Butanol-generating bacterium transformant of genus hydrogenophilus

Abstract

A transformant obtained by introducing the any of genes (1)-(5) to a bacterium belonging to the genus Hydrogenophilus can efficiently produce butanol by using carbon dioxide as the sole carbon source. (1) A gene that encodes an enzyme having butyraldehyde dehydrogenase activity, and a gene that encodes an enzyme having butanol dehydrogenase activity (2) a gene that encodes a multi-functional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity (3) a gene that encodes an enzyme having butyraldehyde dehydrogenase activity, and a gene that encodes a multi-functional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity (4) a gene that encodes an enzyme having butanol dehydrogenase activity, and a gene that encodes a multi-functional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity (5) a gene that encodes an enzyme having butyraldehyde dehydrogenase activity, a gene that encodes an enzyme having butanol dehydrogenase activity, and a gene that encodes a multi-functional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity.

Description

Butanol-producing Hydrogenophilus transformants

[0001] The present invention relates to a transformant of a bacterium belonging to the genus Hydrogenophilus having butanol-producing ability, and a method for producing butanol using the transformant.

[0002] The Paris Agreement, adopted in 2015, calls for rapid reductions in global greenhouse gas emissions. In accordance with this agreement, Japan aims to reduce its greenhouse gas emissions, including carbon dioxide and methane, by 46% by 2030 compared to 2013 levels.

[0003] Globally, the majority of chemical production relies on petroleum as a raw material, resulting in problems such as increasing greenhouse gas emissions. Therefore, there is a need to move away from petroleum-based chemical production, and research and development into biorefineries that produce green chemicals from biomass is being actively conducted in various countries. However, converting biomass into sugars, which can be used as a feedstock for microbial fermentation, requires complex processes and is therefore expensive. Furthermore, using biomass, which can be used as food or feed, in chemical production hinders the stable supply of food and feed. Furthermore, there is also the problem that consuming large amounts of biomass in chemical production actually leads to environmental destruction.

[0004] As part of research into moving away from petroleum, gases such as carbon dioxide, methane, and carbon monoxide are attracting attention as more sustainable carbon feedstocks, and there is growing interest in technologies that use microorganisms to utilize these gases to produce valuable chemicals and biofuels. In particular, there are high hopes for the fixation and effective use of carbon dioxide, which contributes greatly to global warming.

[0005] Butanol is an organic chemical raw material with a wide range of applications in industrial sectors, including the chemical, pharmaceutical, and petroleum industries. It is used in the production of acrylic and methacrylic acid esters, which are used in the production of coatings, plastics, textiles, and adhesives; glycol ethers, which are used in the production of coatings and electronic products; butyl acetate, which is used in the production of paints, inks, coatings, and synthetic fruit-like fragrances; and butylamine, which is used in the production of pesticides and pharmaceuticals, as well as amine resins. Butanol also has direct applications as a solvent in inks and dyes, extraction solvent, antifreeze fluid, cosmetic ingredient, and chromatography eluent component. In particular, butanol, which has four carbon atoms, is attracting attention as a next-generation biofuel following bioethanol because of its many advantages, including higher fuel efficiency than ethanol, which has two carbon atoms, ease of blending with gasoline and diesel, low corrosivity, and low water content.

[0006] Microbial butanol production methods include the acetone-butanol-ethanol fermentation (ABE fermentation) of Clostridium bacteria, such as Clostridium acetobutylicum. ABE fermentation, developed by Weizmann in 1916, is capable of producing butanol from sugars and starches. ABE fermentation declined in popularity due to the development of the petrochemical industry in the 1950s and 1960s. However, in recent years, growing concerns about greenhouse gas (GHG) emissions into the environment have led to a shift to alternative fuels and renewable energy sources. Research and development into the production of butanol as a biofuel using ABE fermentation has once again attracted attention.

[0007] However, this method produces by-products such as solvents like acetone and ethanol, as well as organic acids like butyric acid, acetic acid, and lactic acid. These by-products not only reduce the yield of butanol but also hinder the development of a process for recovering butanol. Furthermore, Clostridium bacteria require strictly anaerobic conditions for growth and butanol production. Cultivation under anaerobic conditions has several drawbacks, including the need for complex procedures, such as replacing the atmosphere in the culture apparatus with an inert gas like nitrogen gas, and the extremely slow growth rate of microorganisms under anaerobic conditions, resulting in a low rate of butanol production associated with the growth of Clostridium bacteria.

[0008] Therefore, techniques for producing butanol using aerobic or facultative anaerobic bacteria have been attempted. Clostridium bacteria have a metabolic pathway for producing butanol from glucose via pyruvate, acetyl-CoA, crotonyl-CoA, and butyryl-CoA, as shown in Figure 1. Therefore, by referring to this metabolic pathway, it is possible to construct a metabolic pathway from pyruvate to butyryl-CoA and then to butanol in bacteria that do not naturally produce butanol. According to Non-Patent Document 1, in aerobic and facultative anaerobic bacteria, the reaction from crotonyl-CoA to butyryl-CoA is involved in the reduction reaction of enoyl-CoA via ubiquitous fatty acid synthesis and fatty acid beta-oxidation pathways, and therefore it is theoretically possible to produce butyryl-CoA from glucose. However, aerobic and facultative anaerobic bacteria are unable to actually produce butanol from butyryl-CoA. For this reason, various methods have been proposed for producing butanol by culturing a transformant in which one or more foreign genes encoding enzymes that catalyze the metabolic pathway for producing butanol from butyryl-CoA are introduced into an aerobic bacterium or a facultative anaerobic bacterium host.

[0009] For example, Patent Document 1 discloses that butanol was produced using a transformant of Escherichia coli into which genes encoding enzymes that catalyze the metabolic pathway from acetyl-CoA to butanol (acetyl-CoA acetyltransferase, β-hydroxybutyl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, butyryl-CoA dehydrogenase, butyrylaldehyde dehydrogenase, and butanol dehydrogenase) were introduced.

[0010] However, the method described in Patent Document 1 is a method for producing butanol using sugars derived from biomass as a carbon raw material. As mentioned above, the use of biomass has drawbacks, such as high costs due to the complex process required to convert biomass into sugars, and the industrial use of biomass actually places a burden on the environment.

[0011] Patent No. 5243748

[0012] Advances in Biochemical Engineering / biotechnology 155:141-163

[0013] An object of the present invention is to provide a Hydrogenophilus bacterium transformant (transformed Hydrogenophilus bacterium) that can efficiently produce butanol using carbon dioxide as a sole carbon source, and a method for efficiently producing butanol using the transformant.

[0014] The present inventors conducted extensive research to solve the above problems and discovered the following: (i) Like other bacterial species, Hydrogenophilus bacteria are capable of producing butyryl-CoA from pyruvate, but do not inherently produce butanol because they lack a gene encoding an enzyme that catalyzes the butanol production reaction. Therefore, to impart butanol production ability, it is necessary to introduce a gene encoding an enzyme that catalyzes the butanol production reaction. (ii) By introducing into Hydrogenophilus bacteria a gene encoding an enzyme with butyraldehyde dehydrogenase activity that produces butyraldehyde from butyryl-CoA (hereinafter sometimes referred to as "butyraldehyde dehydrogenase") and a gene encoding an enzyme with butanol dehydrogenase activity that produces butanol from butyraldehyde (hereinafter sometimes referred to as "butanol dehydrogenase"), these genes are expressed in the Hydrogenophilus bacteria. This allows the Hydrogenophilus bacteria to produce butanol. Similar effects can be obtained by introducing, instead of the above two genes, a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity, which produces butyraldehyde from butyryl-CoA, and butanol dehydrogenase activity, which produces butanol from butyraldehyde (hereinafter sometimes referred to as "butyraldehyde-butanol dehydrogenase").

[0015] The present invention was completed based on the above findings and provides the following [1] to [6]: [1] A transformant (transformed bacterium) obtained by introducing any one of the following genes (1) to (5) into a bacterium belonging to the genus Hydrogenophilus: (1) A gene encoding an enzyme having butyraldehyde dehydrogenase activity, and a gene encoding an enzyme having butanol dehydrogenase activity. (2) A gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity. (3) A gene encoding an enzyme having butyraldehyde dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity. (4) A gene encoding an enzyme having butanol dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity. (5) A gene encoding an enzyme having butyraldehyde dehydrogenase activity, a gene encoding an enzyme having butanol dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity. [2] The transformant according to [1], wherein the gene encoding the multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity is a DNA selected from the group consisting of (a), (b), (c), (d), and (e):(a) DNA comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 9; (b) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 1, 3, 5, 7, or 9, and encoding a polypeptide having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10; (d) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 2, 4, 6, 8, or 10, and having butyraldehyde dehydrogenase activity; (e) DNA encoding a polypeptide having an amino acid sequence in which 1 to 100 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, and having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity [3] The transformant according to [1] or [2], wherein the gene encoding the enzyme having butyraldehyde dehydrogenase activity is a DNA selected from the group consisting of (f), (g), (h), (i), and (j): (f) DNA comprising the nucleotide sequence of SEQ ID NO: 11. (g) DNA comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 11 and encoding a polypeptide having butyraldehyde dehydrogenase activity. (h) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 12. (i) DNA encoding a polypeptide having 90% or more identity to SEQ ID NO: 12 and having butyraldehyde dehydrogenase activity. (j) DNA encoding a polypeptide having butyraldehyde dehydrogenase activity and comprising an amino acid sequence in which 1 to 100 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 12. [4] The transformant according to any of [1] to [3], wherein the DNA of the following (k), (l), (m), (n), or (o) is used as a gene encoding an enzyme having butanol dehydrogenase activity:(k) DNA comprising the nucleotide sequence of SEQ ID NO: 13, 15, 17, or 19. (l) DNA comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 13, 15, 17, or 19, and encoding a polypeptide having butanol dehydrogenase activity. (m) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, 16, 18, or 20. (n) DNA encoding a polypeptide having butanol dehydrogenase activity, and comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 14, 16, 18, or 20. (o) DNA encoding a polypeptide having butanol dehydrogenase activity, and comprising an amino acid sequence in which 1 to 100 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 14, 16, 18, or 20. [5] The transformant according to any of [1] to [4], wherein the bacterium of the genus Hydrogenophilus is Hydrogenophilus thermorteolus. [6] A method for producing butanol, comprising a step of culturing the transformant according to any one of [1] to [5] using carbon dioxide as substantially the only carbon source.

[0016] Measures to curb the increase in carbon dioxide emissions include reducing carbon dioxide emissions and fixing emitted carbon dioxide. To reduce carbon dioxide emissions, solar, wind, geothermal, and other energy sources are being used in place of fossil fuels. However, in reality, the use of these energy sources has not sufficiently curbed the increase in carbon dioxide. Therefore, it is necessary to promote the fixation or resource recovery of emitted carbon dioxide. Carbon dioxide can be fixed physically or chemically, but if it is fixed using living organisms, it can be used to produce organic matter that can be used as food, feed, fuel, and other products. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve both the two problems of global warming caused by increased carbon dioxide and the difficulty in securing food, feed, and fuel. Furthermore, it can produce in-demand chemical raw materials while curbing global warming caused by increased carbon dioxide.

[0017] Hydrogen bacteria, which can grow using carbon dioxide as their sole carbon source, can use the chemical energy generated by the reaction between hydrogen and oxygen to produce chemical products using a mixture of oxygen, hydrogen, and carbon dioxide as raw materials, making it possible to efficiently organicize carbon dioxide and to cultivate them in simple culture media. While hydrogen bacteria generally grow slowly, Hydrogenophilus bacteria have a significantly faster growth rate. The Mitsubishi Research Institute Bulletin No. 34 1999 evaluated Hydrogenophilus bacteria, saying, "Their growth rate is so high that it cannot be compared to the carbon dioxide fixation ability of plants, and clearly demonstrates the high carbon dioxide fixation ability of microorganisms."

[0018] Hydrogenophilus bacteria naturally possess an enzyme gene that catalyzes the reaction that converts pyruvate to butyryl-CoA, but do not possess an enzyme gene that catalyzes the reaction that converts butyryl-CoA to butanol. The present inventors have found that it is difficult to express functional proteins even when foreign genes are introduced into Hydrogenophilus bacteria. However, according to the present invention, by introducing a gene encoding an enzyme with butyraldehyde dehydrogenase activity and a gene encoding an enzyme with butanol dehydrogenase activity into Hydrogenophilus bacteria, these genes are expressed in the Hydrogenophilus bacteria, enabling the Hydrogenophilus bacteria to produce butanol using carbon dioxide as the sole carbon source. Similar effects can be achieved by introducing a gene encoding a multifunctional enzyme with butyraldehyde dehydrogenase activity and butanol dehydrogenase activity instead of the above two genes.

[0019] As described above, among organisms that have the ability to fix carbon dioxide, bacteria of the genus Hydrogenophilus have particularly excellent carbon dioxide fixation ability. Therefore, by using the transformant of the present invention, it is possible to fix carbon dioxide and produce butanol industrially.

[0020] 1 is a diagram showing a metabolic pathway for producing butanol from pyruvic acid, which is present in many microorganisms; 2 is a gas chromatography chromatogram showing that the crude enzyme produced by the transformant prepared in the examples produced butyraldehyde or butanol using butyryl-CoA as a substrate; and 3 is a gas chromatography chromatogram showing that the crude enzyme produced by the transformant prepared in the examples produced butanol using butyraldehyde as a substrate.

[0021] The present invention is described in detail below. (1) Transformant Ability to Produce Butanol The transformant of the present invention is a transformant obtained by introducing any one of the following genes (1) to (5) into a host bacterium belonging to the genus Hydrogenophilus. In other words, the transformant of the present invention has any one of the following exogenous genes (1) to (5): (1) A gene encoding an enzyme having butyraldehyde dehydrogenase activity, and a gene encoding an enzyme having butanol dehydrogenase activity. (2) A gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity. (3) A gene encoding an enzyme having butyraldehyde dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity. (4) A gene encoding an enzyme having butanol dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity. (5) A gene encoding an enzyme having butyraldehyde dehydrogenase activity, a gene encoding an enzyme having butanol dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity.

[0022] The butyraldehyde-butanol dehydrogenase gene, the butyraldehyde dehydrogenase gene, and the butanol dehydrogenase gene may each be DNA of a gene isolated from a naturally occurring bacterium, or may be DNA artificially synthesized using a method known to those skilled in the art.

[0023] One or more of the butyraldehyde-butanol dehydrogenase genes, butyraldehyde dehydrogenase genes, and butanol dehydrogenase genes can be introduced. The present invention also encompasses transformants into which other genes have been introduced in addition to the genes (1) to (5) above.

[0024] Butyraldehyde-butanol dehydrogenase gene The gene encoding butyraldehyde-butanol dehydrogenase does not need to be known to have been identified as a butyraldehyde-butanol dehydrogenase gene, but it may be DNA encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity to produce butyraldehyde from butyryl-CoA and butanol dehydrogenase activity to produce butanol from butyraldehyde. This multifunctional enzyme may be a bifunctional enzyme having only butyraldehyde dehydrogenase activity and butanol dehydrogenase activity.

[0025] Examples of butyraldehyde-butanol dehydrogenase genes that can be used in the present invention include those shown in Table 1. Table 1 shows the bacterial species from which the enzyme is derived, the nucleotide sequence of the gene encoding the enzyme, and the amino acid sequence of the enzyme. Each of the butyraldehyde-butanol dehydrogenases shown in Table 1 is a bifunctional enzyme that has both butyraldehyde dehydrogenase activity and butanol dehydrogenase activity.

[0026] In the present invention, DNA containing the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 9 (particularly, DNA consisting of SEQ ID NO: 1, 3, 5, 7, or 9) can be used. Also usable is DNA containing a nucleotide sequence that has 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to SEQ ID NO: 1, 3, 5, 7, or 9 (particularly, consisting of a nucleotide sequence that has 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to SEQ ID NO: 1, 3, 5, 7, or 9) and that encodes a polypeptide having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity.

[0027] DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 (particularly, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10) can also be used. DNA encoding a polypeptide comprising an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 2, 4, 6, 8, or 10 (particularly, consisting of an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 2, 4, 6, 8, or 10) and having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity can also be used. Furthermore, DNAs that include an amino acid sequence in which 1 to 100, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 (particularly, an amino acid sequence in which 1 to 100, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10), and that encode a polypeptide that has butyraldehyde dehydrogenase activity and butanol dehydrogenase activity, can also be used.

[0028] As shown in the Examples section, it has been confirmed that the bifunctional enzyme having the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 functions in Hydrogenophilus bacteria, as the crude enzyme produced in the bacteria produced butanol using butyryl-CoA as a substrate in the presence of NADH.

[0029] In the present invention, whether a polypeptide has butyraldehyde dehydrogenase activity using butyryl-CoA as a substrate and butanol dehydrogenase activity using butyraldehyde as a substrate is confirmed by reacting the test polypeptide with butyryl-CoA in the presence of NADH and detecting the produced butanol by gas chromatography or the like.

[0030] Butyraldehyde dehydrogenase gene The gene encoding an enzyme having butyraldehyde dehydrogenase activity does not need to be known to have been identified as a butyraldehyde dehydrogenase gene, as long as it is DNA encoding a polypeptide having butyraldehyde dehydrogenase activity.

[0031] An example of a butyraldehyde dehydrogenase gene that can be used in the present invention is the butyraldehyde dehydrogenase gene of Thermus thermophilus consisting of the nucleotide sequence of SEQ ID NO: 11. The butyraldehyde dehydrogenase of Thermus thermophilus is not a multifunctional enzyme that also includes an alcohol dehydrogenase. In the present invention, DNA containing the nucleotide sequence of SEQ ID NO: 11 (particularly, consisting of the nucleotide sequence of SEQ ID NO: 1) can be used. Also usable is DNA containing a nucleotide sequence that is 90% or more, particularly 95% or more, particularly 98% or more, or particularly 99% or more identical to SEQ ID NO: 11 (particularly, a nucleotide sequence that is 90% or more, particularly 95% or more, particularly 98% or more, or particularly 99% or more identical to SEQ ID NO: 11) and that encodes a polypeptide having butyraldehyde dehydrogenase activity.

[0032] Further, examples of aldehyde dehydrogenase genes include DNAs encoding butyraldehyde dehydrogenase from Thermus thermophilus consisting of the amino acid sequence of SEQ ID NO: 12. In the present invention, DNAs encoding polypeptides comprising the amino acid sequence of SEQ ID NO: 12 (particularly consisting of the amino acid sequence of SEQ ID NO: 12) can be used. Also usable are DNAs encoding polypeptides comprising an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 12 (particularly consisting of an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 12) and having butyraldehyde dehydrogenase activity. Furthermore, DNAs that encode a polypeptide having butyraldehyde dehydrogenase activity and that comprise an amino acid sequence in which 1 to 100, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 12 (particularly, an amino acid sequence in which 1 to 100, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 12) can also be used.

[0033] In the present invention, the butyraldehyde dehydrogenase gene can also be a gene encoding an aldehyde-alcohol dehydrogenase that can produce butyraldehyde from butyryl-CoA but cannot produce butanol from butyraldehyde. This enzyme is generally called an aldehyde-alcohol dehydrogenase, but in the present invention it is classified as butyraldehyde dehydrogenase.

[0034] In the present invention, whether a polypeptide has butyraldehyde dehydrogenase activity using butyryl-CoA as a substrate is confirmed by reacting the test polypeptide with butyryl-CoA in the presence of NADH and detecting the produced butyraldehyde by gas chromatography or the like.

[0035] Butanol dehydrogenase gene The gene encoding an enzyme having butanol dehydrogenase activity does not need to be known to have been identified as a butanol dehydrogenase gene, but it may be DNA encoding a polypeptide having butanol dehydrogenase activity that produces butanol from butyraldehyde.

[0036] Butanol dehydrogenase genes that can be used in the present invention include those shown in Table 2. Table 2 shows the bacterial species from which the enzyme is derived, the nucleotide sequence of the gene encoding the enzyme, and the amino acid sequence of the enzyme. mobilis, butanol dehydrogenases having the amino acid sequences of SEQ ID NOs: 14 and 16, respectively, are not multifunctional enzymes having an aldehyde dehydrogenase. Butanol dehydrogenases having the amino acid sequences of SEQ ID NOs: 18 and 20, respectively, from Thermoanaerobacter thermocopriae and Geobacillus stearothermophilus belong to the aldehyde-alcohol dehydrogenase family, but the aldehyde dehydrogenases they possess cannot produce butyryl-CoA from butyryl-CoA.

[0037] In the present invention, DNAs that contain a nucleotide sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identical to SEQ ID NO: 13, 15, 17, or 19 (particularly, DNAs that consist of a nucleotide sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identical to SEQ ID NO: 13, 15, 17, or 19) and that encode a polypeptide that has butanol dehydrogenase activity can also be used.

[0038] In addition, DNA that contains an amino acid sequence that has 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the amino acid sequence of SEQ ID NO: 14, 16, 18, or 20 (particularly, consisting of an amino acid sequence that has 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the amino acid sequence of SEQ ID NO: 14, 16, 18, or 20) and encodes a polypeptide that has butanol dehydrogenase activity can also be used.

[0039] Furthermore, DNAs that contain an amino acid sequence in which 1 to 100, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 14, 16, 18, or 20 (particularly, DNAs that consist of an amino acid sequence in which 1 to 100, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 14, 16, 18, or 20) and that encode a polypeptide that has butanol dehydrogenase activity can also be used.

[0040] As described above, in the present invention, the gene for an enzyme having butanol dehydrogenase activity can also be a gene for an aldehyde-alcohol dehydrogenase that can produce butanol from butyraldehyde but cannot produce butyraldehyde from butyryl-CoA. This enzyme is generally called an aldehyde-alcohol dehydrogenase, but in the present invention it is classified as a butanol dehydrogenase.

[0041] In the present invention, whether a polypeptide has butanol dehydrogenase activity using butyraldehyde as a substrate is confirmed by reacting the test polypeptide with butyraldehyde in the presence of NADH and detecting the produced butanol using a gas chromatograph or the like.

[0042] A DNA sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 may have various base substitutions in the coding region, taking into account codon degeneracy or preferred codons in Hydrogenophilus bacteria, as long as the substitutions do not change the amino acid sequence of the protein expressed from the coding region.

[0043] In the present invention, the identities of base sequences and amino acid sequences are values ​​calculated using GENETYX ver. 17 (GENETYX).

[0044] In the present invention, a DNA that contains a nucleotide sequence that is 90% to less than 100% identical to the nucleotide sequence of a given DNA and encodes a polypeptide that has the same type of activity as the polypeptide encoded by that DNA is referred to as a homolog of that DNA. Also, a DNA that contains an amino acid sequence that is 90% to less than 100% identical to the amino acid sequence of a polypeptide encoded by that DNA and encodes a polypeptide that has the same type of activity as the polypeptide encoded by that DNA is referred to as a homolog of that DNA. Also, a DNA that contains an amino acid sequence in which 1 to 100 amino acids are deleted, substituted, inserted, or added to the amino acid sequence of a polypeptide encoded by that DNA and encodes a polypeptide that has the same type of activity as the polypeptide encoded by that DNA is referred to as a homolog of that DNA. In the present invention, a polypeptide that contains an amino acid sequence that is 90% to less than 100% identical to the amino acid sequence of a given polypeptide and has the same type of activity as the polypeptide is referred to as a homolog of that polypeptide. Also, a polypeptide that contains an amino acid sequence in which 1 to 100 amino acids are deleted, substituted, inserted, or added to the amino acid sequence of a given polypeptide and has the same type of activity as the polypeptide is referred to as a homolog of that polypeptide.

[0045] Examples of Hydrogenophilus bacteria include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidance, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038. Among these, Hydrogenophilus thermoluteolus is preferred because it has the highest growth rate and carbon dioxide fixation ability of any carbon dioxide-fixing microorganism. Hydrogenophilus bacteria can be easily isolated from all over the world. A preferred strain of Hydrogenophilus thermorteolus is the TH-1 (NBRC 14978) strain. Hydrogenophilus thermorteolus TH-1 (NBRC 14978) exhibits the highest growth rate of any carbon-fixing microorganism (Agricultural and Biological Chemistry, 41, 685-690 (1977)) (doubling in one hour). The Hydrogenophilus thermorteolus NBRC 14978 strain has been internationally deposited under the Budapest Treaty and is publicly available.

[0046] The host Hydrogenophilus bacterium may be a bacterium isolated from nature, or may be a bacterium that has been genetically modified from a bacterium isolated from nature. Modification can be performed for purposes such as enabling high expression of an introduced gene. Such modification can be achieved by, for example, removing (curing) the endogenous plasmid in the Hydrogenophilus bacterium. Methods for removing endogenous plasmids are well known, including methods that utilize plasmid incompatibility, such as the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide; destabilizing the endogenous plasmid by introducing a plasmid with the same replication origin as the endogenous plasmid; or destabilizing the endogenous plasmid by disrupting factors involved in the plasmid partition system.

[0047] Method for Preparing Transformants A method for obtaining transformants by introducing the above genes (1) to (5) into bacteria of the genus Hydrogenophilus is described below. These genes can be introduced into bacteria of the genus Hydrogenophilus using standard methods for introducing foreign genes into bacteria. These genes may be introduced directly into bacteria of the genus Hydrogenophilus, or they may be incorporated into a transformation vector (e.g., a plasmid vector, a viral vector, a cosmid, a fosmid, a BAC, etc.) and then introduced into bacteria of the genus Hydrogenophilus. The vector for transformation may contain DNA capable of autonomous replication in Hydrogenophilus bacteria. Examples include broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI Reference Sequence: NC_004444.1), pC194 (NCBI Reference Sequence: NC_002013.1), pK18mobsacB (GenBank: FJ437239.1), and pUB110 (NCBI Reference Sequence: NC_001384.1), as well as genetically modified versions of these vectors (e.g., pCAMO-6). Among these, pCAMO-6 is preferred. pCAMO-6 can be prepared by those skilled in the art according to the Examples. Examples of promoters contained in the vector include the tac promoter, lac promoter, trc promoter, and the OXB1, OXB11 to OXB20 promoters from Oxford Genetics, and examples of terminators contained in the vector include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the bacteriophage λt0 transcription terminator, the T7 terminator, etc. These genes can be introduced (transformed) into Hydrogenophilus bacteria using conventional methods, such as the calcium chloride method, the rubidium chloride method, and the electric pulse method (electroporation).

[0048] (2) Method for Producing Butanol The present invention provides a method for producing butanol using the transformant of the present invention described above. This method includes a step of culturing the transformant of the present invention in an inorganic or organic medium while supplying a carbon dioxide-containing gas, preferably a mixed gas containing hydrogen, oxygen, and carbon dioxide. The gas supplied is preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide, but other gases may be mixed in as long as butanol can be efficiently produced.

[0049] Hydrogenophilus bacteria can grow using hydrogen as an energy source and carbon dioxide as the sole carbon source, and therefore can efficiently fix carbon dioxide by producing butanol using substantially only carbon dioxide as a carbon source (especially using only carbon dioxide). Therefore, in the method of the present invention, it is preferable to use an inorganic medium that does not contain carbon sources such as organic matter or carbonates, i.e., to culture using substantially only carbon dioxide as a carbon source (especially using carbon dioxide as the only carbon source). In the present invention, "using carbon dioxide as substantially the sole carbon source" includes cases where unavoidable amounts of other carbon sources are mixed in.

[0050] The pH of the culture medium used for culture is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, bacterial growth and the solubility of the mixed gas in the culture medium are high, enabling highly efficient production of butanol. When performing batch culture, the mixed gas can be sealed in a sealed culture vessel and cultured by static or shaking. Shaking culture is preferred because it improves the solubility of the mixed gas in the culture medium. When performing continuous culture, the mixed gas can be continuously supplied to the culture vessel while being cultured by shaking, or the mixed gas can be introduced into the culture medium by bubbling, and the transformant can be cultured with stirring. The volume ratio of hydrogen, oxygen, and carbon dioxide (hydrogen:oxygen:carbon dioxide) in the supply gas is preferably 1.75 to 7.5:1:0.25 to 3, more preferably 5 to 7.5:1:1 to 2, and even more preferably 6.25 to 7.5:1:1.5. Within this range, bacterial growth is favorable and butanol can be produced efficiently. The supply rate of the mixed gas or raw material gas is 10 to 60 L / hour, preferably 10 to 40 L / hour, and more preferably 10 to 20 L / hour per liter of medium. Within this range, the growth of the transformant is favorable, butanol can be produced efficiently, and waste of the mixed gas is reduced. The culture temperature is preferably 35 to 55°C, more preferably 37 to 52°C, and even more preferably 50 to 52°C. Within this range, the growth of the transformant is favorable, and butanol can be produced efficiently.

[0051] By culturing as described above, butanol is produced in the culture solution. The butanol can be recovered by recovering the culture solution, but it can also be separated from the reaction solution by known methods. Such known methods include membrane separation and distillation.

[0052] The present invention will now be described with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0053] (1) Construction of the Plasmid Vector (pCAMO-6) The construction method of the plasmid vector pCAMO-6 used to introduce the aldehyde dehydrogenase gene and alcohol dehydrogenase gene is described below. (1-1) Preparation of the tac promoter DNA fragment. To amplify the tac promoter DNA fragment, PCR was performed using the following pair of primers and the plasmid pMAL-c5X (New England BioLabs) as a template. Unless otherwise noted below, PCR was performed according to standard procedures using a Thermo Fisher Scientific 2720 Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as a reaction reagent. Primers for amplifying the tac promoter: (a-1) 5'-TTTTATAACCCGGGCCATCGACTGCACGGTGCACC-3' (SEQ ID NO: 21) (b-1) 5'-TGCTAGCACTGTTTCCTGTGTGAAATTGTTATCCG-3' (SEQ ID NO: 22). Primer (a-1) contained a SmaI restriction enzyme site, as underlined. The reaction mixture prepared above was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.3 kbp corresponding to the tac promoter was detected. This fragment was excised from the agarose gel, and the gel was frozen and thawed to recover the DNA.

[0054] (1-2) Preparation of DNA Fragment of Multicloning Region To prepare the DNA fragment of the multicloning region, PCR was performed using the following pairs of primers to generate the first and second halves. This method does not use template DNA; the 3' ends of each pair of primers anneal and extend to form double-stranded DNA. Primers for preparing the first half of the multicloning region: (a-2) 5'-GGAAACAGTGCTAGCAGATCTGGAGGAGAAACGCATAT-3' (SEQ ID NO: 23) (b-2) 5'-CAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGATATCAGCATATGCGTTTCTCCTCCAGA-3' (SEQ ID NO: 24) The sequences of the 20 bases on the 3' side of primers (a-2) and (b-2) are complementary to each other. Primers for preparing the second half of the multicloning region: (a-3) 5'-ACAAGCTTGCGGCCGCACTGCAGCACCATCACCACCATCATTGATAAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCT-3' (SEQ ID NO: 25) (b-3) 5'-TATTTGAATCGAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGT-3' (SEQ ID NO: 26). The 3'-20 base pairs of primers (a-3) and (b-3) are complementary to each other. The reaction mixture prepared above was subjected to agarose gel electrophoresis, and approximately 0.1 kbp DNA fragments corresponding to the first and second halves of the multicloning region were detected. Each DNA fragment was excised from the agarose gel, and the DNA was recovered from the gel by freezing and thawing.

[0055] Overlap extension PCR was performed using the recovered DNA fragments corresponding to the first and second halves of the multicloning region as templates. The 5' 20 base sequences of primers (b-2) and (a-3) used to amplify this template DNA fragment are complementary to each other. For overlap extension PCR, a combination of primers (a-2) and (b-3) was used to generate DNA for the multicloning region. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.2 kbp corresponding to the multicloning region was detected and recovered from the gel.

[0056] (1-3) Preparation of a DNA fragment containing the rrnB terminator and the replication origin of pUC19 Using the plasmid pMAL-c5X (New England BioLabs) as a template, PCR was performed with the following pair of primers to amplify a DNA fragment of the rrnB terminator. Primers for rrnB terminator amplification: (a-4) 5'-TAACTCGATTCAAATAAAACGAAAGGCTCAGTCGA-3' (SEQ ID NO: 27) (b-4) 5'-CCTAGATCCGCGGAGTTTGTAGAAACGCAAAAAGG-3' (SEQ ID NO: 28) Furthermore, PCR was performed with the following pair of primers to amplify a DNA fragment of the DNA replication origin using the plasmid pUC19 as a template. Primers for amplifying the DNA replication origin of pUC19: (a-5) 5'-ACAAACTCCGCGGATCTAGGTGAAGATCCTTTTTG-3' (SEQ ID NO: 29) (b-5) 5'-CGTCCGCGGCCAGCAAAAGGCCAGGAACCGTAAAA-3' (SEQ ID NO: 30). The resulting reaction solution was subjected to agarose gel electrophoresis, detecting a DNA fragment of approximately 0.3 kbp for the rrnB terminator and an approximately 0.8 kbp for the DNA replication origin of pUC19. Each DNA fragment was excised from the agarose gel, and the gel was frozen and thawed to recover the DNA.

[0057] Overlap extension PCR was performed using the recovered rrnB terminator and a DNA fragment corresponding to the pUC19 DNA replication origin as templates. The 5' 20 base pairs of primers (b-4) and (a-5) used to amplify these template DNA fragments are complementary to each other. For overlap extension PCR, a combination of primers (a-4) and (b-5) was used to generate a DNA fragment linking the rrnB terminator and the pUC19 replication origin. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 1.0 kbp corresponding to the DNA linking the rrnB terminator and the pUC19 replication origin was detected and recovered from the gel.

[0058] (1-4) Preparation of a DNA fragment of the neomycin / kanamycin resistance gene Using the plasmid pK18mobsacB (GenBank: FJ437239.1) [Gene, 145, 69-73 (1994)] containing the neomycin / kanamycin resistance gene (hereinafter sometimes referred to as "nptII") sequence as a template, PCR was performed with the following pair of primers to amplify a DNA fragment containing the nptII gene sequence: Primers for amplifying the nptII gene: (a-6) 5'-TTGCTGGCCGCGGACGTAGAAAGCCTGTCCGCAGA-3' (SEQ ID NO: 31) (b-6) 5'-GGCCCGGGTTATAAAAGCCAGTCATTAGGCCTATC-3' (SEQ ID NO: 32) Primer (b-6) contained an SmaI restriction enzyme site, as underlined. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 1.0 kbp corresponding to the nptII gene was detected and recovered from the gel.

[0059] (1-5) Construction of Circular Plasmids: The DNA fragments prepared in steps (1-1) to (1-4) above share 16-bp homologous sequences with the ends of DNA fragments (1-4) and (1-2), the end of DNA fragment (1-2) shares 16-bp homologous sequences with the ends of DNA fragments (1-1) and (1-3), the end of DNA fragment (1-3) shares 16-bp homologous sequences with the ends of DNA fragments (1-2) and (1-4), and the end of DNA fragment (1-4) shares 16-bp homologous sequences with the ends of DNA fragments (1-3) and (1-1). Therefore, the DNA fragments (1-1), (1-2), (1-3), and (1-4) can be ligated into a circular DNA by Gibson Assembly (Gibson et al., Nature Methods 6(5):343-345), a DNA ligation method that does not use restriction enzymes or ligases. To ligate the DNA fragments prepared in (1-1) to (1-4) into circular DNA, Gibson assembly was performed using New England Biolabs' Gibson Assembly Master Mix. The resulting reaction mixture was used to transform Escherichia coli JM109 using the calcium chloride method and plated on LB solid medium containing 50 μg / mL kanamycin. The strain grown on the medium was cultured in liquid culture using standard methods, and plasmid DNA was extracted from the culture medium and reacted with the restriction enzyme SmaI. Agarose gel electrophoresis of this reaction mixture revealed a DNA fragment of approximately 2.3 kbp, corresponding to the size of the single DNA fragment prepared in (1-1) to (1-4), confirming their ligation. This circular plasmid DNA, which replicates in E. coli, was designated pCAMO-1.

[0060] (1-6) Construction of Plasmid Vector (pCAMO-4) The Hydrogenophilus thermorteolus TH-1 strain was cultured in liquid medium A [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, and CoCl2 6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0). The bacteria were inoculated into a test tube containing 5 mL of PBS using a platinum loop, and the tube was filled with a gas mixture of H2:O2:CO2 = 7.5:1:1.5 and cultured at 50°C. The endogenous plasmid pTH1 [Microbiol Resour Announcement. 2018 Aug 16;7(6)] was extracted from the culture medium using the standard alkaline SDS method. The approximately 66 kbp pTH-1 plasmid was digested with the restriction enzymes ScaI and PvuII, and the plasmid pCAMO-1 was digested with the restriction enzyme SmaI and ligated to each other using T4 DNA ligase (Takara Bio Inc.).

[0061] The resulting ligation solution was used to transform the Hydrogenophilus thermorteolus TH-1 strain (NBRC 14978) by electroporation. The transformants were grown on solid medium A containing 50 μg / mL kanamycin [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, CoCl2 6H2O 4.0 mg, and 15 g of agar dissolved in 1 L of distilled water (pH 7.0). 7.0) and cultured for 60 hours at 50°C in a chamber filled with a gas mixture of H2:O2:CO2 = 7.5:1:1.5. Seven strains grown on solid medium A were inoculated using a platinum loop into test tubes containing 5 mL of liquid medium A containing 50 μg / mL kanamycin. The test tubes were filled with a gas mixture of H2:O2:CO2 = 7.5:1:1.5 and cultured with shaking at 52°C. Plasmid DNA was extracted from the culture medium and subjected to agarose gel electrophoresis. The results confirmed that all seven strains contained the same plasmid DNA of approximately 5.5 kbp.

[0062] Using the extracted plasmid as a template, PCR was performed using the following pair of primers, one on each side of the SmaI restriction site of pCAMO-1, to amplify the DNA fragment of the endogenous plasmid pTH1 inserted into the SmaI restriction site of pCAMO-1. Primers for amplifying the inserted pTH1 DNA fragment: (a-7) 5'-AATTGTCAGATAGGCCTAATGACTGGCTTTTATAA-3' (SEQ ID NO: 33) (b-7) 5'-TGACGCCAGAAGCATTGGTGCACCGTGCAGTCGAT-3' (SEQ ID NO: 34). The resulting reaction solution was subjected to agarose gel electrophoresis, detecting a DNA fragment of approximately 3.2 kbp. In other words, the resulting plasmid contained a portion of pTH1, a DNA fragment of approximately 3.2 kbp, inserted into the SmaI restriction site of the pCAMO-1 plasmid. This approximately 3.2 kbp DNA fragment contains a replication origin that functions in Hydrogenophilus thermorteolus cells, so the resulting plasmid replicates in Hydrogenophilus thermorteolus cells. The constructed plasmid was named pCAMO-4.

[0063] (1-7) Construction of Plasmid Vector (pCAMO-5) The following pair of oligonucleotides were mixed in equimolar amounts and slowly cooled from 98°C to 20°C to prepare a double-stranded DNA fragment to which the oligonucleotides were annealed. The ends of this DNA fragment were equivalent to the protruding ends obtained by cleavage with the restriction enzymes BglII and NdeI. This DNA fragment and a fragment obtained by cleaving pCAMO-4 with the restriction enzymes BglII and NdeI were ligated together using T4 DNA ligase. (a-8) 5'-GATCTGGAGGAGAAACGCA-3' (SEQ ID NO: 35) (b-8) 5'-TATGCGTTTCTCCTCCA-3' (SEQ ID NO: 36) The constructed plasmid was named pCAMO-5.

[0064] (1-8) Construction of Plasmid Vector (pCAMO-6) Using the endogenous plasmid pTH-1 as a template, PCR was performed with the following pair of primers to amplify a DNA fragment of the mazF gene encoding a plasmid stabilization factor. Primers for amplifying the mazF gene: (a-9) 5'-GAGGCCATCTAGGCCATGAGTAAGTCTGACGGAAC-3' (SEQ ID NO: 37) (b-9) 5'-ATCCGGCACCCATATCTGAACCGGACGCAAACCCG-3' (SEQ ID NO: 38)

[0065] PCR was performed using the endogenous plasmid pTH-1 as a template and the following pair of primers to amplify a DNA fragment of the mazE gene encoding a plasmid stabilization factor: Primers for amplifying the mazE gene: (a-10) 5'-TTCAGATATGGGTGCCGGATACCCGCCGCCCGGGC-3' (SEQ ID NO: 39) (b-10) 5'-AAGGCCTTCATGGCCTTATTTCGCGATTCCCAAGA-3' (SEQ ID NO: 40).

[0066] The 3' end of the mazF DNA fragment shares a 20-bp homologous sequence with the 5' end of the mazE DNA fragment. Therefore, the mazF and mazE DNA fragments can be ligated by overlap extension PCR. The mazF and mazE DNA fragments prepared above were mixed, and overlap extension PCR was performed using primers (a-9) and (b-10) as a template to amplify the ligated DNA fragment. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.7 kbp corresponding to the ligated DNA of the mazF and mazE DNA fragments was detected and recovered from the gel.

[0067] The prepared DNA fragment (approximately 0.7 kbp) and the plasmid pCAMO-5 were digested with the restriction enzyme SfiI and ligated together using T4 DNA ligase. The constructed plasmid was designated pCAMO-6.

[0068] (2) Construction of gene expression plasmids Gene expression plasmids were constructed using Gibson Assembly. DNA fragments of the following bacterial genes were amplified by PCR. Butyraldehyde-butanol dehydrogenase gene: DNA fragment of SEQ ID NO: 1 from Thermoanaerobacter masrani DNA fragment of SEQ ID NO: 3 from Fonticella tunisiensis DNA fragment of SEQ ID NO: 5 from Thermoclostridium caenicola DNA fragment of SEQ ID NO: 7 from Thermoanaerobacter ethanolicus DNA fragment of SEQ ID NO: 9 from Bacillus coagulans Butyraldehyde dehydrogenase gene: DNA fragment of SEQ ID NO: 11 from Thermus thermophilus Butanol dehydrogenase gene: DNA fragment of SEQ ID NO: 13 from Bacillus coagulans DNA fragment of SEQ ID NO: 15 from Zymomonas mobilis subsp. mobilis DNA fragment of SEQ ID NO: 17 from Thermoanaerobacter thermocopriae DNA fragment of SEQ ID NO: 19 from Geobacillus stearothermophilus

[0069] The following primers were used for PCR, which contain sequences homologous to the vector pCAMO-6. Primers for amplifying the butyraldehyde-butanol dehydrogenase gene (SEQ ID NO: 1) of Thermoanaerobacter masrani: (a-11) 5'-ATGAAAGTAACAAATGCATGCCTACTTTATTACAAG-3' (SEQ ID NO: 41) (b-11) 5'-GTCGACGGAGCTCGAATTCTTATTCTCCATAGGCTTTTC-3' (SEQ ID NO: 42) Primers for amplifying the butyraldehyde-butanol dehydrogenase gene (SEQ ID NO: 3) of Fonticella tunisiensis: (a-12) 5'-ATCTGGAGGAGAAACGCATATGAAAGTAACAAATGCAGAAGAAC-3' (SEQ ID NO: 43) (b-12) 5'-GTCGACGGAGCTCGAATTCTTATTTATTATTTTCTTCAGTTTCC-3' (SEQ ID NO: 44) Thermoclostridium Primers for amplifying the butyraldehyde-butanol dehydrogenase gene of Thermoanaerobacter ethanolicus (SEQ ID NO: 7): (a-14) 5'-ATGAAAGTAACAAATGCATGCCTAACTTATTACAAGAAAGAAG-3' (SEQ ID NO: 47) (b-14) 5'-GTCGACGGAGCTCGAATTCTTATTCTCCATAGGCTTTTC-3' (SEQ ID NO: 48) Primers for amplifying the butyraldehyde-butanol dehydrogenase gene of Bacillus coagulans (SEQ ID NO: 9): (a-15) 5'-ATCTGGAGGAGAAACGCATATGGCAATCGATGAAAAAGTTGTTGG-3' (SEQ ID NO: 49) (b-15) 5'-GTCGACGGAGCTCGAATTCTTATTTTGCTTCAACGCCTTC-3' (SEQ ID NO: 50) ThermusPrimers for amplifying the butanol dehydrogenase gene of Bacillus coagulans (SEQ ID NO: 13): (a-17) 5'-ATCTGGAGGAGAAACGCATATGAGAGCAGCAGTCGTCAGTTCTG-3' (SEQ ID NO: 53) (b-17) 5'-GTCGACGGAGCTCGAATTCTTATTTCAACGACATATCCAGGACC-3' (SEQ ID NO: 54) Primers for amplifying the butanol dehydrogenase gene of Zymomonas mobilis subsp. mobilis (SEQ ID NO: 15) (a-18) 5'-GATCTGGAGGAGAAACGCATATGAAAGCAGCCGTCATAAC-3' (SEQ ID NO: 55) (b-18) 5'-TGTCGACGGAGCTCGAATTCCTAGTGATGGGTAAAATCAA-3' (SEQ ID NO: 56) Primer for amplifying the butanol dehydrogenase gene (SEQ ID NO: 17) of Thermoanaerobacter thermocopriae (a-19) 5'-ATGAAAGTAACAAATGCATGCCTACCTTATTACAAGAAAAAAAGG -3' (SEQ ID NO: 57) (b-19) 5'-GTCGACGGAGCTCGAATTCCTATATCAGAATTTCCATTATTTG -3' (SEQ ID NO: 58) Primer for amplifying the butanol dehydrogenase gene (SEQ ID NO: 19) of Geobacillus stearothermophilus (a-20) 5'-ATCTGGAGGAGAAACGCATATGGCTGTTGATGAAAAAGTATTAGACC-3' (SEQ ID NO: 59) (b-20) 5'-GTCGACGGAGCTCGAATTCTCATTCAACCCCTTTATACGCTTGGCG-3' (SEQ ID NO: 60)The resulting PCR reaction mixture was subjected to agarose gel electrophoresis, and DNA fragments of approximately 1.0 kbp were detected for the butyraldehyde dehydrogenase gene, approximately 1.1 kbp for the butanol dehydrogenase gene, and approximately 2.7 kbp for the butyraldehyde-butanol dehydrogenase gene. The DNA fragments corresponding to these genes were excised from the gel and recovered.

[0070] To perform DNA ligation using Gibson Assembly, the plasmid vector pCAMO-6 was amplified by PCR. The following primers were used for PCR: Primers for amplification of plasmid vector pCAMO-6: (a-21) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 61) (b-21) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 62) The resulting PCR reaction solution was subjected to agarose electrophoresis, and a DNA fragment of approximately 5.2 kbp corresponding to the vector gene was detected. This was excised from the gel and recovered.

[0071] The DNA fragments of the pCAMO-6 vector synthesized above and the DNA fragments of each dehydrogenase gene were ligated using Gibson Assembly Master Mix (New England Biolabs). The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the resulting mixture was plated on LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C with shaking. Plasmid DNA was extracted from the culture medium. The sequence of the gene inserted into each plasmid was analyzed by Sanger analysis at Eurofins Genomics, and confirmed to match the sequence in the database.

[0072] (3) Hydrogenophilus thermorteolus Transformants (3-1) Gene Introduction The resulting plasmids were transformed into the Hydrogenophilus thermorteolus TH-1 strain by electroporation, and the transformed strains were plated onto LB solid medium containing 50 μg / mL kanamycin and cultured at 52°C for 24 hours. Each strain grown on LB solid medium was inoculated into LB solid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 52°C for 24 hours. Amplification of the insert fragments of each plasmid was confirmed by PCR for each strain grown on LB solid medium. As a result, amplification of DNA fragments of the length corresponding to each gene was confirmed. Plasmids containing genes derived from each bacterium and the Hydrogenophilus thermorteolus TH-1 transformants transformed with these plasmids were named as shown in Table 3. As mentioned above, the genes of SEQ ID NOs: 17 and 19 are known as aldehyde-alcohol dehydrogenase genes, but because they are genes for enzymes that do not produce butyraldehyde from butyryl-CoA, they are referred to as butanol dehydrogenase genes in the present invention.

[0073] (3-2) Measurement of butyraldehyde dehydrogenase and butyraldehyde-butanol dehydrogenase activities of the enzymes produced by the transformants. The transgenic strain of Hydrogenophilus thermorteolus prepared as described above was inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. Cultures were grown at 52°C for 24 hours with shaking, supplying a gas mixture of H2:O2:CO2 = 7.5:1:1.5. Bacteria were harvested from 2 mL of the resulting culture by centrifugation (4°C, 5,000 g, 10 min). The cells were suspended in 300 μL of reaction buffer (100 mM Tris-HCl (pH 7.5), 5 μM FeSO4, 0.2 mM DTT), disrupted by sonication, and then centrifuged (4°C, 20,000 g, 3 min) to obtain the cell disruption supernatant. The cell lysate supernatant was used as a crude enzyme solution to measure butyraldehyde dehydrogenase activity. Reaction buffer was added to the crude enzyme solution, and 20 mM NADH as a coenzyme and 5 mM butyryl-CoA as a substrate were mixed. The reaction was carried out at 52°C for 30 minutes. The reaction solution was analyzed by gas chromatography (Shimadzu Corporation: GC-2014, Polar-WAX column). The butyraldehyde or butanol produced was identified by detecting peaks with the same column retention time as the authentic sample. In the case of a bifunctional enzyme capable of producing butyraldehyde from butyryl-CoA and butanol from butyraldehyde, butanol can be detected by this method.

[0074] The chromatograms obtained for the reaction products of butyryl-CoA and the crude enzyme solution produced by the transformants, BOH-01, BOH-05, BOH-06, BOH-07, and BOH-08, as well as the control strain (empty vector) transformed with pCAMO-6, are shown in the upper panel of Figure 2. Here, STD represents butanol and butyraldehyde standards (2 mM). The enzymes produced by these transformants produced butyryl-CoA as a substrate, indicating that these transformants produced enzymes with butyraldehyde dehydrogenase activity, which produces butyraldehyde from butyryl-CoA, and butanol dehydrogenase activity, which produces butanol from butyraldehyde. The chromatogram obtained for the reaction products of butyryl-CoA and the crude enzyme solution produced by the transformants, BOH-02, is shown in the lower panel of Figure 2. The crude enzyme produced by the BOH-02 strain produced butyraldehyde using butyryl-CoA as a substrate, but did not produce butanol, indicating that an enzyme having butyraldehyde dehydrogenase activity was produced.

[0075] (3-3) Measurement of butanol dehydrogenase activity of the enzyme produced by the transformant. The cell lysate supernatant of the transgenic strain prepared as described above was used as a crude enzyme solution to measure butanol dehydrogenase activity. The crude enzyme solution was mixed with reaction buffer, 20 mM NADH as a coenzyme, and 10 mM butyraldehyde as a substrate, and the reaction was carried out at 52°C for 30 minutes. The reaction solution was analyzed by gas chromatography (Shimadzu: GC-2014, Polar-WAX column), and the butanol produced was identified by detecting a peak showing the same column retention time as the authentic sample.

[0076] Of the transformants listed in Table 3, the chromatograms obtained for the reaction products of butyraldehyde with the crude enzyme solution produced by the transformants, strains BOH-01, BOH-03, BOH-04, BOH-05, BOH-06, BOH-07, BOH-08, BOH-09, and BOH-10, are shown in the upper and lower panels of Figure 3. Here, STD represents the respective standards (2 mM) of butanol and butyraldehyde. The crude enzymes produced by these transformants produced butanol from butyraldehyde, indicating that they produced enzymes with butanol dehydrogenase activity.

[0077] (3-4) Butanol Production: The BOH-01 strain prepared as described above and the control strain carrying the empty vector pCAMO-6 were each inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. A gas mixture of H2:O2:CO2 = 7.5:1:1.5 was supplied during the culture, and the strains were cultured with shaking at 52°C for 24 hours. After the culture, the culture supernatant was obtained by centrifugation (4°C, 5,000 g, 10 minutes). Unlike the culture supernatant of the control strain carrying the empty vector, butanol was produced in the culture supernatant of the BOH-01 strain.

[0078] The transformants of the Hydrogenophilus bacteria of the present invention can be prepared by referring to the description in the Examples. In addition, the other strains described herein are either internationally deposited under the Budapest Treaty, are held by an institution from which they can be obtained without conditions, are commercially available, or can be prepared by those skilled in the art based on the present specification and are publicly available.

[0079] SEQ ID NOs: 1 to 20 are shown below.

[0080] The transformant of the present invention can produce butanol, which is in high demand in the production of chemical products such as plastics, rubber, fibers, polymers, and biofuels, with high efficiency using carbon dioxide as the sole carbon source. Therefore, the transformant of the present invention will contribute to the industrial production of chemical products with high efficiency while resolving global warming caused by increased carbon dioxide.

Claims

1. A transformant obtained by introducing any one of the following genes (1) to (5) into a bacterium of the genus Hydrogenophilus: (1) a gene encoding an enzyme having butyraldehyde dehydrogenase activity, and a gene encoding an enzyme having butanol dehydrogenase activity; (2) a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity; (3) a gene encoding an enzyme having butyraldehyde dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity; (4) a gene encoding an enzyme having butanol dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity; (5) a gene encoding an enzyme having butyraldehyde dehydrogenase activity, a gene encoding an enzyme having butyraldehyde dehydrogenase activity, and a gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity.

2. The transformant according to claim 1, wherein the gene encoding a multifunctional enzyme having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity is selected from the group consisting of the following DNA (a), (b), (c), (d) and (e): (a) DNA comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 9; (b) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 1, 3, 5, 7, or 9, and encoding a polypeptide having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10; (d) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 2, 4, 6, 8, or 10, and encoding a polypeptide having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity; (e) DNA comprising an amino acid sequence in which 1 to 100 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, and encoding a polypeptide having butyraldehyde dehydrogenase activity and butanol dehydrogenase activity.

3. The transformant according to claim 1 or 2, wherein the gene encoding an enzyme having butyraldehyde dehydrogenase activity is a DNA of the following (f), (g), (h), (i), or (j): (f) a DNA comprising the nucleotide sequence of SEQ ID NO: 11; (g) a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 11 and encoding a polypeptide having butyraldehyde dehydrogenase activity; (h) a DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 12; (i) a DNA comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 12 and encoding a polypeptide having butyraldehyde dehydrogenase activity; (j) a DNA comprising an amino acid sequence in which 1 to 100 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 12 and encoding a polypeptide having butyraldehyde dehydrogenase activity.

4. The transformant according to claim 1 or 2, wherein the gene encoding an enzyme having butanol dehydrogenase activity is a DNA of the following (k), (l), (m), (n) or (o): (k) a DNA comprising the nucleotide sequence of SEQ ID NO: 13, 15, 17 or 19; (l) a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 13, 15, 17 or 19 and encoding a polypeptide having butanol dehydrogenase activity; (m) a DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, 16, 18 or 20; (n) a DNA comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 14, 16, 18 or 20 and encoding a polypeptide having butanol dehydrogenase activity; (o) a DNA comprising an amino acid sequence in which 1 to 100 amino acids have been deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 14, 16, 18 or 20, and encoding a polypeptide having butanol dehydrogenase activity.

5. The transformant according to claim 1 or 2, wherein the Hydrogenophilus bacterium is Hydrogenophilus thermorteolus.

6. A method for producing butanol, comprising a step of culturing the transformant according to claim 1 or 2 using carbon dioxide as substantially the only carbon source.