 13, or a gene encoding a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 13; (D4) A gene encoding a polypeptide having an amino acid sequence in which 100 amino acids constitute one unit, with 1 to 10 amino acids deleted, substituted, and / or added per unit, or a gene encoding a polypeptide consisting of an amino acid sequence in which 100 amino acids constitute one unit, with 1 to 10 amino acids deleted, substituted, and / or added per unit, in the amino acid sequence shown in SEQ ID NO: 13; (D5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 13, or a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 13; (D6) A gene having the nucleotide sequence shown in SEQ ID NO: 1, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1; (D7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1, or a gene consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 1; (D8) A gene having a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 1, where 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted and / or added per unit; or a gene consisting of a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 1, where 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted and / or added per unit;(D9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1, or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1.

[0116] In terms of further improving the productivity of isopropanol, gene (D) is preferably, in one or more embodiments, a gene encoding a polypeptide having or comprising the amino acid sequence shown in SEQ ID NO: 13, a gene encoding a polypeptide having or comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 13, a gene having or comprising the base sequence shown in SEQ ID NO: 1, or a gene having or comprising a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 1.

[0117] The microbial host is not particularly limited and, in one or more embodiments, may be a transformant of a bacterium of the genus Corynebacterium, in terms of further improving the productivity of isopropanol. Examples of Corynebacterium bacteria in one or more embodiments include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkanolyticum.

[0118] In this disclosure, the microbial host may, in one or more embodiments, be Corynebacterium glutamicum or a transformant thereof, in view of further improving the productivity of isopropanol. Similarly, it may be Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), or ATCC13869 (DSM1412), or transformants thereof.

[0119] The genetically modified Corynebacterium bacteria of this disclosure, in terms of further improving isopropanol productivity, in one or more embodiments, include (A) a tes gene derived from Pseudomonas putida, (B) an adc gene derived from at least one selected from the group consisting of Bradyrhizobium japonicum, Bradyrhizobium elkanii, Paenibacillus graminis, Paenibacillus polymyxa, Amycolatopsis mediterranei, Bradyrhizobium diazoefficiens, Paraburkholderia caribensis, and Burkholderia glumae, and (C) Limosilactobacillus gastricus, Clostridium tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, Levilactobacillus brevis, Clostridium isatidis, and Blautia Preferably, an adh gene derived from at least one selected from the group consisting of producta and (D) an aacs gene derived from Streptomyces tendae is introduced in an expressible manner, more preferably (A) a tes gene derived from Pseudomonas putida and (B) an adc gene derived from at least one selected from the group consisting of Bradyrhizobium japonicum, Bradyrhizobium elkanii, Paenibacillus graminis, Paenibacillus polymyxa, Amycolatopsis mediterranei, Bradyrhizobium diazoefficiens, Paraburkholderia caribensis, and Burkholderia glumae and (C) Limosilactobacillus gastricus, Clostridium tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, LevilactobacillusThe adh gene derived from brevis and Clostridium isatidis, and the aacs gene derived from (D) Streptomyces tendae are introduced to be expressible, and more preferably the tes gene derived from (A) Pseudomonas putida, the adc gene derived from (B) at least one selected from the group consisting of Bradyrhizobium japonicum, Bradyrhizobium elkanii, Paenibacillus graminis, Paenibacillus polymyxa, Amycolatopsis mediterranei, Bradyrhizobium diazoefficiens, and Paraburkholderia caribensis, the adh gene derived from (C) Limosilactobacillus gastricus, Clostridium tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, or Levilactobacillus brevis, and the aacs gene derived from (D) Streptomyces tendae are introduced to be expressible.

[0120] The genetically modified Corynebacterium bacteria of this disclosure are, in terms of further improving the productivity of isopropanol, preferably (1) to (3) below in one or more embodiments, more preferably (2) and (3), and even more preferably (3). (1) A Corynebacterium glutamicum transformant in which (A) the tes gene from Pseudomonas putida, (B) the adc gene from at least one selected from the group consisting of Bradyrhizobium japonicum, Bradyrhizobium elkanii, Paenibacillus graminis, Paenibacillus polymyxa, Amycolatopsis mediterranei, Bradyrhizobium diazoefficiens, Paraburkholderia caribensis, and Burkholderia glumae, (C) the adh gene from Limosilactobacillus gastricus, and (D) the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformant, or (A) the tes gene from Pseudomonas putida, (B) the adc gene from Paenibacillus polymyxa, and (C) Limosilactobacillus A Corynebacterium glutamicum transformant in which an adh gene derived from at least one selected from the group consisting of gastricus, Clostridium tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, Levilactobacillus brevis, Clostridium isatidis, and Blautia producta, and (D) an aacs gene derived from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformant.(2) Corynebacterium glutamicum transformants having (A) a tes gene derived from Pseudomonas putida, (B) an adc gene derived from at least one selected from the group consisting of Bradyrhizobium japonicum, Bradyrhizobium elkanii, Paenibacillus graminis, Paenibacillus polymyxa, Amycolatopsis mediterranei, Bradyrhizobium diazoefficiens, Paraburkholderia caribensis, and Burkholderia glumae, (C) an adh gene derived from Limosilactobacillus gastricus, and (D) an aacs gene derived from Streptomyces tendae expressed in Corynebacterium glutamicum or its transformants, or (A) a tes gene derived from Pseudomonas putida, (B) an adc gene derived from Paenibacillus polymyxa, and (C) Limosilactobacillus Corynebacterium glutamicum transformants are characterized in that an adh gene derived from at least one selected from the group consisting of gastricus, Clostridium tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, Levilactobacillus brevis, and Clostridium isatidis, and (D) an aacs gene derived from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants.(3) Corynebacterium glutamicum transformants having (A) a tes gene derived from Pseudomonas putida, (B) an adc gene derived from at least one selected from the group consisting of Bradyrhizobium japonicum, Bradyrhizobium elkanii, Paenibacillus graminis, Paenibacillus polymyxa, Amycolatopsis mediterranei, Bradyrhizobium diazoefficiens, and Paraburkholderia caribensis, (C) an adh gene derived from Limosilactobacillus gastricus, and (D) an aacs gene derived from Streptomyces tendae expressed in Corynebacterium glutamicum or its transformants, or (A) a tes gene derived from Pseudomonas putida, (B) an adc gene derived from Paenibacillus polymyxa, and (C) Limosilactobacillus gastricus, Clostridium A Corynebacterium glutamicum transformant in which an adh gene derived from at least one selected from the group consisting of tyrobutyricum, Clostridium cochlearium, Limosilactobacillus fermentum, and Levilactobacillus brevis, and (d) an aacs gene derived from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformant.

[0121] The genetically modified Corynebacterium bacteria of the present disclosure are, in view of further improving the productivity of isopropanol, preferably (1) to (14), more preferably (1) to (13), and even more preferably (1) to (11) in one or more embodiments: (1) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Bradyrhizobium japonicum, the adh gene from Limosilactobacillus gastricus, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (2) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Bradyrhizobium elkanii, the adh gene from Limosilactobacillus gastricus, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (3) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Paenibacillus polymyxa, the adh gene from Clostridium tyrobutyricum, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (4) Corynebacterium glutamicum transformants in which the tes gene derived from Pseudomonas putida, the adc gene derived from Paenibacillus graminis, the adh gene derived from Limosilactobacillus gastricus, and the aacs gene derived from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants.(5) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Amycolatopsis mediterranei, the adh gene from Limosilactobacillus gastricus, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (6) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Paenibacillus polymyxa, the adh gene from Limosilactobacillus gastricus, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (7) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Paenibacillus polymyxa, the adh gene from Clostridium cochlearium, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (8) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Paenibacillus polymyxa, the adh gene from Limosilactobacillus fermentum, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (9) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Bradyrhizobium diazoefficiens, the adh gene from Limosilactobacillus gastricus, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants.(10) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Paenibacillus polymyxa, the adh gene from Levilactobacillus brevis, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (11) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Paraburkholderia caribensis, the adh gene from Limosilactobacillus gastricus, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (12) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Burkholderia glumae, the adh gene from Limosilactobacillus gastricus, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (13) Corynebacterium glutamicum transformants in which the tes gene from Pseudomonas putida, the adc gene from Paenibacillus polymyxa, the adh gene from Clostridium isatidis, and the aacs gene from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants. (14) Corynebacterium glutamicum transformants in which the tes gene derived from Pseudomonas putida, the adc gene derived from Paenibacillus polymyxa, the adh gene derived from Blautia producta, and the aacs gene derived from Streptomyces tendae are expressedly introduced into Corynebacterium glutamicum or its transformants.

[0122] In order to further improve the productivity of isopropanol, the genetically modified Corynebacterium bacteria of the present disclosure preferably, in one or more embodiments, have expressed (A) a gene having or comprising the nucleotide sequence shown in SEQ ID NO: 4, or a gene having or comprising a nucleotide sequence having 90% or more identity to said sequence; (B) a gene having or comprising the nucleotide sequence shown in SEQ ID NOs: 29, 26, 7, 23, 20, 17, 32, or 65, or a gene having 90% or more identity to said sequence; (C) a gene having or comprising the nucleotide sequence shown in SEQ ID NOs: 35, 50, 47, 38, 10, 44, or 41, or a gene having or comprising a nucleotide sequence having 90% or more identity to said sequence; and more preferably, (A) a gene having or comprising the nucleotide sequence shown in SEQ ID NO: 4 (B) A gene having a nucleotide sequence that is 90% or more identical to the sequence shown in (SEQ ID NOs: 29, 26, 7, 23, 20, 17, or 32, or a nucleotide sequence that is 90% or more identical to the sequence shown in (SEQ ID NOs: 35, 50, 47, 38, 10, or 44, or a nucleotide sequence that is 90% or more identical to the sequence shown in (SEQ ID NOs: 1, or a nucleotide sequence that is 90% or more identical to the sequence shown in (SEQ ID NOs: 1) is introduced in an expressible manner, and more preferably (A) A gene having a nucleotide sequence that is 90% or more identical to the sequence shown in (SEQ ID NOs: 4, or a nucleotide sequence that is 90% or more identical to the sequence shown in (SEQ ID NOs: 4, or (B) A gene having a nucleotide sequence that is 90% or more identical to the sequence shown in (SEQ ID NOs: 29, 26, 7, 23, 20, 17, or 32,Alternatively, a gene having a nucleotide sequence that is 90% or more identical to the said sequence, or consisting of such a sequence, (C) a gene having or consisting of the nucleotide sequence shown in SEQ ID NOs. 35, 50, 47, 38, or 10, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to the said sequence, and (D) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO. 1, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to the said sequence, are introduced in an expressible manner.

[0123] The genetically modified Corynebacterium bacteria of this disclosure are, in terms of further improving the productivity of isopropanol, preferably (1) to (3) below in one or more embodiments, more preferably (2) and (3), and even more preferably (3). (1) A Corynebacterium glutamicum transformant having expressedly introduced (A) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 4, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, (B) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 29, 26, 7, 23, 20, 23, 32, or 65, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, (C) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 35, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, and (D) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 1, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, or Corynebacterium glutamicum transformants are characterized in that (A) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; (B) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; (C) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, 50, 47, 38, 10, 44, or 41, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; and (D) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, are expressedly introduced into Corynebacterium glutamicum or its transformants.(2) Corynebacterium glutamicum transformants in which (A) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 4, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, (B) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 29, 26, 7, 23, 20, 23 or 32, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, (C) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 35, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, and (D) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 1, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformants, or Corynebacterium glutamicum transformants are characterized in that (A) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; (B) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; (C) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, 50, 47, 38, 10, or 44, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; and (D) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, are expressedly introduced into Corynebacterium glutamicum or its transformants.(3) Corynebacterium glutamicum transformants in which (A) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, (B) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 29, 26, 7, 23, 20, or 32, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, (C) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 35, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and (D) a gene having or consisting of the nucleotide sequence shown in SEQ ID NO: 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformants, or Corynebacterium glutamicum transformants are characterized in that (A) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; (B) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; (C) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, 50, 47, 38, or 10, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence; and (D) a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence that is 90% or more identical to said sequence, are expressedly introduced into Corynebacterium glutamicum or its transformants.

[0124] The genetically modified Corynebacterium bacteria of the present disclosure are, in terms of further improving the productivity of isopropanol, preferably (1) to (14), more preferably (1) to (13), and even more preferably (1) to (11) in one or more embodiments: (1) A Corynebacterium glutamicum transformant having expressibly introduced into Corynebacterium glutamicum or its transformant a gene having or comprising a gene having or comprising a nucleotide sequence, which is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 4, a gene having or comprising a nucleotide sequence, which is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 29, a gene having or comprising a nucleotide sequence, which is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 35, a gene having or comprising a nucleotide sequence, which is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1, or a gene having or comprising a nucleotide sequence, which is 90% or more identical to the nucleotide sequence. (2) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 26, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant.(3) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 50, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant. (4) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 23, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant.(5) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 20, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant. (6) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant.(7) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 47, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant. (8) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 38, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant.(9) Corynebacterium glutamicum transformant having expressedly introduced a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence; a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 17, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence; a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence; and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence. (10) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 10, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant.(11) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 32, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant. (12) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 65, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 35, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant.(13) Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 44, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant. (14) A Corynebacterium glutamicum transformant in which a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 4, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 7, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 41, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence, and a gene having or consisting of the nucleotide sequence shown in Sequence ID No. 1, or a gene having or consisting of a nucleotide sequence having 90% or more identity to said sequence are expressedly introduced into Corynebacterium glutamicum or its transformant.

[0125] The genetically modified Corynebacterium bacteria of this disclosure may, in one embodiment, optionally have reduced or missing function of one or more or all of the following enzymes (1) to (4), although these are not essential components, in order to further improve isopropanol productivity: (1) phosphoenolpyruvate carboxylase (ppc), (2) lactate dehydrogenase (ldhA), (3) phosphate acetyltransfilase (pta), (4) acetate kinase (ack).

[0126] In one or more embodiments, the genetically modified Corynebacterium bacteria of this disclosure further improve the productivity of isopropanol by increasing the productivity of acetyl-CoA, and therefore, a portion of the genes encoding one or more or all of the enzymes (1) to (4) above may be destroyed or deleted, and a portion of all four genes, including the gene encoding phosphoenolpyruvate carboxylase (e.g., ppc gene), the gene encoding lactate dehydrogenase (e.g., ldhA gene), the gene encoding phosphate acetyltransfilase (e.g., pta gene), and the gene encoding acetate kinase (e.g., ack gene), may be destroyed or deleted.

[0127] The genetically modified Corynebacterium bacteria of this disclosure may, in one embodiment, optionally have enhanced activity of one or more enzymes in at least one of the following pathways, for example, the shikimic acid pathway, the carbohydrate metabolism pathway (iolT1-ppgk), and the nonoxidative pentose phosphate pathway, although this is not an essential configuration. Such enhancement of enzyme activity is not particularly limited and may be achieved, for example, by increasing the expression of the gene encoding the enzyme.

[0128] In one embodiment, the genetically modified Corynebacterium bacteria described herein may be subjected to enhancement of each metabolic pathway by increasing the expression of the above-mentioned metabolic pathway genes or by increasing the activation of enzyme function through the use of heterologous (mutant) genes.

[0129] This disclosure relates, in other embodiments, to vectors for introducing at least one of the genes (A), (B), (C), and (D) described above into a microorganism (host). The vectors relating to this disclosure are not particularly limited and may be, for example, plasmids.

[0130] Vector Construction The introduction of the above-described gene into a microbial host can be carried out by amplifying the gene described above by PCR, cloning it into a suitable vector that can be amplified in a microbial host such as a Corynebacterium, and incubating the microbial host in the presence of the vector. Examples of promoters in one or more embodiments include the promoter of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (also called "glyceraldehyde 3-phosphate dehydrogenase") derived from Corynebacterium glutamicum R (PgapA), the promoter of the mdh gene encoding maleate dehydrogenase (Pmdh), the promoter of the ldhA gene encoding lactate dehydrogenase (PldhA), and the tac promoter (Ptac) obtained by fusing the trp promoter and lac promoter derived from Escherichia coli, among which PgapA is preferred. Examples of terminators in one or more embodiments include the rrnB T1T2 terminator of the E. coli rRNA operon, the trpA terminator of E. coli, and the trp terminator of Brevibacterium lactofermentum, among which the rrnB T1T2 terminator is preferred.

[0131] [Preparation of Transformants of the Disclosure] Transformants of the Disclosure may be obtained in one or more embodiments using known transformation methods. The transformation method may be any known method without limitation. Such known methods include, in one or more embodiments, the calcium chloride / rubidium chloride method, the calcium phosphate method, DEAE-dextran transfection, and electroporation (electric pulse method). When the microbial host is a Coryneform bacterium, the electric pulse method is preferred. The electric pulse method can be performed by known methods [e.g., Kurusu, Y. et al., Electroporation-transformation system for Coryneform bacteria by auxotrophic complementation. Agric. Biol. Chem. 54:443-447 (1990)] and [Vertes AA et al., Presence of mrr- and mcr- like restriction systems in Coryneform bacteria. Res. Microbiol. 144:181-185 (1993)].

[0132] Disruption or Mutation of Host Chromosome Genes When the microbial host is a Corynebacterium or its transformant, genes encoding competitive biosynthetic pathways, repressors of biosynthetic pathways, or efflux transporters may be disrupted or deleted as needed. The function of enzyme proteins encoded by specific genes may be improved by introducing mutations into the chromosome. By ligating DNA fragments before and after the target gene to create a DNA fragment in which the entire target gene is deleted, and transforming the bacteria with this DNA to induce homologous recombination on the chromosome, the target gene on the chromosome can be completely deleted. Alternatively, a deletion-type gene can be created by deleting a partial sequence of the target gene and modifying it so that it does not produce a normally functioning enzyme protein. By transforming the bacteria with DNA containing this gene and inducing homologous recombination between the deletion-type gene and the gene on the chromosome, the target gene on the chromosome can be replaced with the deletion-type or disruption-type gene. Enzyme proteins encoded by deletion-type or disruption-type genes, even if produced, have a different three-dimensional structure from wild-type enzyme proteins and have reduced or absent function. Furthermore, mutations can be introduced at specific locations on a chromosome by inducing homologous recombination between a gene fragment into which a specific mutation has been introduced and the chromosomal region in question. Gene deletion or disruption by gene substitution using such homologous recombination is already established, and includes methods such as using plasmids containing temperature-sensitive origins of replication, plasmids that can be transmitted by conjugation, and suicide vectors that do not have origins of replication in the host (U.S. Patent No. 6,303,383, and Japanese Patent Publication No. 05-007491, etc.). Markerless chromosomal gene transfer vector pCRA725 is a plasmid that cannot replicate in Corynebacterium glutamicum R. In the case of single crossover strains with homologous regions on chromosomes introduced into plasmid pCRA725, kanamycin resistance is exhibited due to the expression of the kanamycin resistance gene on pCRA725, and lethality in sucrose-containing medium is exhibited due to the expression of the sacR-sacB gene of Bacillus subtilis. In contrast, in the case of double crossover strains, kanamycin sensitivity is exhibited due to the loss of the kanamycin resistance gene on pCRA725, and growth in sucrose-containing medium is exhibited due to the loss of the sacR-sacB gene.Therefore, markerless chromosome gene transfection strains exhibit kanamycin sensitivity and growth in sucrose-containing media.

[0133] Growth of Transformants In one or more embodiments, the transformants of this disclosure are preferably grown by culturing under aerobic conditions. In one or more embodiments, the culture conditions for growth are a temperature of about 25°C to about 38°C and a duration of about 12 hours to about 48 hours. In one or more embodiments, the culture medium is a natural or synthetic medium containing a carbon source, a nitrogen source, inorganic salts and other nutrients. In one or more embodiments, the pH of the medium is about 5 to about 8. The culture medium, carbon source, nitrogen source, inorganic salts and other nutrients are as described above.

[0134] The contents of each document cited herein are incorporated by reference as constituting part of this disclosure.

[0135] The disclosure further relates to one or more embodiments described below: [1] A method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified Corynebacterium bacteria having isopropanol-producing ability to produce a fermentation product containing isopropanol; continuously or intermittently withdrawing a portion of the reaction solution containing the fermentation product from the fermenter during the fermentation reaction; separating isopropanol from the withdrawn reaction solution by pervaporation; and supplying the reaction solution from which the isopropanol has been separated to the fermenter, wherein the Corynebacterium bacteria have at least one of the activities of producing isopropanol from acetone and producing acetone from acetoacetic acid enhanced by the genetic modification. [2] The method according to [1], comprising maintaining the weight ratio of isopropanol to acetone (isopropanol / acetone) in the fermenter to 3 or more. [3] The method according to [1] or [2], comprising contacting the Corynebacterium bacteria with fermentation raw materials in a fermenter containing the reaction solution from which the isopropanol has been separated. [4] The method according to any one of [1] to [3], comprising supplying fermentation raw materials to the fermenter during the fermentation reaction and / or discharging a portion of the reaction liquid in the fermenter out of the system. [5] The method according to any one of [1] to [4], wherein the fermentation product further comprises at least one of acetone and acetic acid. [6] The method according to any one of [1] to [5], wherein the separation of isopropanol by pervaporation is carried out under conditions such that the weight ratio of isopropanol production to acetic acid production at 48 hours of culture (isopropanol / acetic acid) is 15 or more. [7] The method according to any one of [1] to [6], wherein the separation of isopropanol by pervaporation is carried out by passing the extracted reaction liquid through a pervaporation module. [8] The method according to any one of [1] to [7], comprising passing the reaction liquid through the pervaporation module such that the reaction liquid passes through the pervaporation module at a linear velocity of 0.0018 m / s or more.[9] The method according to any one of [1] to [8], which is capable of improving the production ratio of isopropanol to by-products.

[10] The method according to any one of [1] to [9], comprising obtaining an aqueous solution of isopropanol having an isopropanol concentration of 30 g / L or more.

[11] The method according to any one of [1] to

[10] , wherein the vacuum on the permeation side of the pervaporation module is 15 kPa or less.

[12] The method according to any one of [1] to

[11] , wherein the pervaporation module is connected to a vacuum pump, a trap is installed between the pervaporation module and the vacuum pump, at least two traps are installed on the exhaust side of the vacuum pump, and at least one of the traps is cooled to 0°C or below.

[13] The method according to any one of [1] to

[12] , comprising setting the temperature in the fermenter to a range of 20°C to 40°C.

[14] The method according to any one of [1] to

[13] , comprising controlling the dissolved oxygen concentration of the reaction liquid in the fermenter to be maintained above 0 ppm.

[15] The method according to any one of [1] to

[14] , comprising: discharging a portion of the reaction solution from the fermenter outside the system; removing microbial cells from the reaction solution; supplying the reaction solution from which the microbial cells have been removed to the pervaporation module; and returning the reaction solution that has passed through the pervaporation module back to the fermenter.

[16] A method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified microorganisms capable of producing isopropanol to produce a fermentation product containing isopropanol; separating isopropanol from at least a portion of the reaction solution containing the fermentation product in the fermenter; supplying the reaction solution from which the isopropanol has been separated to the fermenter; and maintaining the isopropanol concentration in the fermenter at 6 g / L or less.

[17] A method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified microorganisms capable of producing isopropanol to produce a fermentation product containing isopropanol;A method comprising: separating isopropanol from the reaction solution containing the fermentation product in the fermentation tank by passing a portion of the reaction solution through a pervaporation module; supplying the reaction solution that has passed through the pervaporation module to the fermentation tank; and maintaining the isopropanol concentration in the fermentation tank at 6 g / L or less.

[18] A method for producing isopropanol, comprising causing a fermentation reaction in a Corynebacterium bacterium genetically modified to have the ability to produce isopropanol, wherein at least one of gene (A) and gene (B) has been introduced in an expressible manner, gene (A) is at least one gene selected from the group consisting of (A1) to (A9) below, which encodes an enzyme having the activity to produce acetoacetate from acetoacetyl-CoA, and gene (B) is at least one gene selected from the group consisting of (B1) to (B9) below, which encodes an enzyme having the activity to produce acetone from acetoacetate. (A1) The gene from which the genus Pseudomonas originates; (A2) A gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 14; (A3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 14; (A4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in Sequence ID No. 14; (A5) A gene that hybridizes under stringent conditions with a gene having a complementary nucleotide sequence to the gene encoding the polypeptide having the amino acid sequence shown in Sequence ID No. 14; (A6) A gene having the nucleotide sequence shown in Sequence ID No. 4; (A7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 4;(A8) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, with 1 to 10 nucleotides deleted, substituted and / or added per unit; (A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 4; (B1) A gene derived from at least one microorganism selected from the group consisting of the genera Paenibacillus, Bradyrhizobium, Amycolatopsis, Paraburkholderia, and Burkholderia; (B2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, as shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B6) A gene having a nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65; (B7) A gene having a nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65;(B8) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, with 1 to 10 nucleotides being deleted, substituted and / or added per unit, in the nucleotide sequence shown by SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65; (B9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown by SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65.

[19] The Corynebacterium bacterium further has at least one of gene (C) and gene (D) introduced into it so as to be expressible, wherein gene (C) is at least one gene selected from the group consisting of (C1) to (C9) below and is a gene that encodes an enzyme having the activity to produce isopropanol (IPA) from acetone, and gene (D) is at least one gene selected from the group consisting of (D1) to (D9) below and is a gene that encodes an enzyme having the activity to produce acetoacetyl-CoA from acetyl-CoA and malonyl-CoA, according to any one of [1] to

[18] . (C1) A gene derived from the genera Levilactobacillus, Limosilactobacillus, Blautia, or Clostridium; (C2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64; (C3) A gene encoding a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64;(C4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, as shown in SEQ ID NO: 16, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64; (C5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64; (C6) A gene having a nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50; (C7) A gene having a nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50; (C8) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted and / or added per unit, in the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50; (C9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50; (D1) A gene from which the genus Streptomyces originates; (D2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 13; (D3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 13; (D4) A gene encoding a polypeptide having an amino acid sequence in which 100 amino acids constitute one unit, and 1 to 10 amino acids are deleted, substituted and / or added per unit;(D5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 13; (D6) A gene having the nucleotide sequence shown in Sequence ID No. 1; (D7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 1; (D8) A gene having a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 1, with 100 nucleotides as one unit, and 1 to 10 nucleotides being deleted, substituted and / or added per unit; (D9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 1.

[20] The Corynebacterium bacterium is characterized in that the function of at least one enzyme selected from the group consisting of (1) to (4) below is reduced or deleted, according to any one of the methods of [1] to

[19] . (1) Phosphoenolpyruvate carboxylase (ppc), (2) Lactate dehydrogenase (ldhA), (3) Phosphate acetyltransfilase (pta), (4) Acetate kinase (ack).

[21] The method according to any one of [1] to

[20] , wherein the Corynebacterium bacterium is Corynebacterium glutamicum or a transformant of Corynebacterium glutamicum.

[22] The method according to any one of [1] to

[21] , wherein the Corynebacterium bacterium is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof.

[23] A method for producing propylene or acrylic acid or derivatives thereof, characterized by producing isopropanol produced by the method according to any one of [1] to

[22] as a raw material.

[24] A bacterium of the genus Corynebacterium having isopropanol production ability, obtained by introducing at least one of the above genes (A) and (B) into a bacterium of the genus Corynebacterium in an expressible manner.

[25] The bacterium of the genus Corynebacterium according to

[24] , further obtained by introducing at least one of the above genes (C) and (D) in an expressible manner.

[26] The Corynebacterium bacterium described in

[24] or

[25] is characterized by reduced or missing function of at least one enzyme selected from the group consisting of (1) to (4) below: (1) phosphoenolpyruvate carboxylase (ppc), (2) lactate dehydrogenase (ldhA), (3) phosphate acetyltransfilase (pta), (4) acetate kinase (ack).

[27] The Corynebacterium bacterium described in any of

[24] to

[26] is characterized by being Corynebacterium glutamicum or a transformant of Corynebacterium glutamicum.

[28] The Corynebacterium bacterium is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof, as described in any of

[24] to

[27] .

[0136] The present disclosure will be further explained below with reference examples and embodiments. However, the present disclosure shall not be construed as being limited to the following embodiments.

[0137] [Example 1] Construction of isopropanol-producing strains (1) Preparation and acquisition of chromosomal DNA The chromosomal DNA of Levilactobacillus brevis ATCC 367, Paenibacillus polymyxa NBRC 15309, Pseudomonas putida ATCC 700007, Limosilactobacillus gastricus JCM 15952, and Limosilactobacillus fermentum NBRC 3956 was prepared using a DNA genome extraction kit (illustra bacteria genomicPrep Mini Spin Kit, Cytiva) after culturing according to the information of the strain acquisition institution. The Acetoacetyl CoA synthase gene of Streptomyces tendae was produced by artificial synthesis. The acetoacetate decarboxylase genes of Bradyrhizobium diazoefficiens, Amycolatopsis mediterranei, Paenibacillus graminis, Bradyrhizobium elkanii, Bradyrhizobium japonicum, Paraburkholderia caribensis, and Burkholderia glumae were synthesized artificially. The isopropanol dehydrogenase genes of Blautia producta, Clostridium isatidis, Clostridium cochlearium, and Clostridium tyrobutyricum were synthesized artificially. (2) Construction of isopropanol production-related gene expression plasmids The primer sequences used to isolate isopropanol production-related genes are shown in Tables 1-4. PCR was performed using a VeritiPro thermal cycler (Thermo Fisher Scientific, Inc.) and PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) as the reaction reagent. PCR-amplified DNA fragments were introduced into cloning vectors pCRB209 containing the gapA promoter, pCRB214 containing the tac promoter, and pCRB258 containing the ldhA promoter. The introduced cloning vectors and the names of the resulting plasmids are shown in Table 5. The gene sequences, including the promoter and terminator, from the aforementioned plasmids were introduced into the cloning vector pCRB22. The names and summaries of the obtained plasmids are shown in Table 6. (3) Construction of chromosome-modified strains for isopropanol production The markerless chromosome gene modification vector pCRA725 is a plasmid that cannot replicate within Corynebacterium glutamicum R. In the case of a single crossover strain with a homologous region on a chromosome introduced into plasmid pCRA725, it exhibits kanamycin resistance due to the expression of the kanamycin resistance gene on pCRA725 and lethality in sucrose-containing medium due to the expression of the sacR-sacB gene derived from Bacillus subtilis. In contrast, in the case of a double crossover strain, it exhibits kanamycin sensitivity due to the loss of the kanamycin resistance gene on pCRA725 and growth in sucrose-containing medium due to the loss of the sacR-sacB gene. Therefore, markerless chromosome-modified strains exhibit kanamycin sensitivity and growth in sucrose-containing medium. Using the method described above, the gene of Corynebacterium glutamicum RΔ(pta-ack)ΔldhA [Appl Microbiol Biotechnol. Dec;77(4):853-860 (2007)] was disrupted using the ppc gene disruption plasmid LLPEP1 [J Mol Microbiol Biotechnol. 8(4):243-254 (2004)]. An overview of this chromosomal recombination is shown in Table 7. (4) Construction of a strain with isopropanol production-related gene expression plasmid The above-mentioned isopropanol production-related gene expression plasmid was introduced into Corynebacterium glutamicum Ripa1 strain. An overview of this strain is shown in Table 8.

[0138] [Example 2] Production of Isopropanol by Test Tube Culture Isopropanol production was evaluated using strains IPA-1 to IPA-14 (Corynebacterium glutamicum genetically modified to have the isopropanol metabolic pathway) constructed in Example 1. [Method] ・Corynebacterium cells Strains IPA-1 to IPA-14 obtained by conferring the isopropanol metabolic pathway to Corynebacterium glutamicum were used. ・Pre-culture solution Strains IPA-1 to IPA-14 were pre-cultured overnight in test tubes with medium A, which has the composition shown in Table 11 below, to prepare the pre-culture solution. ・IPA production by fermentation reaction 2.5 mL of medium A shown in Table 11 below was added to a 10 mL test tube, 4% glucose was added with an initial OD of 0.2 to 0.3, and culture production was carried out at 30°C and 200 rpm for 28 hours. The production values ​​of each production strain are shown in Table 9.

[0139] As shown in Table 9 above, all of the constructed strains IPA-1 to IPA-14 showed high isopropanol productivity.

[0140] [Reference Example 1] Pervaporation under Aeration Conditions The separation capability of isopropanol by pervaporation under aeration stirring conditions was confirmed using the following apparatus and conditions. [Method] ・Apparatus and experimental conditions The experimental apparatus shown in Figure 1 was used, and the experiment was conducted under the following two conditions. Condition 1: Stirring speed 50 rpm Condition 2: Stirring speed 500 rpm, aeration speed 0.5 vvm In both conditions 1 and 2, 1.5 L of 20 g / L isopropanol aqueous solution was placed in a 2 L jar fermenter, the tank temperature was 30 °C, the vacuum pump was set to 1.5 kPa (controlled by a constant vacuum device), trap 1 was set to -10 °C, trap 2 to room temperature, and trap 3 to 5 °C. A silicone hollow fiber gas separation membrane NAGASEP (M40-B, manufactured by Nagayanagi Kogyo Co., Ltd.) was used as the pervaporation membrane, and the isopropanol aqueous solution was passed through the hollow fiber at a linear velocity of 0.024 m / s using a peristaltic pump. The vacuum pump was stopped every hour, and the permeate collected in traps 1-3 was recovered. Simultaneously, the isopropanol aqueous solution (feed solution) in the jar fermenter was sampled.

[0141] [Analysis Method] ・Quantitative determination of isopropanol concentration Quantitative analysis of isopropanol was performed using liquid chromatography. An Aminex HPX-87H Column (manufactured by Bio-Rad) was used as the column. ・Calculation of permeation flux Permeation flux was calculated according to the following formula 1. • Evaluation of the selectivity of pervaporation membranes The separation selectivity of a pervaporation membrane is usually calculated using a dimensionless separation coefficient α. In the case of a two-component mixture consisting of components A and B, the separation coefficient α is given by the following equation 2. In the above equation 2, y A and y B The composition of the permeate is x A and x B This represents the composition of the feed solution (Encyclopedia of Membranes pp 1-3). In this study, the culture medium was the subject, and both the permeate and feed solution had multi-component compositions. Therefore, the separation count β, which serves as an indicator for evaluating the selectivity of the pervaporation membrane, was calculated using the following formula 3 described in Chemical Engineering Journal 287 (2016) 1-10. In the above equation 3, y i x is the weight fraction of one component in the permeate. i This represents the weight fraction of one component in the supply solution.

[0142] [Results] ・Changes in isopropanol concentration in the tank The isopropanol concentration in the isopropanol aqueous solution (feed liquid) in the sampled jar fermenter was quantified by the above determination of isopropanol concentration, and the changes in isopropanol concentration in the tank under conditions 1 and 2 are shown in Figure 2. Figure 2 is a graph showing the changes in isopropanol concentration in the isopropanol aqueous solution in the tank due to pervaporation separation. As shown in Figure 2, in both conditions 1 and 2, isopropanol decreased at a rate of approximately 3 g / L / h. Since both conditions 1 and 2 showed a similar trend, it was considered that the mixing of air bubbles into the pervaporation membrane due to aeration stirring did not affect the separation of isopropanol. ・Permeate flux The amount of permeate collected in traps 1 to 3 under conditions 1 and 2 is shown in Table 10 below. Under condition 1 (stirring at 50 rpm only), the permeate was collected only in trap 1. On the other hand, under condition 2 (stirring at 500 rpm, aeration velocity 0.5 vvm), the permeate was also collected in traps 2 and 3. This is thought to be because, under condition 2, the aeration (0.5 vvm) introduced air bubbles into the pervaporation membrane, increasing the amount of gas passing through the pervaporation membrane, and trap 1 alone could not capture it all. Next, the permeate flux was calculated from the total amount of permeate collected. The results are shown in Figure 3. Figure 3 is a graph showing the relationship between aeration and stirring conditions and permeate flux. As shown in Figure 3, the permeate velocity under conditions 1 and 2 showed almost the same value. Separation selectivity of the pervaporation membrane The isopropanol concentration of the permeate captured in traps 1 to 3 and the isopropanol concentration of the feed solution (isopropanol concentration in the tank) were analyzed, and the separation coefficient β of isopropanol was calculated from equation 3 above using the obtained isopropanol concentrations. Figure 4 shows the relationship between the separation coefficient β obtained under conditions 1 and 2 and the isopropanol concentration in the tank. Both conditions 1 and 2 showed nearly similar values ​​for the separation coefficient β. The separation coefficient β tended to increase as the isopropanol concentration in the tank decreased. This trend is consistent with the report in Journal of Membrane Science Vol. 74 (1-2), 1992, pp. 171-181.

[0143] [Discussion] The separation capability of isopropanol by pervaporation under aeration and agitation conditions was confirmed, and it was found that the separation rate, permeation flux, and selectivity of isopropanol were not affected by the presence or absence of aeration and agitation. However, under aeration and agitation, trap 1, which was installed in front of the vacuum pump, could not completely capture the isopropanol, and traps 2 and 3, which were installed behind the vacuum pump, also captured it. This is thought to be because air bubbles entered the pervaporation membrane, increasing the amount of gas passing through the pervaporation membrane. The amount of isopropanol charged and the amount recovered in traps 1 to 3 were almost identical, indicating that nearly 100% recovery was possible with traps 1 to 3.

[0144] [Reference Example 2] Gas stripping, a known method for recovering volatile components from culture medium, is applied to the culture of Corynebacterium glutamicum, to which the isopropanol metabolic pathway has been genetically modified, to confirm that simultaneous separation is possible.

[0145] [Method] • Bacterial Cells: IPA-1 strain, obtained by conferring the isopropanol metabolic pathway to Corynebacterium glutamicum through genetic recombination as described in Example 1, was used. • Pre-culture Solution: IPA-1 strain was pre-cultured overnight in a test tube with medium A, which has the composition shown below, to prepare the pre-culture solution. • IPA Production by Fermentation Reaction: Initial amount of medium A was added to the culture apparatus described below, and the apparatus was heated and aerated. When the medium temperature reached 30°C, the obtained pre-culture solution was inoculated to achieve the initial OD610 shown below, and cultivation was started. The culture was then incubated for 48 hours under the following conditions. • Culture Conditions: Medium Used: Medium A (Composition shown in Table 11) Culture apparatus: 1L jar fermenter (ABLE) Initial medium volume: 400mL Initial OD610: 1.365 Aeration rate: Initial aeration rate 0.2L / min (0.5vvm), changed to 0.32L / min (0.8vvm) from 21h, and to 0.35L / min (0.875vvm) from 28h. Stirring speed: DO stud culture with DO 10% setting Temperature: 30℃ pH adjustment: pH controlled to 7 with 10N NH3 Carbon source addition: Glucose was added at 12h (50g), 21h (40g), 28h (20g), and 34h (30g).

[0146] [Measurement Method] ・Gas Stripping Conditions Three collection bottles, each containing 1L of water, were connected in series, and the exhaust line of a jar fermenter was connected to collect volatile components contained in the exhaust. ・Cell Density Measurement Conditions Culture medium was sampled as needed, and the turbidity (OD) of the culture medium at a wavelength of 610nm was measured using a DU 730 UV-visible spectrophotometer (Beckman Coulter) and defined as the cell density. ・Culture Medium Analysis Conditions The production concentrations of isopropanol and by-products contained in the culture medium and gas stripping collection solution were measured using a Nexera X2, Nexera Organic Acid Analysis System (Shimadzu Corporation).

[0147] [Results] - Cell density and culture medium volume: Table 12 shows the changes in cell density (OD) and the volume of culture medium in the jar fermenter. As shown in Table 12, the addition of glucose and NH3 increased the culture medium volume during the culture period, increasing by 70 mL from the initial volume at 48 hours. Considering the increase in culture medium volume, the value obtained by multiplying OD by the culture medium volume (L) was calculated and plotted on the x-axis with the culture time (Figure 5). Analysis of the culture medium results Analysis of the culture medium at 25 hours and 48 hours revealed the presence of isopropanol, as well as by-products acetone, acetic acid, alanine, and valine. The analysis results are shown in Table 13. The amount of glucose, a carbon source, remaining in the culture medium after 48 hours of incubation was analyzed. The result showed that 17.7 g remained after 48 hours. From this result, the amount of glucose consumed during the incubation period (48 hours) was 142.3 g. • Analysis of gas stripping collection solution: The gas stripping collection solution was analyzed after 48 hours of incubation. The analysis results are shown in Table 14. - Based on the analysis results of the culture medium and collected liquid, the production amounts of isopropanol and by-products at 48 hours of cultivation were calculated. The results are shown in Table 15. The glucose consumption after 48 hours of culture was 142.3 g. Therefore, the isopropanol yield relative to glucose was calculated to be 23.6 mol%. Furthermore, the isopropanol productivity per unit of medium, based on an initial medium volume of 400 mL, was 0.58 g / L / h.

[0148] [Example 3] Simultaneous isolation of isopropanol by pervaporation Pervaporation was applied to the culture of Corynebacterium glutamicum, which had been genetically modified to confer the isopropanol metabolic pathway, and isopropanol was simultaneously isolated by pervaporation. [Method] ・Apparatus An apparatus with the same configuration as the experimental apparatus shown in Figure 1 was used, except that the volume of the jar fermenter was set to 1 L. ・Bacterial cells The IPA-1 strain, obtained by conferring the isopropanol metabolic pathway to Corynebacterium glutamicum by genetic modification as described in Example 1, was used. ・Pre-culture solution The IPA-1 strain was pre-cultured overnight in a test tube with medium A, which had the composition shown in Table 11 above, to prepare the pre-culture solution. ・IPA production by fermentation reaction The initial amount of medium A was added to the culture apparatus described below, heated and aerated, and when the temperature of the medium reached 30°C, the obtained pre-culture solution was inoculated to achieve the initial OD610 as shown below, and cultivation was started. The culture was then cultivated for 48 hours under the following conditions. • Culture conditions Culture apparatus: 1L jar fermenter (ABLE) Medium used: Medium A (same as Reference Example 2) Initial volume of medium: 587.5mL (400mL jar fermenter + 187.5mL pervaporation piping) Initial OD: 1.4 Aeration rate: 0.5vvm (DO stud with DO set to 10%) The initial aeration rate was 0.3L / min, changed to 0.32L / min from 22h, and to 0.35L / min from 38.5h. By adjusting the aeration rate, the dissolved oxygen concentration of the reaction solution in the fermenter was maintained above 0ppm. Culture temperature (temperature in the fermenter): 30℃ pH adjustment: pH controlled to 7 with 10N NH3 Additional carbon source: 50g of glucose was added at 13.5h, and 40g each at 22h, 28h, and 38.5h. Pervaporation operating conditions: A silicone hollow fiber gas separation membrane NAGASEP (M40-B, manufactured by Nagayanagi Kogyo Co., Ltd.) was used as the pervaporation membrane. From 16 hours of culture, the culture medium was passed through the outside of the hollow fiber using a peristaltic pump at a linear velocity of 0.007 m / s. The vacuum pump was set to 1.5 kPa (controlled by a constant vacuum device). Trap 1 was cooled to -10°C, and traps 2 and 3 were cooled on ice. The vacuum pump was stopped as needed, and the permeate collected in traps 1 to 3 was recovered.

[0149] [Results] - Cell density and culture medium volume Changes in cell density (OD) and culture medium volume are shown in Table 16. As shown in Table 16, although there was an increase due to the addition of glucose and NH3, the volume of culture medium decreased during the culture period due to evaporation by pervaporation, and at 48 hours it was 415 mL, a decrease of 172 mL from the initial volume. Considering the decrease in the volume of culture medium, the value obtained by multiplying OD by the volume of culture medium (L) was calculated and plotted on the x-axis with the culture time (Figure 6). For comparison, the value obtained by multiplying the OD obtained by simultaneous culture separation by gas stripping in Reference Example 2 by the volume of culture medium (L) was also calculated and is shown in Figure 6. ・Analysis results of the culture medium The culture medium at culture times of 16 to 48 hours was analyzed and the concentrations of isopropanol and acetone were determined. The concentrations of acetic acid, alanine, and valine were also measured at 24 hours and 48 hours. The analysis results are shown in Table 17. As shown in Table 17, the isopropanol concentration in the culture medium was highest at 16h, the start of pervaporation (5.79 g / L), and remained below 6 g / L until 48h of culture with pervaporation. In particular, it fell below 5 g / L after 24h. The by-product acetone also remained at a low concentration of less than 1 g / L after pervaporation, and the production volume was significantly reduced compared to separation by gas stripping. Acetic acid was 13.6 g / L at 48h with simultaneous separation by gas stripping, but the production volume was very suppressed to 1.07 g / L with pervaporation. On the other hand, the production volume of alanine and valine increased compared to gas stripping. Analysis of pervaporation permeate The pervaporation permeate obtained during culture times of 18 to 48h was analyzed. The analysis results are shown in Table 18. - Based on the analysis of the culture medium and permeate, the production amounts of isopropanol and by-products at 48 hours of incubation were calculated. The results are shown in Table 19. The amount of glucose, the carbon source, remaining in the culture medium after 48 hours of incubation was analyzed. The glucose consumption after 48 hours of incubation was 198.3 g. Therefore, the isopropanol yield relative to glucose was calculated to be 28.0 mol%. Furthermore, the isopropanol productivity per unit of medium, based on an initial medium volume of 587.5 mL, was 0.66 g / L / h. • Product yield relative to glucose: The yields (mol%) of isopropanol and by-products relative to glucose were calculated and are shown in Table 20. For comparison, the yields of products obtained by simultaneous culture separation by gas stripping in Reference Example 2 were also calculated and are shown in Table 20. In Example 3, which used pervaporation as a simultaneous culture and separation method, the isopropanol yield improved by more than 20% compared to Reference Example 2, which used gas stripping. Furthermore, the yields of the by-products acetone and acetic acid decreased significantly, with acetone decreasing by more than 75% and acetic acid by more than 99%. The total yield of by-products (acetone, acetic acid, alanine, and valine) was 8.24%, which is approximately one-third of the total yield of by-products in gas stripping (25.42%). Therefore, the simultaneous culture and separation method using pervaporation suppressed the generation of by-products and allowed for the acquisition of isopropanol in high yield. The reason for achieving high isopropanol yield and suppression of by-product generation is attributed to the isopropanol concentration in the culture medium. In Reference Example 2, which used gas stripping, the isopropanol concentration in the culture medium exceeded 10 g / L at 25 hours from the start of cultivation and reached 15 g / L at 48 hours. In contrast, in Example 3, which used pervaporation, the concentration was kept below 6 g / L throughout the entire culture period. It is thought that the inhibition due to the toxicity of isopropanol was reduced, allowing the microorganisms to remain active and leading to a high yield of isopropanol.

[0150] [Reference Example 3] Investigation of the linear velocity of the feed liquid in pervaporation The effect of the linear velocity of the feed liquid flowing through the pervaporation module on pervaporation was investigated. [Method] ・Apparatus and experimental conditions The experimental apparatus shown in Figure 7 was used, and the experiment was conducted under conditions of a stirring speed of 500 rpm and aeration speed of 0.5 vvm. 1.5 L of 5 g / L isopropanol aqueous solution was placed in a 2 L jar fermenter, the tank temperature was 30 °C, the vacuum pump was set to 1.5 kPa (controlled by a constant vacuum device), trap 1 was set to -10 °C, trap 2 to room temperature, and trap 3 to 5 °C. A silicone hollow fiber gas separation membrane NAGASEP (M40-B, manufactured by Nagayanagi Kogyo Co., Ltd.) was used as the pervaporation membrane, and the isopropanol aqueous solution was passed through the outside of the hollow fiber at a linear velocity of 0.007 m / s or 0.0018 m / s using a perister pump. The vacuum pump was stopped every hour, and the permeate collected in traps 1 to 3 was recovered. Simultaneously, an aqueous isopropanol solution (feed solution) was sampled from the jar fermenter. • Analytical methods: The analytical methods, calculation of permeate flux, and evaluation of selectivity followed Reference Example 1.

[0151] [Results] ・Changes in isopropanol concentration in the tank Figure 8 shows the changes in the isopropanol concentration of the isopropanol aqueous solution in the tank after pervaporation separation. As shown in Figure 8, the rate of decrease of isopropanol was 0.13 g / L / h when the liquid was passed through at a linear velocity of 0.0018 m / s, while the rate of decrease of isopropanol was 0.19 g / L / h when the liquid was passed through at a linear velocity of 0.007 m / s. The rate of decrease of isopropanol in the tank was faster when the liquid was passed through at a linear velocity of 0.007 m / s compared to when the liquid was passed through at a linear velocity of 0.0018 m / s. It was also found that the rate of decrease of isopropanol in the tank was more stable at a linear velocity of 0.007 m / s than at a linear velocity of 0.0018 m / s. ・Permeation flux The permeation flux was calculated from the total amount of permeate collected (Figure 9). Figure 9 is a graph showing the relationship between linear velocity and permeation flux. As shown in Figure 9, the permeate flux was higher when the fluid was passed through at a linear velocity of 0.007 m / s compared to when it was passed through at a linear velocity of 0.0018 m / s. The isopropanol concentration in the permeate and feed solution of the pervaporation membrane was analyzed, and the separation coefficient β of isopropanol was calculated from these results. The relationship between the separation coefficient β and the linear velocity is shown in Figure 10. As shown in Figure 10, the separation coefficient β was improved when the fluid was passed through at a linear velocity of 0.007 m / s compared to when it was passed through at a linear velocity of 0.0018 m / s. By changing the linear velocity from 0.0018 m / s to 0.007 m / s, the recovery rate of isopropanol, the permeate flux, and the selectivity of the pervaporation membrane were improved. In addition, it was found that when the linear velocity was 0.007 m / s, the rate of decrease of isopropanol in the tank was more stable compared to when the linear velocity was 0.0018 m / s, and as a result, the recovery of isopropanol was more stable. This is thought to be due to the concentration polarization of the isopropanol aqueous solution. It is believed that if the linear velocity is lowered below 0.0018 m / s, sufficient isopropanol cannot be recovered. By setting the linear velocity to at least 0.0018 m / s, the effects of concentration polarization can be suppressed, and isopropanol can be recovered more stably while suppressing a decrease in the recovery rate, permeation flux, and selectivity of the pervaporation membrane.

[0152] [Reference Example 4] Investigation of Vacuum Level in Pervaporation The effect of the vacuum level on the pervaporation module's transmission side on pervaporation was investigated. [Method] ・Apparatus and experimental conditions The conditions were the same as in Reference Example 3, except that the vacuum level on the transmission side was set to 1.5 kPa, 3.0 kPa, and 5.0 kPa. ・Analysis method Other The analysis method, calculation of permeation flux, and evaluation method of the selectivity of the pervaporation film followed Reference Example 1.

[0153] [Results] Figure 11 shows the relationship between the permeation flux (average value over 3 hours of operation) obtained under each vacuum level and the vacuum level. As shown in Figure 11, the permeation flux decreased linearly as the vacuum level decreased (pressure increased). Under these experimental conditions, it was calculated that the permeation flux became 0 when the vacuum level dropped below 5.35 kPa.

[0154] The abbreviated compound names and their CAS numbers listed in Figure 13 (Overall metabolic pathway diagram illustrating the biosynthesis pathway of isopropanol) are shown below. The "compound names" listed below are examples only, and it goes without saying that synonymous names may exist. Furthermore, it goes without saying that the "CAS numbers" listed below are not necessarily exhaustive.

[0155] The enzymes encoded by the genes shown in Figure 13 and their EC numbers are shown below. The "enzyme names" shown below are examples, and it goes without saying that there may be synonymous names. Also, the "enzymes" encoded by the "genes" shown below are examples, and it goes without saying that there may be "genes" that encode "enzymes" with multiple functions.

[0156]

Claims

1. A bacterium of the genus Corynebacterium having isopropanol production ability, obtained by introducing at least one of the following genes (A) and (B) into a bacterium of the genus Corynebacterium in an expressible manner. Gene (A): At least one gene selected from the group consisting of (A1) to (A9) below, which encodes an enzyme that has the activity to produce acetoacetate from acetoacetyl-CoA; (A1) A gene from which the genus Pseudomonas originates; (A2) A gene encoding a polypeptide having the amino acid sequence shown in Sequence ID No. 14; (A3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in Sequence ID No. 14; (A4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, in the amino acid sequence shown in Sequence ID No. 14; (A5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding the polypeptide having the amino acid sequence shown in Sequence ID No. 14; (A6) A gene having the nucleotide sequence shown in Sequence ID No. 4; (A7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in Sequence ID No. 4; (A8) A gene having a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 4, where 100 nucleotides constitute one unit, with deletions, substitutions, and / or additions of 1 to 10 nucleotides per unit; (A9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 4; Gene (B): At least one gene selected from the group consisting of (B1) to (B9) below, which encodes an enzyme that has the activity of producing acetone from acetoacetate; (B1) A gene that is derived from at least one microorganism selected from the group consisting of the genera Paenibacillus, Bradyrhizobium, Amycolatopsis, Paraburkholderia, and Burkholderia;(B2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit; (B5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 68; (B6) A gene having the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65; (B7) A gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO: 65; (B8) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted and / or added per unit; (B9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, or SEQ ID NO:

65.

2. A Corynebacterium bacterium according to claim 1, further obtained by introducing at least one of gene (C) and gene (D) in an expressible manner. Gene (C): At least one gene selected from the group consisting of (C1) to (C9) below, which encodes an enzyme having the activity to produce isopropanol (IPA) from acetone; (C1) A gene derived from the genera Levilactobacillus, Limosilactobacillus, Blautia, or Clostridium; (C2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64; (C3) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64; (C4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids forming one unit, as shown in SEQ ID NO: 16, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64; (C5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO: 64; (C6) A gene having a nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50; (C7) A gene having a nucleotide sequence that has 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50;(C8) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, and 1 to 10 nucleotides are deleted, substituted and / or added per unit, in the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50; (C9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50; Gene (D): At least one gene selected from the group consisting of (D1) to (D9) below, which encodes an enzyme that has the activity to produce acetoacetyl-CoA from acetyl-CoA and malonyl-CoA; (D1) A gene of the genus Streptomyces; (D2) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 13; (D3) A gene encoding a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 13; (D4) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids forming one unit, as shown in SEQ ID NO: 13; (D5) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the gene encoding the polypeptide having the amino acid sequence shown in SEQ ID NO: 13; (D6) A gene having the nucleotide sequence shown in SEQ ID NO: 1; (D7) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 1; (D8) A gene having a nucleotide sequence in which 1 to 10 bases are deleted, substituted, and / or added per unit, with 100 bases forming one unit, as shown in SEQ ID NO: 1; (D9) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO:

1.

3. The Corynebacterium bacterium according to claim 1 or 2, wherein the function of at least one enzyme selected from the group consisting of (1) to (4) below is reduced or absent: (1) phosphoenolpyruvate carboxylase (ppc), (2) lactate dehydrogenase (ldhA), (3) phosphate acetyltransfilase (pta), (4) acetate kinase (ack).

4. The Corynebacterium bacterium according to any one of claims 1 to 3, wherein the Corynebacterium bacterium is Corynebacterium glutamicum or a transformant of Corynebacterium glutamicum.

5. The Corynebacterium bacterium according to any one of claims 1 to 4, wherein the Corynebacterium bacterium is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof.

6. A method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified Corynebacterium bacteria having isopropanol-producing ability to produce a fermentation product containing isopropanol; continuously or intermittently withdrawing a portion of the reaction solution containing the fermentation product from the fermenter during the fermentation reaction; separating isopropanol from the withdrawn reaction solution by pervaporation; and supplying the reaction solution from which isopropanol has been separated to the fermenter, wherein the Corynebacterium bacteria have at least one of the activities of producing isopropanol from acetone and producing acetone from acetoacetic acid enhanced by the genetic modification.

7. The method according to claim 6, wherein the Corynebacterium bacterium is a Corynebacterium bacterium described in any one of claims 1 to 5.

8. The method according to claim 6 or 7, comprising maintaining the weight ratio of isopropanol to acetone (isopropanol / acetone) in the fermentation tank at 3 or more.

9. The method according to any one of claims 6 to 8, comprising bringing the Corynebacterium bacteria into contact with the fermentation raw material in a fermentation vessel containing the reaction solution from which the isopropanol has been separated.

10. The method according to any one of claims 6 to 9, comprising supplying fermentation raw materials to the fermenter during the fermentation reaction, and / or discharging a portion of the reaction liquid in the fermenter from the system.

11. The method according to any one of claims 6 to 10, wherein the fermentation product further comprises at least one of acetone and acetic acid.

12. The method according to claim 11, wherein the separation of isopropanol by pervaporation is carried out under conditions such that the weight ratio of isopropanol produced to acetic acid produced at 48 hours of incubation (isopropanol / acetic acid) is 15 or more.

13. The method according to any one of claims 6 to 12, wherein the separation of isopropanol by pervaporation comprises passing the extracted reaction solution through a pervaporation module.

14. The method according to claim 13, comprising passing the reaction solution through the pervaporation module such that the reaction solution passes through the pervaporation module at a linear velocity of 0.0018 m / s or more.

15. The method according to any one of claims 6 to 14, which can improve the production ratio of isopropanol to by-products.

16. The method according to any one of claims 6 to 15, comprising obtaining an aqueous isopropanol solution having an isopropanol concentration of 30 g / L or more.

17. The method according to claim 13 or 14, wherein the vacuum level on the permeation side of the pervaporation module is 15 kPa or less.

18. The method according to any one of claims 13, 14, and 17, wherein the pervaporation module is connected to a vacuum pump, a trap is installed between the pervaporation module and the vacuum pump, at least two traps are installed on the exhaust side of the vacuum pump, and at least one of the traps is cooled to 0°C or below.

19. The method according to any one of claims 6 to 18, comprising setting the temperature inside the fermentation tank to a range of 20°C to 40°C.

20. The method according to any one of claims 6 to 19, comprising controlling the dissolved oxygen concentration of the reaction liquid in the fermentation tank to be maintained at a level higher than 0 ppm.

21. The method according to any one of claims 13, 14, 17, and 18, comprising: discharging a portion of the reaction liquid from the fermentation tank outside the system; removing microbial cells from the reaction liquid; supplying the reaction liquid from which the microbial cells have been removed to the pervaporation module; and returning the reaction liquid that has passed through the pervaporation module back to the fermentation tank.

22. A method for producing isopropanol, comprising: causing a fermentation reaction in a fermenter using genetically modified microorganisms capable of producing isopropanol to produce a fermentation product containing isopropanol; separating isopropanol from at least a portion of the reaction solution containing the fermentation product in the fermenter; supplying the reaction solution from which the isopropanol has been separated to the fermenter; and maintaining the isopropanol concentration in the fermenter at 6 g / L or less.

23. The method according to claim 22, wherein the separation of isopropanol comprises separating isopropanol from the reaction solution by passing a portion of the reaction solution containing the fermentation product in the fermentation tank through a pervaporation module.

24. The method according to claim 22 or 23, wherein the microorganism is a bacterium of the genus Corynebacterium as described in any one of claims 1 to 5.

25. A method for producing propylene or acrylic acid or derivatives thereof, characterized by using isopropanol produced by the method described in any one of claims 6 to 24 as a raw material to produce propylene or acrylic acid or derivatives thereof.

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