Ethanol production method and ethanol production system

Abstract

To further increase ethanol fermentation efficiency.SOLUTION: In a method for producing ethanol using ethanol fermentation by yeast, when ethanol fermentation by yeast is performed using a raw material containing acetic acid in addition to sugar as a raw material for ethanol fermentation, and using transformed yeast into which at least an acetaldehyde dehydrogenase gene is introduced as yeast, formic acid is added to a culture solution in which ethanol fermentation is progressing, and the following reactions are caused to proceed: a reduced coenzyme-dependent reaction with acetyl-CoA as a substrate caused by acetaldehyde dehydrogenase obtained by expression of the acetaldehyde dehydrogenase gene introduced into the transformed yeast; an oxidized coenzyme-dependent reaction with formic acid as a substrate caused by formate dehydrogenase obtained by expression of the formate dehydrogenase gene; and an oxidized coenzyme-dependent reaction with glycerol as a substrate caused by glycerol dehydrogenase obtained by expression of the glycerol dehydrogenase gene.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a method for producing ethanol and a system for producing ethanol. [Background technology]

[0002] Conventionally, ethanol fermentation using yeast has been known as one of the metabolic processes for obtaining energy by breaking down sugar. For example, biomass is known as a raw material for ethanol fermentation, and when ethanol produced from biomass is used as fuel, it is expected that carbon dioxide emissions will be reduced compared to when fossil fuels are used. When performing such ethanol fermentation, sugar obtained by breaking down (saccharifying) starch, cellulose, etc. in raw materials such as biomass is generally used. For example, Patent Document 1 discloses a method for making xylose, a pentose obtained from cellulosic biomass, etc., more usable as a substrate for ethanol fermentation, thereby improving the efficiency of ethanol fermentation. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 7078900 Summary of the Invention [Problem to be solved by the invention]

[0004] However, raw materials widely used in ethanol fermentation, such as biomass, and products obtained by subjecting such raw materials to saccharification treatment contain various components other than the sugar used as a substrate for ethanol fermentation. Thus, the raw materials used in ethanol fermentation contain a large amount of organic matter that is not used in ethanol fermentation, and it has been desired to improve the utilization efficiency of the raw materials used in ethanol fermentation and further improve the efficiency of ethanol fermentation. [Means for solving the problem]

[0005] The present disclosure can be realized in the following forms. (1) According to one embodiment of the present disclosure, a method for producing ethanol using ethanol fermentation by yeast is provided. In this method for producing ethanol, a raw material containing acetic acid in addition to sugar is used as the raw material for ethanol fermentation, and a transformed yeast into which at least an acetaldehyde dehydrogenase gene has been introduced is used as the yeast. During ethanol fermentation by the yeast, formic acid is added to a culture solution in which the ethanol fermentation proceeds, and a reduced coenzyme-dependent reaction using acetyl-CoA as a substrate by acetaldehyde dehydrogenase obtained by expression of the acetaldehyde dehydrogenase gene introduced into the transformed yeast, an oxidized coenzyme-dependent reaction using formic acid as a substrate by formate dehydrogenase obtained by expression of at least one of a formate dehydrogenase gene inherent in the transformed yeast and a formate dehydrogenase gene introduced into the transformed yeast, and an oxidized coenzyme-dependent reaction using glycerol as a substrate by glycerol dehydrogenase obtained by expression of at least one of a glycerol dehydrogenase gene inherent in the transformed yeast and a glycerol dehydrogenase gene introduced into the transformed yeast are proceeded. According to this embodiment of the ethanol production method, when ethanol fermentation proceeds, in the presence of formic acid, a reduced coenzyme-dependent reaction by acetaldehyde dehydrogenase with acetyl CoA as a substrate, an oxidized coenzyme-dependent reaction by formate dehydrogenase with formic acid as a substrate, and an oxidized coenzyme-dependent reaction by glycerol dehydrogenase with glycerol as a substrate proceed. Therefore, while the glycerol production reaction consuming NADH is suppressed by glycerol dehydrogenase, the reduced coenzyme-dependent reaction by acetaldehyde dehydrogenase is promoted using NADH produced by the action of formate dehydrogenase, and ethanol can be produced from acetic acid contained in the raw material of ethanol fermentation. As a result, the ethanol production efficiency in ethanol fermentation can be increased. (2) In the above-described ethanol production method, the transformed yeast may be a yeast obtained by transforming a yeast endogenously containing the formate dehydrogenase gene, in which the expression level of the formate dehydrogenase gene has been increased. In this manner, the activity of the oxidized coenzyme-dependent reaction of formate dehydrogenase using formate as a substrate is increased, thereby increasing the amount of NADH produced, and as a result, it is possible to further increase the efficiency of ethanol production in ethanol fermentation. (3) In the above-described ethanol production method, formic acid may be produced using carbon dioxide generated during ethanol fermentation by the yeast, and the produced formic acid may be added to the culture solution in which ethanol fermentation by the yeast progresses. With this configuration, since formic acid is produced using carbon dioxide generated during ethanol fermentation, it is possible to effectively utilize carbon dioxide and reduce the amount of carbon dioxide discharged. Furthermore, since formic acid is produced using carbon dioxide generated during ethanol fermentation, it is possible to eliminate the process of concentrating and purifying carbon dioxide prior to use in formic acid production, and the process of concentrating and purifying formic acid prior to use in ethanol fermentation. (4) In the above-described ethanol production method, formic acid may be added to a culture medium in which the yeast to be used in the ethanol fermentation is cultured prior to use in the ethanol fermentation. With this configuration, contamination due to the proliferation of various bacteria can be suppressed in the culture medium in which the yeast to be used in the ethanol fermentation is cultured prior to use in the ethanol fermentation. Furthermore, the efficiency of ethanol production in ethanol fermentation using the yeast cultured in such a culture medium can be improved. (5) In the above-described ethanol production method, when ethanol fermentation is performed by the yeast, at least one of a glycerol transporter gene present in the transformed yeast and a glycerol transporter gene introduced into the transformed yeast may be expressed. In this configuration, the amount of glycerol taken up by the yeast can be increased by expressing the glycerol transporter gene. As a result, the activity of an oxidized coenzyme-dependent reaction using glycerol as a substrate by glycerol dehydrogenase is increased, increasing the amount of reduced coenzyme produced, and the reaction that produces glycerol during ethanol fermentation is suppressed, thereby increasing the amount of ethanol produced from acetic acid. (6) According to another aspect of the present disclosure, there is provided an ethanol production system utilizing ethanol fermentation by yeast, the ethanol production system including a fermenter in which ethanol fermentation by yeast proceeds, a raw material supply unit that supplies a raw material containing acetic acid in addition to sugar to the fermenter as a raw material for the ethanol fermentation, and a formic acid supply unit that supplies formic acid to the fermenter, the yeast included in the fermenter being a transformed yeast into which at least an acetaldehyde dehydrogenase gene has been introduced and which expresses formate dehydrogenase and glycerol dehydrogenase. According to this form of ethanol production system, when ethanol fermentation proceeds in the fermenter, a reduced coenzyme-dependent reaction by acetaldehyde dehydrogenase with acetyl CoA as a substrate, an oxidized coenzyme-dependent reaction by formate dehydrogenase with formic acid as a substrate, and an oxidized coenzyme-dependent reaction by glycerol dehydrogenase with glycerol as a substrate can proceed in the presence of formic acid. Therefore, while suppressing the glycerol production reaction that consumes NADH with glycerol dehydrogenase, the reduced coenzyme-dependent reaction by acetaldehyde dehydrogenase can be promoted using the reduced coenzyme produced by the action of formate dehydrogenase to produce ethanol from acetic acid contained in the raw material for ethanol fermentation. As a result, the ethanol production efficiency in ethanol fermentation can be improved. Furthermore, the addition of formic acid can suppress contamination due to the growth of various bacteria in the fermenter. The present disclosure can be realized in various forms other than those described above, for example, in the form of an ethanol fermentation yeast or a method for producing ethanol from acetic acid. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 is an explanatory diagram illustrating a schematic configuration of an ethanol production system according to a first embodiment. [Diagram 2] FIG. 1 is an explanatory diagram showing an overview of the reaction proceeding in a fermenter. [Diagram 3] FIG. 1 is an explanatory diagram showing a schematic configuration of an ethanol production system according to a second embodiment. [Figure 4] FIG. 1 is an explanatory diagram showing a schematic configuration of an artificial photosynthesis reactor. [Diagram 5] FIG. 1 is an explanatory diagram showing a schematic configuration of an artificial photosynthesis reactor. [Figure 6] FIG. 1 is an explanatory diagram showing a schematic configuration of an artificial photosynthesis reactor. [Figure 7] FIG. 2 is an explanatory diagram showing the molecular structure of a reduction catalyst. [Figure 8A] FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 8B]FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 9A] FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 9B] FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 10A] FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 10B] FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 11] FIG. 13 is an explanatory diagram showing a destination vector for level 0. [Figure 12] FIG. 13 is an explanatory diagram showing a destination vector for level 0. [Figure 13] FIG. 13 is an explanatory diagram showing a destination vector for level 1. [Figure 14] Schematic diagram showing the introduction of DNA sequences into the yeast genome. [Figure 15] FIG. 2 is an explanatory diagram showing the configuration of each level 1 module. [Figure 16A] FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 16B] FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 16C] FIG. 1 is an explanatory diagram showing the primers used for cloning the DNA fragments. [Figure 17] FIG. 2 is an explanatory diagram showing the content of each component in the fermentation liquid obtained in the fermentation test. [Figure 18] FIG. 2 is an explanatory diagram showing the content of each component in the fermentation liquid obtained in the fermentation test. [Figure 19] FIG. 2 is an explanatory diagram showing the content of each component in the fermentation liquid obtained in the fermentation test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] A. First embodiment: (A-1) Configuration of ethanol production system: 1 is an explanatory diagram showing a schematic configuration of an ethanol production system 110 according to a first embodiment of the present disclosure. The ethanol production system 110 is a system for producing ethanol by utilizing ethanol fermentation by yeast. The ethanol production system 110 includes a raw material supply unit 11, a fermenter 16, a bacterial culture tank 15, a formic acid supply unit 20, and a distillation unit 18.

[0008] The raw material supply unit 11 is a device that supplies raw materials for ethanol fermentation to the fermenter 16. The raw material supply unit 11 of this embodiment is a device that uses biomass to generate raw materials for ethanol fermentation (raw materials containing acetic acid in addition to sugar), and includes a pretreatment device 12 and a saccharification reaction tank 14. As the biomass, for example, herbaceous biomass such as rice straw, wheat straw, rice husk, bagasse, etc., and lignocellulosic biomass such as woody biomass such as waste wood and wood chips can be used. The pretreatment device 12 is a device that processes biomass to make it in a state that is easy to saccharify and obtains a pretreatment liquid, and can include, for example, a crushing device that crushes biomass, a device that acid-treats biomass, and a hydrothermal treatment device that performs hydrolysis using hot water. The saccharification reaction tank 14 is connected to the pretreatment device 12 via a pretreatment liquid supply path 30, receives a supply of pretreatment liquid from the pretreatment device 12, and decomposes the pretreatment liquid using enzymes such as cellulase and amylase to generate sugar such as glucose. During such a process in the raw material supplying unit 11, various secondary products containing acetic acid are generated by decomposition or condensation of biomass components, etc. As a result, the raw material supplying unit 11 produces a raw material (saccharified solution) containing acetic acid in addition to sugar.

[0009] The fermenter 16 is connected to the saccharification reaction tank 14 via a saccharified liquid supply line 32, and is a reaction tank in which ethanol is produced by fermentation with yeast using sugar in the saccharified liquid as a carbon source to obtain a fermented liquid. The yeast used in the fermenter 16 may have ethanol fermentation ability, and for example, yeast belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, etc. may be used. Yeast of the genus Saccharomyces or Schizosaccharomyces, which can perform ethanol fermentation even under aerobic conditions, are preferred, and in particular, Saccharomyces cerevisiae and Schizosaccharomyces pombe can be preferably used. In addition to the ethanol fermentation, the fermenter 16 may also carry out a reaction to further advance saccharification of the biomass-derived raw material supplied to the fermenter 16.

[0010] The bacteria culture tank 15 is a device for pre-culturing yeast to be used for ethanol fermentation in the fermentation tank 16. The bacteria culture tank 15 is connected to the fermentation tank 16 via a yeast supply path 38, and is capable of supplying the cultured yeast to the fermentation tank 16.

[0011] The formic acid supplying unit 20 is a device that supplies formic acid to the fermentation tank 16, that is, to the culture liquid undergoing ethanol fermentation. The formic acid supplying unit 20 includes a formic acid releasing unit 22 for releasing formic acid, a first formic acid inlet 24 connecting the formic acid releasing unit 22 and the fermentation tank 16, and a second formic acid inlet 26 connecting the formic acid releasing unit 22 and the bacteria culture tank 15. The formic acid releasing unit 22 may be, for example, a device that promotes a reaction to generate formic acid from water, carbon dioxide, etc., or may be a formic acid tank that stores formic acid that has been generated in advance. The configuration of the device that promotes a reaction to generate formic acid from water, carbon dioxide, etc. will be described in detail in the second embodiment. From the formic acid releasing unit 22, formic acid is supplied to the culture liquid in the fermentation tank 16 via the first formic acid inlet 24, and formic acid is supplied to the culture liquid in the bacteria culture tank 15 via the second formic acid inlet 26. If the formic acid concentration in the fermenter 16 or the bacterial culture tank 15 is too high, the formic acid may inhibit the growth of yeast and fermentation by the yeast. Therefore, it is desirable to control the amount of formic acid supplied from the formic acid release unit 22 so that the formic acid concentration in the fermenter 16 or the bacterial culture tank 15 is, for example, 3 g / L or less.

[0012] The distillation unit 18 is connected to the fermenter 16 via a fermentation liquid supply path 34. The distillation unit 18 is a device for recovering ethanol from the fermentation liquid, which is the product of the ethanol fermentation that proceeds in the fermenter 16, and increases the ethanol concentration through the processes of distillation and dehydration.

[0013] In the ethanol production system 110 of this embodiment, when ethanol fermentation progresses in the fermenter 16, formic acid is supplied from the formic acid supply unit 20 to the fermenter 16, and acetaldehyde dehydrogenase (EC 1.2.1.10), glycerol dehydrogenase (EC 1.1.1.6), and formate dehydrogenase (EC 1.17.1.9) act in the yeast in the fermenter 16. Here, acetaldehyde dehydrogenase is an enzyme that promotes a reduced coenzyme-dependent reaction with acetyl CoA as a substrate, glycerol dehydrogenase is an enzyme that promotes an oxidized coenzyme-dependent reaction with glycerol as a substrate, and formate dehydrogenase is an enzyme that promotes an oxidized coenzyme-dependent reaction with formic acid as a substrate. The combination of reduced coenzyme and oxidized coenzyme used when the above three types of enzymes act may be a combination of NADH and NAD, or a combination of NADPH and NADP. In the following, a configuration in which a combination of NADH and NAD is used as an example of a combination of a reduced coenzyme and an oxidized coenzyme will be described.

[0014] Here, since yeasts that perform ethanol fermentation generally do not have an acetaldehyde dehydrogenase gene, in this embodiment, a transformed yeast into which an acetaldehyde dehydrogenase gene has been introduced is used as the yeast used for ethanol fermentation. The acetaldehyde dehydrogenase gene introduced into the yeast is not particularly limited as long as it is a gene for an enzyme defined by EC number 1.2.1.10, and genes derived from various organisms can be used. Specifically, examples of the acetaldehyde dehydrogenase gene include the adhE gene and eutE gene in Escherichia coli, the acetaldehyde dehydrogenase gene derived from Clostridium beijerinckii, and the acetaldehyde dehydrogenase gene derived from Chlamydomonas reinhardtii.

[0015] In addition, when the yeast used for ethanol fermentation does not have a formate dehydrogenase gene, the formate dehydrogenase gene may be further introduced into the yeast used for ethanol fermentation. Here, "endogenous" means that the yeast, such as a wild strain, that has not undergone special gene introduction naturally has the gene. When the yeast used for ethanol fermentation has a formate dehydrogenase gene, such as Saccharomyces cerevisiae, the formate dehydrogenase obtained by expression of the endogenous formate dehydrogenase gene can be used, but the expression level of the formate dehydrogenase gene in such yeast may be further increased. As a method for increasing the expression level of the formate dehydrogenase gene, for example, a method of introducing a formate dehydrogenase gene into yeast to increase the copy number of the formate dehydrogenase gene in the yeast may be mentioned. In addition to or in addition to the method of introducing the formate dehydrogenase gene, the promoter of the formate dehydrogenase gene endogenous to the yeast may be replaced with a promoter for high expression. The formate dehydrogenase gene to be introduced into yeast is not particularly limited as long as it is a gene for an enzyme defined by the EC number EC 1.17.1.9, and genes derived from various organisms can be used. Specifically, examples of formate dehydrogenase genes include the FDH1 gene and FDH2 gene of Saccharomyces cerevisiae.

[0016] In addition, when the yeast used for ethanol fermentation does not substantially have an NAD-dependent glycerol dehydrogenase gene, such as Saccharomyces cerevisiae, the yeast used for ethanol fermentation may further include an NAD-dependent glycerol dehydrogenase gene. When the yeast used for ethanol fermentation has an NAD-dependent glycerol dehydrogenase gene, such as Schizosaccharomyces pombe, the glycerol dehydrogenase obtained by expression of the endogenous glycerol dehydrogenase gene can be used, and the expression level of the glycerol dehydrogenase gene in such yeast may be further increased. As a method for increasing the expression level of the glycerol dehydrogenase gene, for example, a method of introducing a glycerol dehydrogenase gene into yeast to increase the copy number of the glycerol dehydrogenase gene in the yeast can be mentioned. Alternatively, instead of or in addition to the method of introducing a glycerol dehydrogenase gene, the promoter of the glycerol dehydrogenase gene endogenous to yeast may be replaced with a promoter for high expression. The glycerol dehydrogenase gene to be introduced into yeast is not particularly limited as long as it is a gene for an enzyme defined by EC number EC1.1.1.6, and genes derived from various organisms can be used. Specifically, examples of the glycerol dehydrogenase gene include the gld1 gene of fission yeast Schizosaccharomyces pombe, the Gld1 gene of Schizosaccharomyces octosporus (NCBI accession number: XP_013020646), the gldA gene derived from Escherichia coli, and the gldA gene derived from Klebsiella pneumoniae or Thermoanaerobacterium thermosaccharolyticum.

[0017] When an acetaldehyde dehydrogenase gene, a formate dehydrogenase gene, or a glycerol dehydrogenase gene is introduced into yeast, it is preferable to use a gene whose base sequence has been modified to match the codon usage frequency of the yeast to be introduced into. Furthermore, the acetaldehyde dehydrogenase gene, the formate dehydrogenase gene, and the glycerol dehydrogenase gene to be introduced into yeast do not need to code for an enzyme having the same amino acid sequence as the enzyme derived from the above-mentioned organisms, and may have sequence similarity that allows them to exhibit activity as each enzyme.

[0018] (A-2) Reaction taking place in the fermenter: FIG. 2 is an explanatory diagram showing an overview of the reaction that proceeds in the fermenter 16. In the following, a reaction that proceeds in the fermenter 16 by supplying formic acid from the formic acid supply unit 20 to the fermenter 16 when ethanol fermentation proceeds in the fermenter 16 will be described. FIG. 2 illustrates a reaction in which glucose is used as sugar, which is a carbon source for ethanol fermentation. As shown in FIG. 2, ethanol fermentation using yeast produces ethanol from glucose via fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, acetaldehyde, and the like. At this time, as a side reaction of ethanol fermentation, a reaction proceeds in which glycerol is produced from fructose-1,6-bisphosphate via dihydroxyacetone phosphate, and the like. As described above, the raw material for ethanol fermentation supplied from the raw material supply unit 11 to the fermenter 16 contains acetic acid, so in the fermenter 16, a reaction proceeds in which acetyl CoA is produced from acetic acid.

[0019] In the present embodiment, the transformed yeast used for ethanol fermentation in the fermenter 16 has an acetaldehyde dehydrogenase gene introduced therein, as described above. The transformed yeast of the present embodiment can proceed with a reaction to produce acetaldehyde from acetyl-CoA produced from acetic acid as described above by introducing an acetaldehyde dehydrogenase gene into the transformed yeast to express acetaldehyde dehydrogenase. The reaction promoted by acetaldehyde dehydrogenase is shown below as formula (1). The reaction shown in formula (1) is a reaction that can proceed reversibly. As shown in FIG. 2, ethanol is also produced from acetaldehyde produced from acetyl-CoA by the reaction of formula (1). In FIG. 2, the acetaldehyde dehydrogenase gene is shown as eutE, which is an example of an acetaldehyde dehydrogenase gene.

[0020] Acetyl CoA + NADH + H + → Acetaldehyde + CoA + NAD + …(1)

[0021] Here, since the above-mentioned acetaldehyde dehydrogenase acts as an NADH-dependent enzyme when the reaction of formula (1) proceeds, NADH, which is a reduced coenzyme, is required to proceed with the reaction of producing acetaldehyde from acetyl-CoA. In this embodiment, formic acid is further supplied to the fermenter 16 to proceed with the reaction of producing carbon dioxide from formic acid, thereby producing NADH. The reaction proceeded by the formate dehydrogenase contained in the yeast when formic acid is added is shown below as formula (2). Note that the reaction shown in formula (2) is a reaction that can proceed reversibly. By utilizing NADH produced by the reaction of formula (2), which is an NAD-dependent reaction using formic acid as a substrate, the reaction efficiency of formula (1) can be improved. In FIG. 2, formate dehydrogenase is represented as FDH1. When using yeast that contains an endogenous formate dehydrogenase gene, such as Saccharomyces cerevisiae, the expression level of the formate dehydrogenase gene can be increased, as described above, to enhance the activity that promotes the reaction of equation (2), thereby increasing the amount of NADH produced.

[0022] Formate + NAD + → CO2 + NADH … (2)

[0023] As described above, the transformed yeast of this embodiment is provided with a glycerol dehydrogenase gene. Here, by supplying formic acid to the fermentation tank 16 as described above, NADH is generated by the action of formate dehydrogenase, but when ethanol fermentation proceeds, as described above, a reaction proceeds in which glycerol is generated from glucose via fructose-1,6-bisphosphate and dihydroxyacetone phosphate, etc. Here, the reaction in which glycerol is generated from dihydroxyacetone phosphate is an NADH-dependent reaction. Therefore, even if NADH is generated by proceeding the reaction of formula (2) by adding formic acid, the generated NADH is also consumed in the reaction in which glycerol is generated, making it difficult to increase the reaction efficiency of formula (1). In this embodiment, as described above, the transformed yeast expresses glycerol dehydrogenase, and the reaction shown in the following formula (3) proceeds, and the reaction proceeds in the opposite direction to the reaction in which glycerol is generated, and NADH is regenerated. Note that the reaction shown in formula (3) is a reaction that can proceed reversibly. The production of NADH by reaction (3), which is an NAD-dependent reaction using glycerol as a substrate, reduces the consumption of NADH resulting from the reaction producing glycerol, and makes it possible to efficiently use the NADH produced by reaction (2) using formic acid to advance reaction (1). In Figure 2, the glycerol dehydrogenase gene is shown as the gld1 gene, which is an example of a glycerol dehydrogenase gene.

[0024] Glycerol + NAD + → Dihydroxyacetone + NADH + H + … (3)

[0025] According to the ethanol production method and ethanol production system 110 of the first embodiment configured as described above, when ethanol fermentation proceeds in the fermenter 16, formic acid is supplied from the formic acid supply unit 20 to the fermenter 16, and acetaldehyde dehydrogenase, glycerol dehydrogenase, and formate dehydrogenase act in the yeast in the fermenter 16. That is, when ethanol fermentation proceeds in the fermenter 16, in the presence of formic acid, an NADH-dependent (reduced coenzyme-dependent) reaction by acetaldehyde dehydrogenase with acetyl-CoA as a substrate, an NAD-dependent (oxidized coenzyme-dependent) reaction by formate dehydrogenase with formic acid as a substrate, and an NAD-dependent (oxidized coenzyme-dependent) reaction by glycerol dehydrogenase with glycerol as a substrate proceed. Therefore, while glycerol dehydrogenase suppresses the glycerol production reaction that consumes NADH (reduced coenzyme), NADH (reduced coenzyme) produced by the action of formate dehydrogenase is used to advance the NADH-dependent (reduced coenzyme-dependent) reaction by acetaldehyde dehydrogenase, and ethanol can be produced from the acetic acid contained in the raw material for ethanol fermentation. As a result, the efficiency of ethanol production in ethanol fermentation can be increased.

[0026] Furthermore, in this embodiment, in addition to the fermenter 16, formic acid is also supplied from the formic acid supply unit 20 to the bacteria culture tank 15 in which yeast for use in ethanol fermentation is cultured prior to use in ethanol fermentation. Therefore, contamination due to the proliferation of unwanted bacteria can be suppressed in the fermenter 16 and the bacteria culture tank 15. Specifically, yeast used in ethanol fermentation generally grows slower than bacteria (unwanted bacteria) that can cause contamination, so that it may be contaminated by bacteria during cultivation. The main unwanted bacteria that can cause contamination in ethanol fermentation are lactic acid bacteria. When lactic acid bacteria proliferate during ethanol fermentation, sugar is converted to lactic acid by the lactic acid bacteria, so that the reaction that produces lactic acid competes with ethanol fermentation, and the productivity of ethanol may decrease. Here, it is known that formic acid exhibits relatively higher antibacterial properties against lactic acid bacteria than against yeast (Journal of the Japanese Society of Food Engineering, 1994, 41, 687, https: / / doi.org / 10.3136 / nskkk1962.41.687). Therefore, the growth of unwanted bacteria such as lactic acid bacteria can be suppressed by adding formic acid to the culture medium in the fermentation tank 16 or the bacterial culture tank 15. As a result, sugar consumption by unwanted bacteria such as lactic acid bacteria is suppressed, and the ethanol production efficiency can be improved.

[0027] As described above, a combination of NADPH and NADP may be used as a combination of a reduced coenzyme and an oxidized coenzyme. That is, when the reaction shown in FIG. 2 is carried out in the fermenter 16, acetaldehyde dehydrogenase that promotes an NADPH-dependent reaction using acetyl CoA as a substrate, formate dehydrogenase that promotes an NADP-dependent reaction using formic acid as a substrate, and glycerol dehydrogenase that promotes an NADP-dependent reaction using glycerol as a substrate may be used. As for NADPH-dependent acetaldehyde dehydrogenase, for example, it is known that acetaldehyde dehydrogenase derived from Clostridium beijerinckii has NADPH-dependent activity as well as NAD-dependent activity (Appl. Environ. Microbiol., 56, 2591-2599(1990) https: / / doi.org / 10.1128 / aem.56.9.2591-2599.1990). Regarding NADP-dependent formate dehydrogenase, for example, it is known that the FDH1 of Saccharomyces cerevisiae is converted to NADP-dependent by introducing two point mutations (D196A and Y197R) into the amino acid sequence (Biochem. J., 367:841-847(2002). doi: 10.1042 / BJ20020379). Similarly, it is known that the NAD-dependent FDH gene of Candida boidinii is converted to an NADP-dependent enzyme by introducing a mutation (FEBS J. 75:3859-69(2008). doi: 10.1111 / j.1742-4658.2008.06533.x.). Such an acetaldehyde dehydrogenase having NADPH-dependent activity or a formate dehydrogenase having NADP-dependent activity can be introduced into yeast. Regarding NADP-dependent glycerol dehydrogenase, eukaryotes such as Saccharomyces cerevisiae generally have endogenous NADP-dependent glycerol dehydrogenase.Therefore, as described above, glycerol dehydrogenase produced by expressing an endogenous NADP-dependent glycerol dehydrogenase gene or an NADP-dependent glycerol dehydrogenase gene further introduced into yeast may be used. By adopting such a configuration, the same effects as those of the first embodiment may be obtained.

[0028] Furthermore, when the gene for formate dehydrogenase, which promotes an NADP-dependent reaction using formate as a substrate, is introduced into yeast, the formate dehydrogenase produces NADPH, suppressing the reaction that produces NADPH in the pentose phosphate cycle that functions in yeast. The reaction that produces NADPH in the pentose phosphate cycle produces carbon dioxide along with NADPH, which results in a reduction in carbon dioxide emissions and an improvement in the ethanol yield in yeast.

[0029] B. Second embodiment: 3 is an explanatory diagram showing a schematic configuration of an ethanol production system 210 as a second embodiment. The ethanol production system 210 is a system that produces ethanol by utilizing ethanol fermentation by yeast, and the same reference numbers are used for parts common to the ethanol production system 110 of the first embodiment. The ethanol production system 210 of the second embodiment differs from the first embodiment in that it includes a formic acid supply unit 220 instead of the formic acid supply unit 20.

[0030] The formic acid supply unit 220 is an apparatus that supplies formic acid to the fermenter 16 and the bacterial culture tank 15, similar to the formic acid supply unit 20. The formic acid supply unit 220 includes a formic acid production unit 222 that produces formic acid using carbon dioxide, a first formic acid introduction path 24 that connects the formic acid production unit 222 to the fermenter 16, a second formic acid introduction path 26 that connects the formic acid production unit 222 to the bacterial culture tank 15, and a carbon dioxide supply path 28 that connects the formic acid production unit 222 to the fermenter 16. The formic acid production unit 222 may be an apparatus that produces formic acid using carbon dioxide supplied from the fermenter 16 via the carbon dioxide supply path 28. The formic acid production unit 222 can also be called a "formic acid release unit" that releases formic acid.

[0031] The process of generating formic acid from carbon dioxide proceeding in the formic acid generating unit 222 can be, for example, a process that uses a reaction using an organic metal complex or an inorganic metal as a catalyst and electricity, heat, or light as an energy source. Specifically, there are electrochemical processes that use electric energy using metal electrodes and metal electrodes modified with conductive polymers or organic metal complexes, thermochemical processes that supply raw carbon dioxide as a catalyst in a reactor and heat it, photochemical processes that irradiate a catalyst with light of a certain wavelength or natural sunlight and use the light energy, and photoelectrochemical processes that use a combination of electric energy and light energy. An example of a photochemical process is an artificial photosynthesis process using a photocatalyst. An example of a photoelectrochemical process is an artificial photosynthesis process that combines a solar cell and an electrochemical reactor.

[0032] 4 to 6 are explanatory diagrams showing the schematic configuration of an artificial photosynthesis reactor. As an example of a device that can be used as the formic acid generator 222, an overview of an artificial photosynthesis reactor will be described with reference to FIGS. 4 to 6. There are three types of artificial photosynthesis reactors that execute the process of generating formic acid from carbon dioxide, as shown in FIGS. 4 to 6. These artificial photosynthesis reactors combine an electrode that performs an oxidation reaction of water with an electrode that performs a reduction reaction of carbon dioxide (CO2) to generate formic acid, which is an organic substance, at room temperature and pressure.

[0033] Figure 4 shows a structure using a photocatalyst (PC) made of semiconductor particles, in which holes (h + ) to oxidize water to generate oxygen (O2), while at the same time reducing CO2 with excited electrons to generate formic acid. This type of device absorbs only the short-wavelength components of the solar spectrum and uses them in the reaction, and is highly practical in that it is low-cost.

[0034] Figure 5 shows the configuration of a photoelectrochemical reactor (PEC) that combines a semiconductor and a molecular catalyst such as a Ru complex polymer. In the photoelectrochemical reactor in Figure 5, holes (h +) oxidizes water on an oxidation catalyst to generate oxygen (O2), and at the same time, excited electrons reduce CO2 on a reduction catalyst to generate formic acid. This type of device increases the efficiency of electron-hole generation and separation by using a SiGe triple-junction (SiGe-3jn) semiconductor, and can reduce the energy required for CO2 reduction (low overvoltage) by molecular design of the reduction catalyst (molecular catalyst) described below. For example, the conversion efficiency from sunlight to chemical energy (η STC ), it is possible to obtain values ​​that exceed the efficiency of photosynthesis in plants.

[0035] FIG. 6 shows a reactor combining an electrochemical reactor (EC) and a solar cell (PV), which uses a reduction catalyst (molecular catalyst) similar to that of the reactor in FIG. 5. FIG. 7 is an explanatory diagram showing the molecular structure of Ru complex polymer (RuCP, [Ru[4,4'-di(1-H-1-pyrrolypropyl carbonate)-2,2'-bipyridine](CO)2Cl2]), which is a reduction catalyst used in the cathode of the reactor shown in FIG. 5 and FIG. 6. In the reactor in FIG. 6, the electrochemical reactor (EC) and the solar cell (PV) are connected with electric wires to separate their functions, and the PV power is used to electrochemically cause the oxidation reaction of water and the reduction reaction of CO2. In addition, a separator that allows hydrogen ions necessary for formic acid synthesis to pass but does not allow oxygen, which inhibits formic acid synthesis, is placed between the anode and the cathode. With this configuration, it is possible to achieve both high efficiency and large area. For example, in a reactor combining EC and PV as shown in Figure 6, higher conversion efficiency has been achieved by using an EC reactor structure in which multiple anodes and cathodes are stacked (ACS Sustainable Chem. Eng., 2021, 9, 2348).

[0036] According to the ethanol production system 210 of the second embodiment, carbon dioxide discharged from the fermenter 16 by ethanol fermentation is utilized in the process of producing formic acid in the formic acid supply unit 220, so that it is possible to reduce the amount of carbon dioxide emission. The reaction in which ethanol is produced from glucose that proceeds in the fermenter 16 is shown below as formula (4). When the reaction of formula (4) proceeds, approximately half the weight of the sugar is converted to carbon dioxide. In the past, carbon dioxide generated during ethanol fermentation was generally released to the atmosphere, but according to this embodiment, carbon dioxide generated during ethanol fermentation is utilized to produce formic acid, so that it is possible to effectively utilize carbon dioxide and reduce the amount of carbon dioxide emission.

[0037] C6H 12 O6 → 2C2H5OH + 2CO2… (4)

[0038] In addition, it is desirable that the raw material gas used in the process of generating formic acid from carbon dioxide in the formic acid generating unit 222 has a sufficiently high carbon dioxide concentration to ensure the efficiency of formic acid generation, and a sufficiently low oxygen concentration so as not to inhibit the formic acid generating reaction. For example, when carbon dioxide is dissolved in water and supplied to the electrochemical reactor described above (CO2 liquid phase supply), the carbon dioxide concentration to be supplied is desirably 50% or more, assuming that the carbon dioxide saturation concentration is 100%. When carbon dioxide gas is directly supplied to the electrochemical reactor (CO2 gas phase supply), the carbon dioxide concentration is desirably 10% or more. In that case, the oxygen concentration is desirably 5% or less. In the second embodiment, a gas with a sufficiently high carbon dioxide concentration and a suppressed oxygen concentration for the above purpose is generated with the ethanol fermentation in the fermenter 16. Therefore, it is possible to supply and use the gas generated in the fermenter 16 to the formic acid generating unit 222 without subjecting it to special treatment, which simplifies the system configuration and improves the energy efficiency of the system. On the other hand, for example, combustion gas is considered as another carbon dioxide-containing gas that can be used for generating formic acid in the formic acid generating unit 222 and is relatively easy to obtain. However, since the carbon dioxide concentration in the combustion gas is generally about 10%, it is necessary to concentrate the carbon dioxide and further remove oxygen in order to ensure the efficiency of generating formic acid in the formic acid generating unit 222, which complicates the device configuration and requires energy consumption for the above-mentioned processes.

[0039] According to the second embodiment, the formic acid-containing liquid obtained in the formic acid generating unit 222 can be supplied to the fermenter 16 or the bacterial culture tank 15 without any special treatment, making the purification process of formic acid unnecessary, simplifying the system configuration, and suppressing a decrease in the energy efficiency of the system. For example, the formic acid concentration in the liquid obtained from the formic acid generating unit 222 as described with reference to Figs. 4 to 6 is relatively low, and impurities (such as potassium phosphate and phosphoric acid, which are solutes of the electrolyte, and organic amines, which are organic bases) may also be contained. However, when used to supply formic acid to the fermenter 16 as a substrate for formate dehydrogenase, or to supply formic acid to suppress contamination of the fermenter 16 or the bacterial culture tank 15, the formic acid-containing liquid discharged from the formic acid generating unit 222 can be used without any special treatment for purifying formic acid.

[0040] C. Other embodiments: In each of the above-mentioned embodiments, in order to suppress the consumption of NADH due to the production of glycerol accompanying ethanol fermentation, the gene of glycerol dehydrogenase is expressed to allow the reaction of regenerating NADH to proceed. Here, in order to suppress the consumption of NADH accompanying the production of glycerol, a glycerol transporter gene, specifically STL1 (Mol. Biol. Cell, 2005, 16, 2068), may be used. By expressing the glycerol transporter gene, the amount of glycerol taken up by yeast can be increased. As a result, the activity of the reaction of formula (3) by glycerol dehydrogenase is increased, the amount of NADH regenerated is increased, and the reaction of producing glycerol accompanying ethanol fermentation is suppressed, and the amount of ethanol produced from acetic acid can be increased. The above-mentioned glycerol transporter gene may be a gene inherent to the yeast used for ethanol fermentation, or may be a gene introduced into the yeast. For example, Saccharomyces cerevisiae has an endogenous STL1 gene, and it is desirable to increase the expression level of the STL1 gene by further introducing an STL1 gene, etc. Alternatively, the promoter of the glycerol transporter gene endogenous to the yeast may be replaced with a promoter for high expression.

[0041] In each of the above-described embodiments, formic acid is supplied from the formic acid supply unit 20 or the formic acid supply unit 220 to the bacteria culture tank 15 in addition to the fermenter 16, but a different configuration may be used, and formic acid may not be supplied to the bacteria culture tank 15. Even in this case, by supplying formic acid to the fermenter 16, it is possible to obtain the similar effect of promoting ethanol production from acetic acid to increase the efficiency of ethanol production and suppressing contamination of the fermenter 16 by unwanted bacteria.

[0042] In the above-mentioned embodiments, examples of using glucose as a substrate for ethanol fermentation have been shown, but a different configuration may be used. For example, instead of glucose, or in addition to glucose, ethanol fermentation may be performed using a different type of sugar, such as a pentose such as xylose, as a substrate. For example, when using xylose and using a yeast that does not have the ability to assimilate xylose as a yeast for ethanol fermentation, a yeast into which a gene for an enzyme required for assimilation of xylose has been introduced, specifically, a transformed yeast into which a gene for xylose isomerase (XI) or a gene for xylose reductase (XR) and a gene for xylitol dehydrogenase (XDH) have been introduced may be used. Xylose enters the pentose phosphate cycle, but ultimately enters the glycolysis system, so that glycerol is produced as a metabolic product in xylose fermentation as in glucose fermentation. In this way, sugar from which glycerol is produced as a metabolic product is used as a substrate for ethanol fermentation, and by adding formic acid and activating acetaldehyde dehydrogenase, formate dehydrogenase, and glycerol dehydrogenase, the same effects as those of the above-mentioned embodiments can be obtained.

[0043] In each of the above-mentioned embodiments, the raw material for ethanol fermentation containing sugar and acetic acid is obtained by processing biomass, but a different configuration may be used. The raw material for ethanol fermentation is not limited to a raw material derived from biomass, and if a raw material containing sugar and acetic acid is used, the same effects as those of each of the embodiments can be obtained by applying the ethanol fermentation method and ethanol production system disclosed herein. EXAMPLES

[0044] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the description of these examples. In the examples described below, yeast capable of metabolizing formic acid and acetic acid was prepared and a fermentation test was performed.

[0045] <Creation of formic acid / acetic acid metabolizing yeast> (Construction of a plasmid for overexpressing FDH1·eutE) While leaving the PFK1 gene at the PFK1 locus, homologous recombination was performed near the PFK1 locus to create the plasmid "FDH1·eutE overexpression plasmid: pUC-5U_PFK1-PFK1-T_PFK1-P_FBA1-FDH1-T_RPL41B-P_TDH3-eutE-T_RPL3-loxP71-P_TEF1-SAT-T_LEU2-P_GAL1-Crei-T_CYC1-loxP66-3U_PFK1," which contains the sequences required for introducing the formate dehydrogenase gene (FDH1) derived from S. cerevisiae and the acetaldehyde dehydrogenase gene (eutE) derived from E. coli into yeast. The nucleotide sequence of the FDH1 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the FDH1 gene is shown in SEQ ID NO:2. The nucleotide sequence of the eutE gene is shown in SEQ ID NO:3, and the amino acid sequence of the protein encoded by the eutE gene is shown in SEQ ID NO:4.

[0046] This plasmid contains the FDH1 gene with the FBA1 promoter and RPL41B terminator added, the eutE gene with the TDH3 promoter and RPL3 terminator added, a 4000 bp upstream DNA sequence (5U_PFK1-PFK1-T_PFK1) containing the PFK1 gene CDS from approximately 300 bp downstream from the 3' end of the PFK1 gene, a 500 bp downstream DNA sequence (3U300_PFK1) from approximately 300 bp downstream from the 3' end of the PFK1 gene, a gene sequence (natR marker) containing the nourseothricin resistance gene (natR) (GenBank ID S60706.1) with the TEF1 promoter and LEU2 terminator added, the GAL1 promoter and CYC1 terminator, and the CDS (CoDing The vector was constructed to contain a Cre-inducible expression cassette with an intron of COX5B added so that the COX5B protein coding region (protein coding region) was split. The marker gene and Cre gene were sandwiched between two mutant loxP sites (Plant J.1995, 7, 649 https: / / doi.org / 10.1046 / j.1365-313X.1995.7040649.x), and the marker gene and Cre gene could be simultaneously removed by expressing the Cre gene.

[0047] Figures 8A and 8B are explanatory diagrams showing the primers used for cloning the DNA fragments necessary for the construction of the above-mentioned FDH1·eutE overexpression plasmid. Using these primers, each of the "amplified DNA fragments" shown in Figures 8A and 8B was amplified by PCR. Each of the above-mentioned primers was designed by adding a DNA sequence so that it overlaps with the adjacent DNA sequence by 15 bp or more in order to bind each of the amplified DNA fragments. As templates for amplification, S. cerevisiae OC-2 strain genomic DNA, E. coli K-12 strain genomic DNA, pUC19, and synthetic genes were used. Each of the target DNA fragments was amplified as described above, and the DNA fragments were sequentially bound using NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs NEB) or the like, and cloned into the plasmid pUC19 to prepare the final "FDH1·eutE overexpression plasmid."

[0048] (Construction of a plasmid for overexpressing eutE) We created a plasmid for overexpressing eutE (PDC6 locus): pUC-5U500_PDC6-T_DIT1-eutE-P_HOR7-loxP71-P_TEF1-SAT-T_LEU2-P_GAL1-Crei-T_CYC1-loxP66-5U_PDC6, which contains the sequence required for introducing the eutE gene into yeast by homologous recombination near the PDC6 locus while leaving the PDC6 gene at the PDC6 locus.

[0049] This plasmid was constructed to contain the eutE gene with the HOR7 promoter and DIT1 terminator added, a DNA sequence (5U500_PDC6) in the region of approximately 500-950 bp upstream from the 5' end of the PDC6 gene as a region for homologous recombination into the yeast genome, a DNA sequence (5U_PDC6) in the region of approximately 450 bp upstream from the 5' end of the PDC6 gene, a nat marker, and a Cre inducible expression cassette. The marker gene and the Cre inducible expression cassette are sandwiched between two mutant loxP sequences, and it is possible to simultaneously remove the marker and Cre gene by expressing the Cre gene.

[0050] 9A and 9B are explanatory diagrams showing primers used for cloning DNA fragments necessary for the preparation of the above-mentioned eutE overexpression plasmid (PDC6 locus), and each of the "amplified DNA fragments" shown in FIG. 9A and FIG. 9B was amplified by PCR using these primers. Each of the above-mentioned primers was designed by adding a DNA sequence so as to overlap with the adjacent DNA sequence by 15 bp or more in order to bind each of the amplified DNA fragments. As templates for amplification, S. cerevisiae OC-2 strain genomic DNA, E. coli K-12 strain genomic DNA, pUC19, and synthetic genes were used. The target DNA fragments were amplified as described above, and the DNA fragments were sequentially bound using NEBuilder HiFi DNA Assembly Cloning Kit or the like, and cloned into the plasmid pUC19 to prepare the final "eutE overexpression plasmid (PDC6 locus)".

[0051] We created a plasmid for overexpressing eutE (ADH2 locus): pUC-5U_ADH2-ADH2-3U_ADH2-T_DIT1-eutE-P_HOR7-loxP71-P_TEF1-SAT-T_LEU2-P_GAL1-Crei-T_CYC1-loxP66-3U300_ADH2, which contains the sequence required for introducing the eutE gene into yeast by homologous recombination near the ADH2 locus while leaving the ADH2 gene at the ADH2 locus.

[0052] This plasmid was constructed to contain the eutE gene with the HOR7 promoter and DIT1 terminator added, a DNA sequence of about 2300 bp from about 300 bp downstream of the 3' end of the ADH2 gene to 500 bp upstream of the ADH2 gene (5U_ADH2-ADH2-3U_ADH2) as a homologous recombination region into the yeast genome, a DNA sequence of about 300-800 bp downstream of the 3' end of the ADH2 gene (3U300_ADH2), a nat marker, and a Cre induction expression cassette. The marker gene and the Cre induction expression cassette are sandwiched between two mutant loxP sequences, and it is possible to simultaneously remove the marker and Cre gene by expressing the Cre gene.

[0053] 10A and 10B are explanatory diagrams showing primers used for cloning DNA fragments necessary for the preparation of the above-mentioned eutE overexpression plasmid (ADH2 locus), and each of the "amplified DNA fragments" shown in FIG. 10A and FIG. 10B was amplified by PCR using these primers. Each of the above-mentioned primers was designed by adding a DNA sequence so as to overlap with the adjacent DNA sequence by 15 bp or more in order to bind each of the amplified DNA fragments. As templates for amplification, S. cerevisiae OC-2 strain genomic DNA, E. coli K-12 strain genomic DNA, pUC19, and synthetic genes were used. The target DNA fragments were amplified as described above, and the DNA fragments were sequentially bound using NEBuilder HiFi DNA Assembly Cloning Kit or the like, and cloned into the plasmid pUC19 to prepare the final "eutE overexpression plasmid (ADH2 locus)".

[0054] (Construction of a plasmid for overexpressing gld1·STL1) We constructed a "gld1·STL1 overexpression plasmid," which is a plasmid containing sequences required for introducing the glycerol dehydrogenase gene (gld1) and the glycerol transporter gene (STL1) into yeast. The nucleotide sequence of the gld1 gene is shown in SEQ ID NO: 95, and the amino acid sequence of the protein encoded by the gld1 gene is shown in SEQ ID NO: 96. The nucleotide sequence of the STL1 gene is shown in SEQ ID NO: 97, and the amino acid sequence of the protein encoded by the STL1 gene is shown in SEQ ID NO: 98.

[0055] This plasmid was constructed by the Golden Gate method (PLoS ONE 6, e16765(2011) https: / / doi.org / 10.1371 / journal.pone.0016765). The Golden Gate method is a well-known gene integration method in which multiple DNA fragments are inserted into a vector in a pre-designed order using Type IIS restriction enzymes and T4 DNA ligase. Vectors equivalent to the level 0 and level 1 destination vectors were prepared as subcloning vectors required for the Golden Gate method. Below, the methods for constructing the level 0 and level 1 destination vectors are explained in order.

[0056] 11 and 12 are explanatory diagrams showing destination vectors for level 0. As shown in FIG. 11 and FIG. 12, the destination vector for level 0 is a vector containing a BsaI recognition site. When incorporating one fragment into the destination vector for level 0, the vector shown in FIG. 11 was used, and when incorporating three fragments, the vector set (A) to (C) shown in FIG. 12 was used. When preparing the destination vector for level 0, the target DNA fragment was amplified by PCR using pUC19 or synthetic DNA as a template. The primers used in PCR were artificially synthesized, with DNA sequences added so as to overlap with adjacent DNA sequences by 15 bp or more in order to bind each DNA fragment. The amplified PCR fragments were sequentially bound using the NEBuilder HiFi DNA Assembly Cloning Kit to complete the destination vector for level 0.

[0057] FIG. 13 is an explanatory diagram showing a destination vector for level 1. As shown in FIG. 13, the destination vector for level 1 is a set of multiple types of vectors, each of which includes a fluorescent protein zFP538 gene (Nat. Biotechnol. 17, 969-973 (1999)) and BsaI recognition sites arranged in opposite directions on either side of the gene. In addition, each of the destination vectors for level 1 differs in the presence or absence, combination, and arrangement of a mutant loxP sequence (Plant J., 7, 649-659 (1995) https: / / doi.org / 10.1046 / j.1365-313X.1995.7040649.x), sequences α to δ described below, and a restriction enzyme site and a cutting site used in the Golden Gate reaction. These destination vectors for level 1 were prepared by amplifying the target DNA fragments using pUC19 and synthetic DNA (including a kanamycin resistance gene) as templates, and sequentially joining the DNA fragments using the NEBuilder HiFi DNA Assembly Cloning Kit. Here, the above-mentioned sequences α to δ are homologous sequences of about 57 bp provided at the ends of each amplified fragment obtained by PCR amplification in order to join the amplified fragments in the desired order. Sequences α to δ can be any sequences as long as they have sufficiently low sequence similarity to each other. Here, we set artificial sequences that have little similarity to the yeast genome sequence. For sequences α and β, we used the sequences described in Microb Cell Fact 12, 47(2013)https: / / doi.org / 10.1186 / 1475-2859-12-47, and for sequences γ and δ, we used the sequences described in Nucleic Acids Res. 43, 6620-6630(2015)https: / / doi.org / 10.1093 / nar / gkv466.

[0058] (Preparation of yeast vector set) FIG. 14 is an explanatory diagram showing the introduction of a DNA sequence into the ALD4 locus of the yeast genome by homologous recombination of a level 1 module prepared using the above-mentioned destination vector for level 0 and destination vector for level 1. As shown in FIG. 14, all vectors tested for yeast were designed so that by mixing five fragment sets and transforming them, recombination occurs between the homologous regions of each fragment (any of sequences α to δ), and the resulting fragment is finally introduced into the yeast genome as one fragment. In FIG. 14, each fragment constituting the five fragment set for obtaining one fragment to be introduced into the yeast genome is shown as level 1 modules A to E in the order from the 5' side after ligation.

[0059] Figure 15 is an explanatory diagram showing the configuration of each of the level 1 modules used for introduction into the yeast genome shown in Figure 14. That is, Figure 15 shows, for each level 1 module, the type of level 1 destination vector used (see Figure 13), the type of level 0 module, and the configuration of the level 0 module insert. In Figure 15, the level 1 module V2P238 corresponds to the level 1 module A, the level 1 module V2P644 corresponds to the level 1 module B, the level 1 module V2P306 corresponds to the level 1 module C, the level 1 module V2P118 corresponds to the level 1 module D, and the level 1 module V2P86 corresponds to the level 1 module E.

[0060] In the following, the method of constructing each level 1 module will be described. First, the sequence of the region (CDS) coding for the ALD4 protein for performing homologous recombination with the genome, the full length of the ORF or the ATG41 terminator, was amplified by PCR using the S. cerevisiae BY4742 strain genome or synthetic DNA as a template. The primers used are shown in FIG. 16A. The target DNA fragments were sequentially linked using the NEBuilder HiFi DNA Assembly Cloning Kit and subcloned into a vector corresponding to the destination vector for level 0 of the Golden Gate method, and the obtained modules were named level 0 modules V1P1030 and V1P153. Note that a short spacer sequence was introduced as an insert into V1P20. These level 0 modules are used to obtain level 1 module A (V2P238).

[0061] In addition, the nat1 resistance gene fused with the PMA1 promoter and LEU2 terminator, the INO1 promoter, and the Cre gene fused with the COX5B intron so that the CYC1 terminator and CDS are split were subcloned into a destination vector for level 0, and the resulting module was named V1P263, a level 0 module. This will be used to obtain level 1 module B (V2P644).

[0062] In addition, the FBA1 promoter, gld1 (GenBank ID 2542881) derived from S. pombe, and the DIT1 terminator were subcloned into the destination vector for level 0, and the resulting modules were named level 0 modules V1P105, V1P1031, and V1P137. These were used to obtain level 1 module C (V2P306).

[0063] In addition, the TDH3 promoter, STL1, and RPL41B terminator were subcloned into the destination vector for level 0, and the resulting modules were named level 0 modules V1P154, V1P1032, and V1P109, which were used to obtain level 1 module D (V2P118).

[0064] In addition, a DNA fragment containing a homologous recombination region extending from 301 bp on the 3'-end of the CDS of the ALD4 gene in the yeast genomic DNA to be introduced to approximately 850 bp was subcloned into a destination vector for level 0, and the resulting module was named level 0 module V1P73. This level 0 module was used to obtain level 1 module E (V2P86).

[0065] Each of the level 0 module vectors described above was subjected to Golden Gate reaction (restriction enzyme BsaI) with the corresponding level 1 destination vector shown in Figure 15 to generate level 1 modules. Five types of the obtained level 1 modules were combined and introduced into the yeast genome.

[0066] (Creation of formate / acetate metabolizing strains) The fragment between the homologous recombination sites of the "FDH1·eutE overexpression plasmid: pUC-5U_PFK1-PFK1-T_PFK1-P_FBA1-FDH1-T_RPL41B-P_TDH3-eutE-T_RPL3-loxP71-P_TEF1-SAT-T_LEU2-P_GAL1-Crei-T_CYC1-loxP66-3U_PFK1" constructed as described above was amplified by PCR and used to transform S. cerevisiae OC-2 strain. Transformation was performed according to the method of Akada et al. (BioTechniques 28,852,2000 https: / / doi.org / 10.2144 / 00285bm05).

[0067] Then, yeast transformed with a fragment obtained from the above-mentioned "FDH1·eutE overexpression plasmid" was spread on YPD agar medium containing nourseothricin, and the grown colonies were purified. Next, this strain was allowed to form spores on sporulation medium (1% potassium phosphate, 0.1% yeast extract, 0.05% glucose, 2% agar), and the cells were doubled by utilizing homothallic property. The strain was then inoculated on YPGa agar medium (yeast extract 10g / L, peptone 20g / L, galactose 20g / L), and expression of the Cre gene was induced, and the nat marker was deleted by site-specific recombination. This strain was named Uz4184.

[0068] Similarly, the above Uz4184 strain was transformed with a fragment amplified by PCR between the homologous recombination sites of the "eutE overexpression plasmid (PDC6 locus): pUC-5U500_PDC6-T_DIT1-eutE-P_HOR7-loxP71-P_TEF1-SAT-T_LEU2-P_GAL1-Crei-T_CYC1-loxP66-5U_PDC6" prepared as described above. Then, the yeast further transformed with a fragment obtained from the above-mentioned "eutE overexpression plasmid (PDC6 locus)" was spread on a YPD agar medium containing nourseothricin, and the grown colonies were purified. Next, this strain was allowed to form spores in a sporulation medium, and doubled by utilizing homothallic properties. It was further inoculated on a YPGa agar medium, expression of the Cre gene was induced, and the nat marker was deleted by site-specific recombination. This was named strain Uz4264.

[0069] Similarly, the above Uz4264 strain was transformed with a fragment amplified by PCR between the homologous recombination sites of the "eutE overexpression plasmid (ADH2 locus): pUC-5U_ADH2-ADH2-3U_ADH2-T_DIT1-eutE-P_HOR7-loxP71-P_TEF1-SAT-T_LEU2-P_GAL1-Crei-T_CYC1-loxP66-3U300_ADH2" prepared as described above. Thereafter, the yeast further transformed with a fragment obtained from the above-mentioned "eutE overexpression plasmid (ADH2 locus)" was spread on a YPD agar medium containing nourseothricin, and the grown colonies were purified. Next, this strain was allowed to form spores in a sporulation medium, and doubling was performed using homothallic properties. The yeast was then inoculated onto YPGa agar medium, and expression of the Cre gene was induced to delete the nat marker by site-specific recombination. The purified yeast was designated Uz4273.

[0070] Similarly, the above Uz4273 strain was transformed using a fragment amplified by PCR between the homologous recombination sites of five types of level 1 modules (level 1 modules A to E, V2P238, V2P644, V2P306, V2P118, and V2P86) prepared as described above. Then, the yeast further transformed with the above Uz4273 strain was spread on YPD agar medium containing nourseothricin, and the grown colony was purified. Next, this strain was allowed to form spores in a sporulation medium, and doubled by utilizing homothallic properties. It was further inoculated on YPGa agar medium, expression of the Cre gene was induced, and the nat marker was deleted by site-specific recombination. The purified yeast was named Uz4288 strain.

[0071] <Fermentation test> The test strains were inoculated into 50 mL cell reactor tubes (Greiner) containing 10 mL of bacterial growth medium, and cultured at 32 ° C for 24 hours. The test strains were the Uz4273 and Uz4288 strains, which are the transformed yeasts described above. In addition, the OC-2 strain, a wild-type strain of S. cerevisiae, was used as a control. Next, 270 μl of each cultured bacterium was inoculated into a 24-hole deep well plate containing 3.6 mL of ethanol fermentation medium, and a fermentation test was performed by shaking culture (230 rpm, amplitude 25 mm) at a temperature of 32 ° C for 40 hours. In addition, a silicon lid with a check valve was placed on each treatment area of ​​the 24-hole deep well plate, and the generated carbon dioxide gas was released to the outside air, but oxygen was not allowed to enter from the outside, so that each treatment area was kept in an anaerobic state. In addition, since the electrolytes used in the artificial photosynthesis process often contain high concentrations of salts (ACS Sustainable Chem. Eng. 2021,9, 16031, J. CO2 Util. 2017, 18, 222, J. CO2 Util. 2014, 7, 1), when conducting fermentation tests, potassium salts were added to the ethanol fermentation medium in addition to formic acid to mimic the conditions when using formic acid obtained from the artificial photosynthesis process.

[0072] As the bacterial growth media used in the fermentation test, a medium without formic acid and a medium containing formic acid were used. As the ethanol fermentation media, three types of media with different formic acid concentrations were used, a medium without formic acid and a medium containing formic acid. The composition of each medium is shown below. -Bacteria growth medium (without formic acid): molasses concentrate 100g / L, potassium dihydrogen phosphate 0.5g / L, urea 3.12g / L -Bacteria growth medium (with formic acid): Molasses stock solution 100g / L, potassium dihydrogen phosphate 0.5g / L, urea 3.12g / L, formic acid 3.0g / L Ethanol fermentation medium (without formic acid): glucose 200g / L, peptone 20g / L, yeast extract 10g / L, potassium phosphate 0.1M, acetic acid 3g / L Ethanol fermentation medium (with formic acid): glucose 200g / L, peptone 20g / L, yeast extract 10g / L, potassium phosphate 0.1M, acetic acid 3g / L, formic acid 1.0g / L Ethanol fermentation medium (with formic acid): glucose 200g / L, peptone 20g / L, yeast extract 10g / L, potassium phosphate 0.1M, acetic acid 3g / L, formic acid 2.0g / L Ethanol fermentation medium (with formic acid): glucose 200g / L, peptone 20g / L, yeast extract 10g / L, potassium phosphate 0.1M, acetic acid 3g / L, formic acid 3.0g / L

[0073] In the fermentation test, the test strain and the composition of each medium were changed and fermentation (cultivation) was performed, and then the content of each component in the fermentation liquid was measured using HPLC (Prominence; Shimadzu Corporation). The measurement conditions are as follows: Column: Aminex HPX-87H, Mobile phase: 0.01N H2SO4, Flow rate: 0.6mL / min, Temperature: 50℃, Detector: Differential refractive index detector RID-10A

[0074] <Fermentation test results> 17 to 19 are explanatory diagrams showing the results of measuring the content (unit: g / L) of each component in the fermentation liquid obtained by the fermentation test. In Fig. 17 to Fig. 19, the measured content (concentration) of each component is shown, along with the magnitude relationship of the content compared to the results of other test strains, by the size of the hatched rectangle.

[0075] In the fermentation test shown in FIG. 17, the test strains used were Uz4273, which overexpressed FDH1 and eutE, Uz4288, which overexpressed STL1 and gld1 in addition to FDH1 and eutE, and the wild-type strain OC-2. A medium without formic acid was used as the bacterial growth medium, and a medium with a formic acid concentration of 2.0 g / L was used as the ethanol fermentation medium. As shown in FIG. 17, in Uz4273, which overexpressed FDH1 and eutE, the concentrations of formic acid and acetate were reduced compared to the wild-type strain OC-2. However, the concentration of glycerol was increased, and the metabolism of formic acid and acetate did not lead to an improvement in ethanol productivity. In contrast, in Uz4288, which overexpressed STL1 and gld1 in addition to FDH1 and eutE, the concentrations of formic acid, acetate, and glycerol were all reduced, and ethanol productivity was improved. At the end of the fermentation in this test, all of the glucose had been consumed. As described above, when performing ethanol fermentation using a raw material containing acetic acid in addition to sugar, it was confirmed that in order to efficiently utilize the reducing power (NADH, a reducing agent) obtained by adding formic acid and expressing the formate dehydrogenase gene (FDH1) for ethanol production, it is necessary to control the metabolism of glycerol and reduce the amount of glycerol produced.

[0076] In the fermentation test shown in FIG. 18, Uz4288 and OC-2 were used as test strains, and a medium without formic acid and a medium with a formic acid concentration of 2.0 g / L were used as the ethanol fermentation medium. Furthermore, in an example where a medium without formic acid was used as the ethanol fermentation medium, a medium without formic acid and a medium with a formic acid concentration of 3.0 g / L were used as the bacterial growth medium. In the fermentation test shown in FIG. 18, n=3 was used for each condition. As shown in FIG. 18, Uz4288 consumes more acetic acid and glycerol than the wild-type strain OC-2, and the ethanol yield is improved. The ethanol yield was further increased by adding formic acid to the bacterial growth medium and the ethanol fermentation medium. For OC-2, the ethanol yield may be improved by adding formic acid to the bacterial growth medium and the ethanol fermentation medium, but no significant effect of improving the ethanol yield was observed as with Uz4288.

[0077] In the fermentation test shown in FIG. 19, Uz4288 was used as the test strain, a medium without formic acid and a medium with formic acid were used as the bacterial growth medium, and a medium without formic acid and a medium with a formic acid concentration of 1.0 g / L, 2.0 g / L, and 3.0 g / L were used as the ethanol fermentation medium. In the fermentation test shown in FIG. 19, n=3 was used for each condition. As shown in FIG. 19, when using an ethanol fermentation medium containing 1.0 to 3.0 g / L of formic acid, it was confirmed that the ethanol yield was improved compared to the condition in which formic acid was not added to the ethanol fermentation medium, regardless of whether or not formic acid was added to the bacterial growth medium.

[0078] In addition, although the addition of formic acid to the bacterial growth medium is not directly related to the consumption of formic acid in the ethanol fermentation medium in which ethanol fermentation proceeds, it has been found that there are conditions under which the addition of formic acid to the bacterial growth medium improves the ethanol yield. Although the reason why the addition of formic acid to the bacterial growth medium improves the ethanol yield is unknown, since formic acid can cause growth inhibition at high concentrations, it is possible that exposure to formic acid at low concentrations during yeast growth before ethanol fermentation improved formic acid resistance during ethanol fermentation and improved ethanol yield. Alternatively, exposure to formic acid during yeast growth before ethanol fermentation may cause an oxidized coenzyme-dependent reaction in the yeast using formic acid as a substrate by formate dehydrogenase to generate a reduced coenzyme, which may increase the reducing power in the yeast prior to ethanol fermentation, making it easier for a reduced coenzyme-dependent reaction using acetyl CoA as a substrate by acetaldehyde dehydrogenase to proceed.

[0079] The present disclosure is not limited to the above-mentioned embodiments, etc., and can be realized in various configurations without departing from the spirit of the present disclosure. For example, the technical features in the embodiments corresponding to the technical features in each form described in the Summary of the Invention column can be appropriately replaced or combined in order to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

[0080] The present disclosure can also be realized in the following forms. [Application example 1] A method for producing ethanol using ethanol fermentation by yeast, comprising: As a raw material for the ethanol fermentation, a raw material containing acetic acid in addition to sugar is used, As the yeast, a transformed yeast into which at least an acetaldehyde dehydrogenase gene has been introduced is used, When ethanol fermentation is performed by the yeast, formic acid is added to the culture solution in which the ethanol fermentation is progressing, and a reduced coenzyme-dependent reaction using acetyl-CoA as a substrate by acetaldehyde dehydrogenase obtained by expression of the acetaldehyde dehydrogenase gene introduced into the transformed yeast, an oxidized coenzyme-dependent reaction using formic acid as a substrate by formate dehydrogenase obtained by expression of at least one of a formate dehydrogenase gene inherent to the transformed yeast and a formate dehydrogenase gene introduced into the transformed yeast, and an oxidized coenzyme-dependent reaction using glycerol as a substrate by glycerol dehydrogenase obtained by expression of at least one of a glycerol dehydrogenase gene inherent to the transformed yeast and a glycerol dehydrogenase gene introduced into the transformed yeast. How ethanol is produced. [Application example 2] The method for producing ethanol according to Application Example 1, As the transformed yeast, a yeast obtained by transforming a yeast endogenously containing the formate dehydrogenase gene, in which the expression level of the formate dehydrogenase gene is increased, is used. How ethanol is produced. [Application example 3] The method for producing ethanol according to Application Example 1 or 2, further comprising: producing formic acid using carbon dioxide generated in association with ethanol fermentation by the yeast; The produced formic acid is added to a culture solution in which ethanol fermentation by the yeast progresses. How ethanol is produced. [Application example 4] The method for producing ethanol according to any one of Application Examples 1 to 3, further comprising: Formic acid is added to a culture medium in which the yeast for use in the ethanol fermentation is cultured prior to use in the ethanol fermentation. How ethanol is produced. [Application example 5] The method for producing ethanol according to any one of Application Examples 1 to 4, When ethanol fermentation is performed by the yeast, at least one of a glycerol transporter gene present in the transformed yeast and a glycerol transporter gene introduced into the transformed yeast is further expressed. How ethanol is produced. [Application Example 6] An ethanol production system using ethanol fermentation by yeast, a fermenter in which ethanol fermentation by yeast proceeds; A raw material supply unit that supplies a raw material containing acetic acid in addition to sugar as a raw material for the ethanol fermentation to the fermenter; A formic acid supply unit that supplies formic acid to the fermenter; Equipped with The yeast in the fermenter is a transformed yeast into which at least an acetaldehyde dehydrogenase gene has been introduced, and is a yeast that expresses formate dehydrogenase and glycerol dehydrogenase. Ethanol production system. [Application Example 7] The ethanol production system according to Application Example 6, The formic acid supply unit is a formic acid generating unit that generates formic acid using carbon dioxide; A carbon dioxide supply line that supplies carbon dioxide generated during the ethanol fermentation process in the fermenter to the formic acid production unit; A first formic acid inlet passage for introducing the formic acid produced in the formic acid production section into the fermenter; An ethanol production system comprising: [Application Example 8] The ethanol production system according to Application Example 6, further comprising: A bacterial culture tank for culturing yeast for use in the ethanol fermentation; A yeast supply line for supplying the yeast cultured in the bacteria culture tank to the fermentation tank; Equipped with The formic acid supply unit is a formic acid release section for releasing formic acid; A first formic acid inlet passage for introducing formic acid from the formic acid release section to the fermenter; A second formic acid inlet passage for introducing formic acid from the formic acid release section to the bacteria culture tank; An ethanol production system comprising: [Application Example 9] The ethanol production system according to Application Example 7, The formic acid generating unit is an artificial photosynthetic reactor Ethanol production system. [Explanation of symbols]

[0081] 11...Raw material supply section 12...Pretreatment device 14...Saccharification reactor 15...Bacteria culture tank 16...Fermentation tank 18…Distillation section 20,220…Formic acid supply section 22...Formic acid release section 24...First formic acid inlet 26...Second formic acid introduction channel 28...Carbon dioxide supply channel 30...Pretreatment liquid supply path 32...Saccharified liquid supply path 34...Fermentation liquid supply channel 38...Yeast supply path 110,210…Ethanol production system 222...Formic acid generating unit

Claims

1. A method for producing ethanol using ethanol fermentation by yeast, comprising: As a raw material for the ethanol fermentation, a raw material containing acetic acid in addition to sugar is used, As the yeast, a transformed yeast into which at least an acetaldehyde dehydrogenase gene has been introduced is used, When ethanol fermentation is performed by the yeast, formic acid is added to the culture solution in which the ethanol fermentation is progressing, and a reduced coenzyme-dependent reaction using acetyl CoA as a substrate by acetaldehyde dehydrogenase obtained by expression of the acetaldehyde dehydrogenase gene introduced into the transformed yeast, an oxidized coenzyme-dependent reaction using formic acid as a substrate by formate dehydrogenase obtained by expression of at least one of a formate dehydrogenase gene inherent to the transformed yeast and a formate dehydrogenase gene introduced into the transformed yeast, and an oxidized coenzyme-dependent reaction using glycerol as a substrate by glycerol dehydrogenase obtained by expression of at least one of a glycerol dehydrogenase gene inherent to the transformed yeast and a glycerol dehydrogenase gene introduced into the transformed yeast. How ethanol is produced.

2. The method for producing ethanol according to claim 1, As the transformed yeast, a yeast obtained by transforming a yeast endogenously containing the formate dehydrogenase gene, in which the expression level of the formate dehydrogenase gene is increased, is used. How ethanol is produced.

3. The method for producing ethanol according to claim 1, further comprising: producing formic acid using carbon dioxide generated in association with ethanol fermentation by the yeast; The produced formic acid is added to a culture solution in which ethanol fermentation by the yeast progresses. How ethanol is produced.

4. The method for producing ethanol according to claim 1, further comprising: Formic acid is added to a culture medium in which the yeast for use in the ethanol fermentation is cultured prior to use in the ethanol fermentation. How ethanol is produced.

5. The method for producing ethanol according to claim 1, When ethanol fermentation is performed by the yeast, at least one of a glycerol transporter gene present in the transformed yeast and a glycerol transporter gene introduced into the transformed yeast is further expressed. How ethanol is produced.

6. An ethanol production system using ethanol fermentation by yeast, a fermenter in which ethanol fermentation by yeast proceeds; A raw material supply unit that supplies a raw material containing acetic acid in addition to sugar as a raw material for the ethanol fermentation to the fermenter; A formic acid supply unit that supplies formic acid to the fermenter; Equipped with The yeast in the fermenter is a transformed yeast into which at least an acetaldehyde dehydrogenase gene has been introduced, and is a yeast that expresses formate dehydrogenase and glycerol dehydrogenase. Ethanol production system.

7. The ethanol production system according to claim 6, The formic acid supply unit is a formic acid generating unit that generates formic acid using carbon dioxide; A carbon dioxide supply line that supplies carbon dioxide generated during the ethanol fermentation process in the fermenter to the formic acid production unit; A first formic acid inlet passage for introducing the formic acid produced in the formic acid production section into the fermenter; An ethanol production system comprising:

8. The ethanol production system according to claim 6, further comprising: A bacterial culture tank for culturing yeast for use in the ethanol fermentation; A yeast supply line for supplying the yeast cultured in the bacteria culture tank to the fermentation tank; Equipped with The formic acid supply unit is a formic acid release section for releasing formic acid; A first formic acid inlet passage for introducing formic acid from the formic acid release section to the fermenter; A second formic acid inlet passage for introducing formic acid from the formic acid release section to the bacteria culture tank; An ethanol production system comprising:

9. The ethanol production system according to claim 7, The formic acid generating unit is an artificial photosynthetic reactor Ethanol production system.

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