Crotyl alcohol-generating bacterium transformant of genus hydrogenophilus

Abstract

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

Description

Transformants of Hydrogenophilus sp. producing crotyl alcohol

[0001] The present invention relates to a transformant of a bacterium belonging to the genus Hydrogenophilus having the ability to produce crotyl alcohol, and a method for producing crotyl alcohol 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 significantly to global warming.

[0005] Butadiene is used as a raw material for synthetic rubbers such as butadiene rubber, styrene-butadiene rubber, acrylonitrile-butadiene rubber, and chloroprene rubber, and synthetic resins such as styrene-butadiene-acrylonitrile (ABS) resin and styrene-butadiene methacrylate (MBS) resin, as well as for other chemical products such as adiponitrile, 1,4-butanediol, cyclododecatriene, chloroprene, and sulfolane.

[0006] Butadiene is mainly produced by catalytic cracking naphtha with steam at a high temperature of over 900°C, producing butadiene together with other olefins and aromatic hydrocarbon compounds. In recent years, it has also been produced using a method that uses bioethanol as a raw material in consideration of the environment, but this method does not sufficiently reduce the environmental impact because the conversion of ethanol to acetaldehyde and the conversion of the ethanol-acetaldehyde mixture to butadiene are carried out by chemical reactions.

[0007] Therefore, attempts have been made to develop technologies for producing butadiene using microorganisms. Many microorganisms possess most of the metabolic pathway (shown in Figure 1) that converts pyruvate to crotonyl-CoA and crotyl alcohol (crotonyl alcohol) to produce butadiene, but no microorganisms that produce butadiene have been identified. For this reason, various methods have been proposed for producing butadiene by culturing a transformant in which one or more foreign genes encoding the enzymes that make up this metabolic pathway have been introduced into the host.

[0008] For example, Patent Document 1 discloses a method for producing butadiene, which includes a step of producing crotyl alcohol by culturing an Escherichia coli transformant and a step of chemically converting this crotyl alcohol into butadiene (Claim 1). The transformant used here has introduced therein the following exogenous genes (A) and (B) that catalyze the pathway for producing crotyl alcohol from crotonyl-CoA: (A) (1) aldehyde-forming crotonyl-CoA reductase (5 to 7 in Fig. 1) and alcohol-forming crotonaldehyde reductase (7 to 6 in Fig. 1), (2) alcohol-forming crotonyl-CoA reductase (5 to 6 in Fig. 1), or (3) crotonyl-CoA hydrolase (5 to 8 in Fig. 1), crotonyl-CoA synthetase (5 to 8 in Fig. 1), or crotonyl-CoA transferase (5 to 8 in Fig. 1), crotonate reductase (8 to 7 in Fig. 1), and alcohol-forming crotonaldehyde reductase (7 to 6 in Fig. 1) (B) Acetyl-CoA:acetyl-CoA acyltransferase (2 and 3 in Figure 1), acetoacetyl-CoA reductase (3 and 4 in Figure 1), and 3-hydroxybutyryl-CoA dehydratase (4 and 5 in Figure 1). However, the method of Patent Document 1 uses glucose or starch derived from biomass as a carbon source (paragraph 0087). As mentioned above, the use of biomass requires complex processes to convert biomass into sugars, which results in high costs, and the industrial use of biomass has the drawback of placing a burden on the environment.

[0009] Furthermore, Patent Document 2 discloses a method for producing butadiene by introducing an exogenous gene (A) or (B) below into bacteria of the genus Burkholderia, Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus, and contacting the resulting transformant with a carbon source in a medium (claims 1 and 2): (A) Crotonyl-CoA reductase (5 to 7 in Figure 1), and linalool dehydratase, a type of crotonyl alcohol dehydratase (6 to 11 in Figure 1). (B) Crotonaldehyde dehydrogenase (5 to 7 in Figure 1), crotonyl alcohol dehydrogenase (7 to 6 in Figure 1), and linalool dehydratase, a type of crotonyl alcohol dehydratase (6 to 11 in Figure 1). However, the method of Patent Document 2 is a method for producing butadiene by growing the above-mentioned microorganisms using sugars or the like as carbon raw materials (paragraph 0088, etc.), and there are drawbacks associated with using biomass.

[0010] Also, a method for producing crotyl alcohol, which is a raw material for butadiene, using microorganisms has been proposed. For example, Patent Document 3 discloses a technology for producing crotyl alcohol from a carbon-1 compound selected from methane, methanol, methylamine, formic acid, formaldehyde, and formamide as a raw material using a transformant obtained by introducing into a host, which is a methylotrophic strain such as a methanol-assimilating yeast, at least one selected from a gene encoding acetoacetyl-CoA thiolase (see sections 2 and 3 in Figure 1), a gene encoding 3-hydroxybutyryl-CoA dehydrogenase (see sections 3 and 4 in Figure 1), a gene encoding 3-hydroxybutyryl-CoA dehydratase (see sections 4 and 5 in Figure 1), a crotonyl-CoA hydrolase (see sections 5 and 8 in Figure 1), a crotonate reductase (see sections 8 and 7 in Figure 1), and an alcohol-producing crotonaldehyde reductase (see sections 7 and 6 in Figure 1), in addition to a gene encoding an enzyme that converts methanol and / or formic acid to formaldehyde and a gene that imparts formaldehyde fixation ability (claim 11, paragraph 0001). However, since compounds with one carbon atom such as methane are mainly synthesized using natural gas, coal, oil shale, etc. as raw materials, it is difficult to say that the method of Patent Document 3 is effective in reducing greenhouse gases.

[0011] Patent No. 5960729 Patent No. 6415326 JP2014-155455

[0012] An object of the present invention is to provide a Hydrogenophilus bacterium transformant (in other words, a transformed Hydrogenophilus bacterium) that can efficiently produce crotyl alcohol using carbon dioxide as a sole carbon source, and a method for efficiently producing crotyl alcohol using this transformant.

[0013] The present inventors conducted extensive research to solve the above problems and discovered the following: (i) Hydrogenophilus bacteria do not inherently produce crotyl alcohol because they do not possess a gene encoding an enzyme that catalyzes the crotyl alcohol production reaction. Therefore, in order to confer the ability to produce crotyl alcohol, it is necessary to introduce a gene encoding an enzyme that catalyzes the crotyl alcohol production reaction. (ii) By introducing into Hydrogenophilus bacteria a gene encoding an enzyme with crotonaldehyde dehydrogenase activity that produces crotonaldehyde from crotonyl-CoA (hereinafter sometimes referred to as "crotonaldehyde dehydrogenase") and a gene encoding an enzyme with crotyl alcohol dehydrogenase activity that produces crotyl alcohol from crotonaldehyde (hereinafter sometimes referred to as "crotyl alcohol dehydrogenase"), these genes are expressed in the Hydrogenophilus bacteria. This allows the Hydrogenophilus bacteria to produce crotyl alcohol. Similar effects can be obtained by introducing, instead of the above two genes, a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity, which produces crotonaldehyde from crotonyl-CoA, and crotyl alcohol dehydrogenase activity, which produces crotyl alcohol from crotonaldehyde (hereinafter sometimes referred to as "crotonaldehyde-crotyl alcohol dehydrogenase").

[0014] The present invention was completed based on the above findings and provides the following [1] to [5]: [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) Genes encoding enzymes with crotonaldehyde dehydrogenase activity and genes encoding enzymes with crotyl alcohol dehydrogenase activity. (2) Genes encoding multifunctional enzymes with crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (3) Genes encoding enzymes with crotonaldehyde dehydrogenase activity and genes encoding multifunctional enzymes with crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (4) Genes encoding enzymes with crotyl alcohol dehydrogenase activity and genes encoding multifunctional enzymes with crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (5) Genes encoding enzymes with crotonaldehyde dehydrogenase activity, genes encoding enzymes with crotyl alcohol dehydrogenase activity, and genes encoding multifunctional enzymes with crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. [2] The transformant described in [1] uses the DNA of (a), (b), (c), (d), or (e) below as a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity.(a) DNA comprising the base sequence of SEQ ID NO: 1; (b) DNA comprising a base sequence having 90% or more identity with SEQ ID NO: 1 and encoding a polypeptide having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (d) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 2 and having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity; (e) DNA encoding a polypeptide having an amino acid sequence in which 1 to 80 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2 and having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. [3] The transformant according to [1] or [2], wherein the DNA of (f), (g), (h), (i), or (j) below is used as the gene encoding an enzyme having crotyl alcohol dehydrogenase activity. (f) DNA containing the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17. (g) DNA containing a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, and encoding a polypeptide having crotyl alcohol dehydrogenase activity. (h) DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18. (i) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18, and having crotyl alcohol dehydrogenase activity. (j) DNA encoding a polypeptide having SEQ ID NO: 4, 6, 8, 10, 12, 14, [4] A transformant described in any of [1] to [3], wherein the Hydrogenophilus bacterium is Hydrogenophilus thermorteolus.[5] A method for producing crotyl alcohol, comprising a step of culturing the transformant according to any one of [1] to [4] using carbon dioxide as substantially the only carbon source.

[0015] 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.

[0016] 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, allowing them to efficiently organicize carbon dioxide and can be cultured in a simple medium. While hydrogen bacteria generally grow slowly, the growth rate of the Hydrogenophilus genus is particularly fast. 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."

[0017] Hydrogenophilus bacteria naturally possess an enzyme gene that catalyzes the reaction of producing crotonyl-CoA from pyruvate, but not the enzyme gene that catalyzes the reaction of producing crotonyl-CoA from crotonyl-CoA. 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 into Hydrogenophilus bacteria a gene encoding an enzyme with crotonaldehyde dehydrogenase activity that produces crotonaldehyde from crotonyl-CoA and a gene encoding an enzyme with crotyl alcohol dehydrogenase activity that produces crotyl alcohol from crotonaldehyde, these genes are expressed in the Hydrogenophilus bacteria, enabling the Hydrogenophilus bacteria to produce crotyl alcohol using carbon dioxide as the sole carbon source. Similar effects can be achieved by introducing a gene encoding a multifunctional enzyme with crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity instead of the above two genes.

[0018] 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 crotyl alcohol industrially.

[0019] Crotyl alcohol can be used as an intermediate for butadiene production. Crotyl alcohol produced using the transformant of the present invention can be used as a raw material for butadiene production using a microorganism. It can also be used as a raw material for butadiene production by an enzymatic reaction or a chemical reaction.

[0020]

[0023] Figure 1 shows a metabolic pathway for producing crotonyl-CoA from pyruvate, which is present in many microorganisms, as well as a pathway for producing butadiene from crotonyl-CoA by introducing a foreign gene.

[0024] Figure 2 shows a gas chromatogram demonstrating that the crude enzyme produced by the transformant prepared in the examples produced crotyl alcohol using crotonyl-CoA as a substrate.

[0025] Figure 3 shows a gas chromatogram demonstrating that the crude enzyme produced by the transformant prepared in the examples produced crotyl alcohol using crotonaldehyde as a substrate.

[0021] The present invention is described in detail below. (1) Transformant capable of producing crotyl alcohol 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 an exogenous gene selected from the following genes (1) to (5): (1) A gene encoding an enzyme having crotonaldehyde dehydrogenase activity, and a gene encoding an enzyme having crotyl alcohol dehydrogenase activity. (2) A gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (3) A gene encoding an enzyme having crotonaldehyde dehydrogenase activity, and a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (4) A gene encoding an enzyme having crotyl alcohol dehydrogenase activity, and a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (5) A gene encoding an enzyme having crotonaldehyde dehydrogenase activity, a gene encoding an enzyme having crotyl alcohol dehydrogenase activity, and a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity.

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

[0023] One or more of the crotonaldehyde-crotyl alcohol dehydrogenase genes, the crotonaldehyde dehydrogenase gene, and the crotyl alcohol dehydrogenase gene 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] The gene encoding crotonaldehyde-crotyl alcohol dehydrogenase does not need to have been identified as a crotonaldehyde-crotyl alcohol dehydrogenase gene, but may be DNA encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity that produces crotonaldehyde from crotonyl-CoA and crotyl alcohol dehydrogenase activity that produces crotyl alcohol from crotonaldehyde. This multifunctional enzyme may be a bifunctional enzyme having only crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity.

[0025] An example of a crotonaldehyde-crotyl alcohol dehydrogenase gene that can be used in the present invention is the crotonaldehyde-crotyl alcohol dehydrogenase gene of Clostridium akagii, which consists of the nucleotide sequence of SEQ ID NO: 1. The crotonaldehyde-crotyl alcohol dehydrogenase of Clostridium akagii is a bifunctional enzyme that possesses both crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. As shown in the Examples section, the fact that this bifunctional enzyme of Clostridium akagii functions in bacteria of the genus Hydrogenophilus has been confirmed by the fact that the crude enzyme produced intracellularly by Clostridium akagii produced crotyl alcohol using crotonyl-CoA as a substrate in the presence of NADH.

[0026] In the present invention, DNA can be used that contains the nucleotide sequence of SEQ ID NO: 1 (particularly, that consists of the nucleotide sequence of SEQ ID NO: 1). Also usable is DNA that contains a nucleotide sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and particularly 99% or more identical to SEQ ID NO: 1 (particularly, that consists of a nucleotide sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and particularly 99% or more identical to SEQ ID NO: 1) and encodes a polypeptide that has crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity.

[0027] Further, examples of aldehyde-alcohol dehydrogenase genes include DNA encoding the crotonaldehyde-crotyl alcohol dehydrogenase of Clostridium Akagi, which has the amino acid sequence of SEQ ID NO: 2. In the present invention, DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (particularly consisting of the amino acid sequence of SEQ ID NO: 2) can be used. Furthermore, DNA encoding a polypeptide 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: 2 (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: 2) and having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity can also be used. Furthermore, DNAs that include an amino acid sequence in which 1 to 100, 1 to 80, 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 (particularly, an amino acid sequence in which 1 to 100, 1 to 80, 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) and that encode a polypeptide that has crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity can also be used.

[0028] In the present invention, whether a polypeptide has crotonaldehyde dehydrogenase activity using crotonyl-CoA as a substrate and crotyl alcohol dehydrogenase activity using crotonaldehyde as a substrate is confirmed by reacting the test polypeptide with crotonyl-CoA in the presence of NADH and detecting the produced crotyl alcohol by gas chromatography or the like.

[0029] Crotonaldehyde dehydrogenase gene The gene encoding an enzyme having crotonaldehyde dehydrogenase activity does not need to be known to have been identified as a crotonaldehyde dehydrogenase gene, but may be DNA encoding a polypeptide having crotonaldehyde dehydrogenase activity.

[0030] In the present invention, whether a polypeptide has crotonaldehyde dehydrogenase activity using crotonyl-CoA as a substrate is confirmed by reacting the test polypeptide with crotonyl-CoA in the presence of NADH and detecting the produced crotonaldehyde by gas chromatography or the like.

[0031] In the present invention, the crotonaldehyde dehydrogenase gene can also be a gene encoding an aldehyde-alcohol dehydrogenase that can produce crotonaldehyde from crotonyl-CoA but cannot produce crotyl alcohol from crotonaldehyde. This enzyme is generally called an aldehyde-alcohol dehydrogenase, but in the present invention it is classified as a crotonaldehyde dehydrogenase.

[0032] In the present invention, whether a polypeptide has aldehyde dehydrogenase activity using crotonyl-CoA as a substrate is confirmed by reacting the test polypeptide with crotonyl-CoA in the presence of NADH and detecting the produced crotonaldehyde by gas chromatography or the like.

[0033] Crotyl alcohol dehydrogenase gene The gene encoding an enzyme having crotyl alcohol dehydrogenase activity does not need to be known to have been identified as a crotyl alcohol dehydrogenase gene, but may be DNA encoding a polypeptide having crotyl alcohol dehydrogenase activity that produces crotyl alcohol from crotonaldehyde.

[0034] Examples of crotyl alcohol 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.

[0035] The crotyl alcohol dehydrogenases of Bacillus coagulans and Zymomonas mobilis subsp. pomaceae, which have the amino acid sequences of SEQ ID NOS: 4 and 6, respectively, are not multifunctional enzymes that include an aldehyde dehydrogenase. Furthermore, the crotyl alcohol dehydrogenases of Thermoclostridium caenicola, Thermoanaerobacter masrani, Thermoanaerobacter ethanolicus, Bacillus coagulans, Geobacillus thermoglucosidasius, and Fonticella tunisiensis, which have the amino acid sequences of SEQ ID NOS: 8, 10, 12, 14, 16, and 18, respectively, belong to the aldehyde-alcohol dehydrogenase family, but the aldehyde dehydrogenases they possess cannot produce crotonaldehyde from crotonyl-CoA.

[0036] 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: 3, 5, 7, 9, 11, 13, 15, or 17 (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: 3, 5, 7, 9, 11, 13, 15, or 17) and that encode a polypeptide having crotyl alcohol dehydrogenase activity can also be used.

[0037] Also usable is a 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: 4, 6, 8, 10, 12, 14, 16, or 18 (particularly, 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: 4, 6, 8, 10, 12, 14, 16, or 18), and encodes a polypeptide that has crotyl alcohol dehydrogenase activity.

[0038] Furthermore, DNAs that contain an amino acid sequence in which 1 to 100, 1 to 90, 1 to 80, 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: 4, 6, 8, 10, 12, 14, 16, or 18 (particularly, an amino acid sequence in which 1 to 100, 1 to 90, 1 to 80, 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: 4, 6, 8, 10, 12, 14, or 16) and that encode a polypeptide having crotyl alcohol dehydrogenase activity can also be used.

[0039] As described above, in the present invention, the gene for an enzyme having crotyl alcohol dehydrogenase activity can also be a gene for an aldehyde-alcohol dehydrogenase that can produce crotyl alcohol from crotonaldehyde but cannot produce crotonaldehyde from crotonyl-CoA. Although this enzyme is generally called an aldehyde-alcohol dehydrogenase, in the present invention it is classified as a crotyl alcohol dehydrogenase.

[0040] In the present invention, whether a polypeptide has crotyl alcohol dehydrogenase activity using crotonaldehyde as a substrate is confirmed by reacting the test polypeptide with crotonaldehyde in the presence of NADH and detecting the produced crotyl alcohol by gas chromatography or the like.

[0041] A DNA sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18 may have various base substitutions in the coding region, taking into account codon degeneracy or preferred codons in Hydrogenophilus bacteria, as long as the amino acid sequence of the protein expressed from the coding region is not changed.

[0042] In the present invention, the identities of base sequences and amino acid sequences are values ​​calculated using GENETYX ver. 17 (GENETYX).

[0043] In the present invention, a DNA that contains a nucleotide sequence that is 90% to 100% identical to the nucleotide sequence of a certain DNA and that encodes a polypeptide with the same type of activity is referred to as a "homolog" of that DNA. A polypeptide that contains an amino acid sequence that is 90% to 100% identical to the amino acid sequence of a certain polypeptide and has the same type of activity is referred to as a homolog of that polypeptide. A DNA that contains an amino acid sequence in which 1 to 100 amino acids have been deleted, substituted, inserted, or added to the amino acid sequence of a certain polypeptide and encodes a polypeptide with the same type of activity is referred to as a homolog of that DNA.

[0044] Examples of Hydrogenophilus bacteria include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidans, 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.

[0045] 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.

[0046] Method for Preparing Transformants The 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, a YAC, 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 vectors include the tac promoter, lac promoter, trc promoter, and the OXB1, OXB11 to OXB20 promoters from Oxford Genetics, and examples of terminators contained in vectors 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, calcium phosphate method, DEAE-dextran-mediated transfection method, and electric pulse method (electroporation method).

[0047] (2) Method for Producing Crotyl Alcohol The present invention provides a method for producing crotyl alcohol 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 crotyl alcohol can be efficiently produced.

[0048] 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 crotyl alcohol 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" and "using carbon dioxide as the sole carbon source" include cases where unavoidable amounts of other carbon sources are mixed in.

[0049] 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 crotyl alcohol. 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 a sealed culture vessel while the culture is shaken, or the transformant can be cultured in a sealed culture vessel while the mixed gas is introduced into the culture medium by bubbling. 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 crotyl alcohol can be efficiently produced. 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 L of medium. Within this range, the growth of the transformant is favorable, crotyl alcohol 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 crotyl alcohol can be produced efficiently.

[0050] By culturing as described above, crotyl alcohol is produced in the culture solution. Crotyl alcohol 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.

[0051] 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.

[0052] (1) Construction of Plasmid Vector (pCAMO-6) The construction method of the plasmid vector pCAMO-6 used for gene introduction is described below. (1-1) Preparation of tac Promoter DNA Fragment To amplify the tac promoter DNA fragment, PCR was performed using the following pair of primers, using the New England BioLabs plasmid pMAL-c5X as a template. Unless otherwise noted, PCR was performed using a Thermo Fisher Scientific 2720 Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as the reaction reagent, according to standard procedures. Primers for tac promoter amplification: (a-1) 5'-TTTTATAACCCGGGCCATCGACTGCACGGTGCACC-3' (SEQ ID NO: 19) (b-1) 5'-TGCTAGCACTGTTTCCTGTGTGAAATTGTTATCCG-3' (SEQ ID NO: 20) Primer (a-1) contained a SmaI restriction enzyme site, as underlined. The reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.3 kbp corresponding to the tac promoter was detected. The agarose gel containing this fragment was excised, and the DNA was recovered from the gel by freezing and thawing.

[0053] (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: 21) (b-2) 5'-CAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGATATCAGCATATGCGTTTCTCCTCCAGA-3' (SEQ ID NO: 22) 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: 23) (b-3) 5'-TATTTGAATCGAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGT-3' (SEQ ID NO: 24) The 3'-20 base sequences of primers (a-3) and (b-3) are complementary to each other. The reaction solution prepared above was subjected to agarose gel electrophoresis, and DNA fragments of approximately 0.1 kbp each corresponding to the first and second halves of the multicloning region were detected and recovered from the gel.

[0054] 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.

[0055] (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: 25) (b-4) 5'-CCTAGATCCGCGGAGTTTGTAGAAACGCAAAAAGG-3' (SEQ ID NO: 26) 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 region of pUC19: (a-5) 5'-ACAAACTCCGCGGATCTAGGTGAAGATCCTTTTTG-3' (SEQ ID NO: 27) (b-5) 5'-CGTCCGCGGCCAGCAAAAGGCCAGGAACCGTAAAA-3' (SEQ ID NO: 28) The resulting reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.3 kbp was detected for the rrnB terminator and approximately 0.8 kbp for the DNA replication origin region of pUC19, which were recovered from the gel.

[0056] 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.

[0057] (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: 29) (b-6) 5'-GGCCCGGGTTATAAAAGCCAGTCATTAGGCCTATC-3' (SEQ ID NO: 30) 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.

[0058] (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.

[0059] (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.).

[0060] The resulting ligation reaction mixture was used to transform Hydrogenophilus thermorteolus strain TH-1 (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.

[0061] Using the extracted plasmid as a template, PCR was performed with 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: 31) (b-7) 5'-TGACGCCAGAAGCATTGGTGCACCGTGCAGTCGAT-3' (SEQ ID NO: 32). 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.

[0062] (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: 33) (b-8) 5'-TATGCGTTTCTCCTCCA-3' (SEQ ID NO: 34) The constructed plasmid was named pCAMO-5.

[0063] (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: 35) (b-9) 5'-ATCCGGCACCCATATCTGAACCGGACGCAAACCCG-3' (SEQ ID NO: 36)

[0064] 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: 37) and (b-10) 5'-AAGGCCTTCATGGCCTTATTTCGCGATTCCCAAGA-3' (SEQ ID NO: 38).

[0065] 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 PCR. The DNA fragments prepared above were mixed, and PCR was performed using primers (a-9) and (b-10) to amplify the ligated DNA fragment as a template. 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.

[0066] 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.

[0067] (2) Construction of gene expression plasmids Gene expression plasmids were constructed using Gibson Assembly. DNA fragments of each gene derived from the following bacteria were amplified by PCR using the genomic DNA of each bacterium as a template. Crotonaldehyde-crotyl alcohol dehydrogenase gene: DNA fragment of SEQ ID NO: 1 derived from Clostridium Akagi Crotyl alcohol dehydrogenase gene: DNA fragment of SEQ ID NO: 3 derived from Bacillus coagulans DNA fragment of SEQ ID NO: 5 derived from Zymomonas mobilis subsp. pomaceae DNA fragment of SEQ ID NO: 7 derived from Thermoclostridium caenicola DNA fragment of SEQ ID NO: 9 derived from Thermoanaerobacter masrani DNA fragment of SEQ ID NO: 11 derived from Thermoanaerobacter ethanolicus DNA fragment of SEQ ID NO: 13 derived from Bacillus coagulans DNA fragment of SEQ ID NO: 15 derived from Geobacillus thermoglucosidasius DNA fragment of SEQ ID NO: 17 derived from Fonticella tunisiensis

[0068] The following primers were used for PCR: Primers for amplifying the crotonaldehyde-crotyl alcohol dehydrogenase gene (SEQ ID NO: 1) of Clostridium Akagi: (a-11) 5'-GATCTGGAGGAGAAACGCATATGAAAGTAACAAACATAGAAGAG-3' (SEQ ID NO: 39) (b-11) 5'-TGTCGACGGAGCTCGAATTCTTATTTTTCTCCCATTTTAATTTCACC-3' (SEQ ID NO: 40) Primers (a-11) and (b-11) contained sequences homologous to the vector pCAMO-6. Primers for amplifying the crotyl alcohol dehydrogenase gene (SEQ ID NO: 3) of Bacillus coagulans: (a-12) 5'-GATCTGGAGGAGAAACGCATATGAGAGCAGCAGTCGTCAGTTCT-3' (SEQ ID NO: 41) (b-12) 5'-TGTCGACGGAGCTCGAATTCTTATTTCAACGACATATCCAGGAC-3' (SEQ ID NO: 42) Primers (a-12) and (b-12) contain sequences homologous to the vector pCAMO-6. Primers for amplifying the crotyl alcohol dehydrogenase gene (SEQ ID NO: 5) of Zymomonas mobilis subsp. pomaceae: (a-13) 5'-GATCTGGAGGAGAAACGCATATGAAAGCAGCCGTCATAACTAAA-3' (SEQ ID NO: 43) (b-13) 5'-TGTCGACGGAGCTCGAATTCCTAGTGATGGGTAAAATCAACAAC-3' (SEQ ID NO: 44) Primers (a-13) and (b-13) contain sequences homologous to the vector pCAMO-6. Primers for amplifying the crotyl alcohol dehydrogenase gene (SEQ ID NO: 7) of Thermoclostridium caenicola: (a-14) 5'-GATCTGGAGGAGAAACGCATATGGAAAACAAAGAAAGAAAGATT-3' (SEQ ID NO: 45) (b-14) 5'-TGTCGACGGAGCTCGAATTCTCATGCTCTCTTCGCTCCGACTCT-3' (SEQ ID NO: 46) Primers (a-14) and (b-14) contain sequences homologous to the vector pCAMO-6.Primers for amplifying the crotyl alcohol dehydrogenase gene (SEQ ID NO: 9) of Thermoanaerobacter masrani: (a-15) 5'-GATCTGGAGGAGAAACGCATATGCCTACTTTATTACAAGAAAAA-3' (SEQ ID NO: 47) (b-15) 5'-TGTCGACGGAGCTCGAATTCTTATTCTCCATAGGCTTTTCTATA-3' (SEQ ID NO: 48) Primers (a-15) and (b-15) contain sequences homologous to the vector pCAMO-6. Primers for amplifying the crotyl alcohol dehydrogenase gene (SEQ ID NO: 11) of Thermoanaerobacter ethanolicus: (a-16) 5'-GATCTGGAGGAGAAACGCATATGCCTAACTTATTACAAGAAAGA-3' (SEQ ID NO: 49) (b-16) 5'-TGTCGACGGAGCTCGAATTCTTATTCTCCATAGGCTTTTCTATA-3' (SEQ ID NO: 50) Primers (a-16) and (b-16) contain sequences homologous to the vector pCAMO-6. Primers for amplifying the crotyl alcohol dehydrogenase gene (SEQ ID NO: 13) of Bacillus coagulans: (a-17) 5'-GATCTGGAGGAGAAACGCATATGGCAATCGATGAAAAAGTTGTT-3' (SEQ ID NO: 51) (b-17) 5'-TGTCGACGGAGCTCGAATTCTTATTTTGCTTCAACGCCTTCAAA-3' (SEQ ID NO: 52). Primers (a-17) and (b-17) contain sequences homologous to those of the vector pCAMO-6. Primers for amplifying the crotyl alcohol dehydrogenase gene (SEQ ID NO: 15) of Geobacillus thermoglucosidasius: (a-18) 5'-GATCTGGAGGAGAAACGCATATGGCTGTGGAGGAAAGAGTCGTC-3' (SEQ ID NO: 53) (b-18) 5'-TGTCGACGGAGCTCGAATTCTTAAACTCCTTTAAACGCTTGGCG-3' (SEQ ID NO: 54) Primers (a-18) and (b-18) contain sequences homologous to the vector pCAMO-6.Primers for amplifying the Fonticella tunisiensis crotyl alcohol dehydrogenase gene (SEQ ID NO: 17): (a-19) 5'-GATCTGGAGGAGAAACGCATATGAGGGTTGCAGAACCTATAGGA-3' (SEQ ID NO: 55) and (b-19) 5'-TGTCGACGGAGCTCGAATTCTTATTTATTATTTTCTTCAGTTTC-3' (SEQ ID NO: 56). Primers (a-19) and (b-19) contain sequences homologous to the pCAMO-6 vector. The resulting reaction mixture was subjected to agarose gel electrophoresis, detecting a DNA fragment of approximately 1.0 kbp for the crotyl alcohol dehydrogenase gene and an approximately 2.7 kbp for the crotonaldehyde-crotyl alcohol dehydrogenase gene. The agarose gel containing these DNA fragments was excised, and the DNA fragments were recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0069] To perform DNA ligation using Gibson Assembly, the plasmid vector pCAMO-6 was amplified by PCR. The following primers were used for PCR: Amplification primers for plasmid vector pCAMO-6: (a-20) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 57) (b-20) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 58). The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 5.5 kbp was detected. The agarose gel containing this vector DNA fragment was excised, and the vector DNA fragment was recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0070] 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 transformants were plated onto 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 nucleotide sequences of the genes inserted into each plasmid were analyzed by Sanger analysis at Eurofins Genomics, and were confirmed to match the sequences in the database.

[0071] (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 for each strain grown on LB solid medium by PCR. This confirmed the amplification of DNA fragments of lengths corresponding to the respective genes. The Hydrogenophilus thermorteolus TH-1 transformants transformed with plasmids containing genes derived from each bacterium were named as shown in Table 2.

[0072]

[0073] (3-2) Measurement of crotonaldehyde dehydrogenase and crotyl alcohol dehydrogenase activities of the transformants. The hydrogenophilus thermorteolus transformant CA-1 strain prepared as described above was 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 cells were cultured with shaking for 24 hours. From 2 mL of the culture, cells were harvested by centrifugation (4°C, 5,000 g, 10 min). The cells were suspended in 200 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, 10 min) to obtain the cell disruption supernatant. The cell lysate supernatant was used as a crude enzyme solution to measure crotonaldehyde dehydrogenase activity. The crude enzyme solution was mixed with reaction buffer, 10 mM NADH as a coenzyme, and 1 mM crotonyl-CoA as a substrate, and the reaction was carried out at 52°C. The reaction solution was analyzed by gas chromatography (Shimadzu Corporation: GC-2014, Polar-WAX column), and the crotonaldehyde or crotyl alcohol produced was identified by detecting peaks with the same column retention time as the authentic sample. In the case of a bifunctional enzyme with both crotonaldehyde dehydrogenase and crotyl alcohol dehydrogenase activities, crotyl alcohol was detected by this method.

[0074] Figure 2 shows a chromatogram of the reaction product between crotonyl-CoA and the crude enzyme solution produced by the transformant CA-1 strain. Here, NC represents a negative control strain transformed with an empty vector, and Std represents crotyl alcohol and crotonaldehyde preparations (1 mM). The crude enzyme produced by transformant CA-1, in which the DNA of SEQ ID NO: 1 was introduced into the Hydrogenophilus thermorteolus TH-1 strain, produced crotyl alcohol using crotonyl-CoA as a substrate, indicating the production of a bifunctional enzyme possessing crotonaldehyde dehydrogenase and crotyl alcohol dehydrogenase activities. Thus, the Hydrogenophilus thermorteolus CA-1 strain produced functional crotonaldehyde-crotyl alcohol dehydrogenase by growing using carbon dioxide as the sole carbon source, demonstrating its ability to produce crotyl alcohol using carbon dioxide as the sole carbon source.

[0075] (3-3) Measurement of crotyl alcohol dehydrogenase activity of the enzyme produced by the transformants. The cell lysate supernatants of the Hydrogenophilus thermorteolus transformants CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, and CA-8 prepared as described above were used as crude enzyme solutions to measure crotyl alcohol dehydrogenase activity. The crude enzyme solutions were mixed with reaction buffer, 10 mM NADH as a coenzyme, and 10 mM crotonaldehyde as a substrate, and the reaction was carried out at 52°C for 15 minutes. The reaction mixture was analyzed by gas chromatography (Shimadzu Corporation: GC-2014, Polar-WAX column). The crotyl alcohol produced was identified by detecting a peak with the same column retention time as the authentic sample.

[0076] Chromatograms obtained for the reaction products of crotonaldehyde with the crude enzyme solution produced by each transformant are shown in Figure 3. Here, NC represents the negative control strain transformed with an empty vector, and Std represents the respective crotyl alcohol and crotonaldehyde standards (1 mM). The crude enzyme produced by the transformant in which DNA of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17 was introduced into the Hydrogenophilus thermorteolus TH-1 strain produced crotyl alcohol using crotonaldehyde as a substrate, indicating that an enzyme with crotyl alcohol dehydrogenase activity was produced. Thus, the Hydrogenophilus thermorteolus strains CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, and CA-8 produced functional crotyl alcohol dehydrogenase when grown using carbon dioxide as a sole carbon source. This indicates that crotyl alcohol can be produced using carbon dioxide as a sole carbon source by further introducing a gene encoding an enzyme having crotonaldehyde dehydrogenase activity or a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity.

[0077] (3-4) Production of crotyl alcohol by transformants The CA-1 strain and the NC strain containing the empty vector prepared as described above were 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 transformants 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 NC strain containing the empty vector, crotyl alcohol was produced in the culture supernatant of the CA-1 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 18 are shown below.

[0080] The transformant of the present invention can highly efficiently produce crotyl alcohol, a raw material for the production of butadiene, which is in high demand in the rubber, resin, and other chemical industries, using carbon dioxide as the sole carbon source. Therefore, the transformant of the present invention can contribute to the highly efficient industrial production of chemical products 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 belonging to the genus Hydrogenophilus: (1) A gene encoding an enzyme having crotonaldehyde dehydrogenase activity, and a gene encoding an enzyme having crotyl alcohol dehydrogenase activity. (2) A gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (3) A gene encoding an enzyme having crotonaldehyde dehydrogenase activity, and a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (4) A gene encoding an enzyme having crotyl alcohol dehydrogenase activity, and a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. (5) A gene encoding an enzyme having crotonaldehyde dehydrogenase activity, a gene encoding an enzyme having crotyl alcohol dehydrogenase activity, and a gene encoding a multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity.

2. The transformant according to claim 1, wherein the gene encoding the multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity is a DNA of the following (a), (b), (c), (d), or (e): (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; (b) a DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 1 and encoding a polypeptide having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity; (c) a DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (d) a DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 2 and having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity; (e) a DNA encoding a polypeptide having an amino acid sequence in which 1 to 80 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2 and having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity.

3. The transformant according to claim 1 or 2, wherein the gene encoding an enzyme having crotyl alcohol dehydrogenase activity is a DNA selected from the group consisting of (f), (g), (h), (i) and (j) below. (f) DNA containing the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17. (g) DNA containing a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17 and encoding a polypeptide having crotyl alcohol dehydrogenase activity. (h) DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18. (i) DNA encoding a polypeptide containing an amino acid sequence having 90% or more identity with SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18 and having crotyl alcohol dehydrogenase activity. (j) DNA encoding a polypeptide containing an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, A DNA encoding a polypeptide having crotyl alcohol dehydrogenase activity, comprising an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of 16 or 18.

4. The transformant according to any one of claims 1 to 3, wherein the Hydrogenophilus bacterium is Hydrogenophilus thermorteolus.

5. A method for producing crotyl alcohol, comprising a step of culturing the transformant according to any one of claims 1 to 4 using carbon dioxide as substantially the only carbon source.

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