Transformants of Hydrogenophilus sp. producing crotyl alcohol

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

[Technical Field]

[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. [Background technology]

[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 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 a 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 (Figure 1, sections 5 to 7) and alcohol-forming crotonaldehyde reductase (Figure 1, sections 7 to 6), (2) alcohol-forming crotonyl-CoA reductase (Figure 1, sections 5 to 6), or (3) crotonyl-CoA hydrolase (Figure 1, sections 5 to 8), crotonyl-CoA synthetase (Figure 1, sections 5 to 8), or crotonyl-CoA transferase (Figure 1, sections 5 to 8), crotonate reductase (Figure 1, sections 8 to 7), and alcohol-forming crotonaldehyde reductase (Figure 1, sections 7 to 6). (B) Acetyl-CoA:acetyl-CoA acyltransferase (2 to 3 in Figure 1), acetoacetyl-CoA reductase (3 to 4 in Figure 1), and 3-hydroxybutyryl-CoA dehydratase (4 to 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 (6 to 11 in Figure 1), a type of crotonyl alcohol dehydratase. (B) Crotonaldehyde dehydrogenase (Figure 1, 5 to 7), crotonyl alcohol dehydrogenase (Figure 1, 7 to 6), and linalool dehydratase, a type of crotonyl alcohol dehydratase (Figure 1, 6 to 11). 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 has drawbacks associated with the use of 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 lines 2 and 3 in Figure 1), a gene encoding 3-hydroxybutyryl-CoA dehydrogenase (see lines 3 and 4 in Figure 1), a gene encoding 3-hydroxybutyryl-CoA dehydratase (see lines 4 and 5 in Figure 1), a crotonyl-CoA hydrolase (see lines 5 and 8 in Figure 1), a crotonate reductase (see lines 8 and 7 in Figure 1), and an alcohol-producing crotonaldehyde reductase (see lines 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. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent No. 5960729 [Patent Document 2] Patent No. 6415326 [Patent Document 3] Patent Publication No. 2014-155455 Summary of the Invention [Problem to be solved by the invention]

[0012] An objective 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. [Means for solving the problem]

[0013] The present inventors have conducted extensive research to solve the above problems and have obtained the following findings. (i) Hydrogenophilus bacteria do not have a gene encoding an enzyme that catalyzes the crotyl alcohol production reaction and do not naturally produce crotyl alcohol. Therefore, in order to impart 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, which 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 has been 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) 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 with crotonaldehyde dehydrogenase and crotyl alcohol dehydrogenase activities (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 described in [1], wherein the gene encoding the multifunctional enzyme having crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity is the DNA of (a), (b), (c), (d), or (e) below: (a) DNA containing the base sequence of SEQ ID NO: 1 (b) DNA containing 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) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (d) DNA encoding a polypeptide comprising 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) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 80 amino acids are 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 gene encoding the enzyme having crotyl alcohol dehydrogenase activity is a DNA selected from the group consisting of (f), (g), (h), (i), and (j): (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 comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18 and having crotyl alcohol dehydrogenase activity. (j) DNA encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18 in which 1 to 30 amino acids have been deleted, substituted, inserted, or added, and having crotyl alcohol dehydrogenase activity. [4] The transformant according to any one of [1] to [3], wherein the bacterium of the genus Hydrogenophilus 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. [Effects of the Invention]

[0015] Measures to curb the increase in carbon dioxide include reducing carbon dioxide emissions and fixing emitted carbon dioxide. To reduce carbon dioxide emissions, energy sources such as solar, wind, and geothermal energy are being used in place of fossil fuels. However, in reality, the use of these types of energy sources has not been enough to sufficiently curb 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, etc. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve two problems: global warming caused by increased carbon dioxide levels, and the difficulty in securing food, feed, and fuel. It can also produce in-demand chemical raw materials while suppressing global warming caused by increased carbon dioxide levels.

[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 have the gene for the enzyme that catalyzes the reaction of producing crotonyl-CoA from pyruvate, but do not have the gene for the enzyme that catalyzes the reaction of producing crotyl alcohol from crotonyl-CoA. The present inventors have found that it is difficult to express a functional protein even when a foreign gene is introduced into a Hydrogenophilus bacterium. However, according to the present invention, by introducing into a Hydrogenophilus bacterium 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 bacterium, enabling the Hydrogenophilus bacterium to produce crotyl alcohol using carbon dioxide as the sole carbon source. The same effect can be obtained by introducing a gene encoding a multifunctional enzyme having 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. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 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. [Figure 2] 1 is a gas chromatogram showing that the crude enzyme produced by the transformant prepared in the example produced crotyl alcohol using crotonyl-CoA as a substrate. [Figure 3] 1 is a gas chromatography chromatogram showing that the crude enzyme produced by the transformant prepared in the Examples produced crotyl alcohol using crotonaldehyde as a substrate. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention will be described in detail below. (1) Transformants 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 any one of the following exogenous 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 with crotonaldehyde dehydrogenase and crotyl alcohol dehydrogenase activities (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] Crotonaldehyde-crotyl alcohol dehydrogenase gene 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 containing the base sequence of SEQ ID NO: 1 (particularly consisting of the base sequence of SEQ ID NO: 1) can be used. In addition, DNA that contains a base sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identical to SEQ ID NO: 1 (particularly, consisting of a base sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identical to SEQ ID NO: 1) and encodes a polypeptide that has crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity can also be used.

[0027] Also, Croton aldehyde- Crotyl An example of an alcohol dehydrogenase gene is 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. In addition, DNA can also be used that contains an amino acid sequence that has 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity with SEQ ID NO: 2 (particularly, consisting of an amino acid sequence that has 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity with SEQ ID NO: 2), and encodes a polypeptide that has crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity. 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] The gene for crotyl alcohol dehydrogenase 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. [Table 1]

[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 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 contained in these strains cannot produce crotonaldehyde from crotonyl-CoA.

[0036] In the present invention, DNA can also be used that contains 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: 3, 5, 7, 9, 11, 13, 15, or 17 (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: 3, 5, 7, 9, 11, 13, 15, or 17) and encodes a polypeptide that has crotyl alcohol dehydrogenase activity.

[0037] Also usable is a DNA that encodes a polypeptide having crotyl alcohol dehydrogenase activity and that contains an amino acid sequence that is 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identical to the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18 (particularly, consisting of an amino acid sequence that is 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identical to the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18).

[0038] Furthermore, DNAs that encode a polypeptide having crotyl alcohol dehydrogenase activity and that comprise 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), 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 base sequence that is 90% to less than 100% identical to the base 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. Also, a polypeptide that contains an amino acid sequence that is 90% to less than 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. Also, a polypeptide that contains an amino acid sequence in which 1 to 100 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of a certain polypeptide and has the same type of activity is referred to as a homolog of that polypeptide. Do the Polypeptides It is called a homolog of.

[0044] Hydrogenophilus bacteria 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 fixation 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. The TH-1 (NBRC 14978) strain exhibits the highest growth rate among carbon dioxide-fixing microorganisms [Agricultural and Biological Chemistry, 41, 685-690 (1977)] (doubling in one hour). The NBRC 14978 strain has been deposited internationally 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 isolated from nature that has been genetically modified. The modification may be carried out for the purpose of enabling high expression of an introduced gene, for example. Such modification can be achieved by removing (curing) endogenous plasmids in Hydrogenophilus bacteria. Techniques for removing endogenous plasmids are well known, and include methods that utilize plasmid incompatibility, such as the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide, the introduction of a plasmid with the same origin of replication as the endogenous plasmid to destabilize it, and the disruption of factors involved in the plasmid partition system to destabilize it.

[0046] Method for preparing transformants A method for introducing the above genes (1) to (5) into a bacterium belonging to the genus Hydrogenophilus to obtain a transformant will be described below. These genes can be introduced into Hydrogenophilus bacteria using standard methods for introducing foreign genes into bacteria. These genes may be introduced directly into Hydrogenophilus bacteria, or they may be introduced into transformation vectors (e.g., plasmid vectors, viral vectors, cosmids, fosmids, BACs, YACs, etc.) containing these genes, which are then introduced into Hydrogenophilus bacteria. The vector used for transformation may contain DNA capable of autonomously replicating in Hydrogenophilus bacteria. Examples of such vectors 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 each of the OXB1, OXB11 to OXB20 promoters from Oxford Genetics. Examples of terminators contained in the vector include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the bacteriophage λt0 transcription terminator, and the T7 terminator. These genes can be introduced (transformed) into Hydrogenophilus bacteria using common techniques, such as the calcium chloride method, calcium phosphate method, DEAE-dextran-mediated transfection method, and electric pulse method (electroporation method).

[0047] (2) Manufacturing method of 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" encompass cases where unavoidable amounts of other carbon sources are mixed in.

[0049] The pH of the medium used for the 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, the growth of the bacteria and the solubility of the mixed gas in the medium are high, and crotyl alcohol can be produced with high efficiency. In the case of batch culture, the mixed gas can be sealed in a sealed culture vessel and cultured by static culture or shaking, with shaking culture being preferred since it improves the dissolution of the mixed gas into the medium. In the case of continuous culture, the transformant can be cultured by shaking culture while continuously supplying the mixed gas to a sealed culture vessel, or by introducing the mixed gas into the medium by bubbling using a sealed culture vessel. The volume ratio of hydrogen, oxygen, and carbon dioxide in the feed gas (hydrogen:oxygen:carbon dioxide) 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, the growth of the bacteria is favorable, and crotyl alcohol can be produced efficiently. The supply rate of the mixed gas or raw material gas may be 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 transformant grows well 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. [Example]

[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 the 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 PCR was performed using the following pair of primers to amplify the tac promoter DNA fragment, using the plasmid pMAL-c5X (New England Biolabs) 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. Primers for amplifying the tac promoter (a-1) 5'-TTTTATAA CCCGGG CCATCGACTGCACGGTGCACC-3' (SEQ ID NO: 19) (b-1) 5'-TGCTAGCACTGTTTCCTGTGTGAAATTGTTATCCG-3' (SEQ ID NO: 20) The primer (a-1) has an SmaI restriction enzyme site added thereto as shown by the underline. 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 fragments from the 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, and the 3' ends of each pair of primers anneal and extend to form double-stranded DNA. Primer 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. Primer for preparing the latter half of the multicloning region (a-3)5'-ACAAGCTTGCGGCCGCACTGCAGCACCATCACCACCATCATTGATAAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCT-3' (SEQ ID NO: 23) (b-3) 5'-TATTTGAATCGAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGT-3' (SEQ ID NO: 24) The sequences of the 20 bases on the 3' side of primers (a-3) and (b-3) are complementary to each other. The reaction mixture 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 these template DNA fragments are complementary to each other. For overlap extension PCR, a combination of primers (a-2) and (b-3) was used to prepare 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 PCR was performed using the following pair of primers to amplify the DNA fragment of the rrnB terminator, using the plasmid pMAL-c5X (New England Biolabs) as a template. Primers for amplifying the rrnB terminator (a-4) 5'-TAACTCGATTCAAATAAAACGAAAGGCTCAGTCGA-3' (SEQ ID NO: 25) (b-4) 5'-CCTAGATCCGCGGAGTTTGTAGAAACGCAAAAAGG-3' (SEQ ID NO: 26) Furthermore, PCR was carried out using the following pair of primers to amplify a DNA fragment of the DNA replication origin region using the plasmid pUC19 as a template. Primers for amplifying the DNA replication origin of pUC19 (a-5) 5'-ACAAACTCCGCGGATCTAGGTGAAGATCCTTTTTG-3' (SEQ ID NO: 27) (b-5) 5'-CGTCCGCGGCCAGCAAAAGGCCAGGAACCGTAAAA-3' (SEQ ID NO: 28) The resulting reaction mixture 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 DNA replication origin of pUC19 as templates. The 5' 20 base sequences 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 replication origin of pUC19. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 1.0 kbp corresponding to the DNA in which the rrnB terminator and the replication origin region of pUC19 were linked was detected and recovered from the gel.

[0057] (1-4) Preparation of neomycin / kanamycin resistance gene DNA fragment PCR was performed using the following pair of primers to amplify a DNA fragment containing the neomycin / kanamycin resistance gene (hereinafter sometimes referred to as "nptII") sequence, plasmid pK18mobsacB (GenBank: FJ437239.1) [Gene, 145, 69-73 (1994)], which contains the neomycin / kanamycin resistance gene sequence. Primers for amplifying the nptII gene (a-6) 5'-TTGCTGGCCGCGGACGTAGAAAGCCTGTCCGCAGA-3' (SEQ ID NO: 29) (b-6)5'-GG CCCGGG TTATAAAAGCCAGTCATTAGGCCTATC-3' (SEQ ID NO: 30) The primer (b-6) has an SmaI restriction enzyme site added thereto as shown by the underline. 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 plasmid Regarding the DNA fragments prepared in (1-1) to (1-4) above, the end of DNA fragment (1-1) has a 16-bp homologous sequence to the ends of DNA fragments (1-4) and (1-2), the end of DNA fragment (1-2) has a 16-bp homologous sequence to the ends of DNA fragments (1-1) and (1-3), the end of DNA fragment (1-3) has a 16-bp homologous sequence to the ends of DNA fragments (1-2) and (1-4), and the end of DNA fragment (1-4) has a 16-bp homologous sequence to 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 require restriction enzymes or ligases. To ligate the DNA fragments prepared in (1-1) to (1-4) into a circular DNA, Gibson Assembly was performed using Gibson Assembly Master Mix (New England BioLabs). The resulting reaction mixture was used to transform Escherichia coli JM109 by the calcium chloride method and plated on LB solid medium containing 50 μg / mL kanamycin. The strains grown on the medium were cultured in liquid culture using standard methods, and plasmid DNA was extracted from the culture medium and reacted with the restriction enzyme SmaI. This reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 2.3 kbp was observed, which corresponds to the size of the single DNA fragment formed by ligating the DNA fragments prepared in (1-1) to (1-4), confirming that they had been ligated. This circular plasmid DNA, which replicates in E. coli, was designated pCAMO-1.

[0059] (1-6) Construction of plasmid vector (pCAMO-4) Hydrogenophilus thermorteolus strain TH-1 was inoculated into a test tube containing 5 mL of liquid medium A (3.0 g of (NH4)2SO4, 1.0 g of KH2PO4, 2.0 g of K2HPO4, 0.25 g of NaCl, 0.014 g of FeSO4·7H2O, 0.5 g of MgSO4·7H2O, 0.03 g of CaCl2, 4.0 mg of MoO3, 28 mg of ZnSO4·7H2O, 2.0 mg of CuSO4·5H2O, 4.0 mg of H3BO3, 4.0 mg of MnSO4·5H2O, and 4.0 mg of CoCl2·6H2O dissolved in 1 L of distilled water (pH 7.0)) using a platinum loop. The test tube was then filled with a 7.5:1:1.5 H2:O2:CO2 gas mixture 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 prepared pTH-1 of about 66 kbp was cleaved with restriction enzymes ScaI and PvuII, and the plasmid pCAMO-1 was cleaved with restriction enzyme SmaI, and the two were ligated together 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 at 50°C for 60 hours in a chamber filled with a mixed gas of H2:O2:CO2 = 7.5:1:1.5. The 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 then 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. This 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 using the following pair of primers corresponding to both sides of the SmaI restriction enzyme site of pCAMO-1 to amplify the DNA fragment of the endogenous plasmid pTH1 inserted into the SmaI restriction enzyme 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 mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 3.2 kbp was detected. In other words, the resulting plasmid had a 3.2 kbp DNA fragment, a part of pTH1, inserted into the SmaI restriction enzyme site of the pCAMO-1 plasmid. Because this 3.2 kbp DNA fragment contains a replication origin that functions in Hydrogenophilus thermorteolus cells, the resulting plasmid replicates in Hydrogenophilus thermorteolus cells. The constructed plasmid was designated pCAMO-4.

[0062] (1-7) Construction of plasmid vector (pCAMO-5) A mixture of the following pairs of oligonucleotides in equimolar amounts was slowly cooled from 98°C to 20°C to prepare a double-stranded DNA fragment to which the oligonucleotides were annealed. Both ends of this DNA fragment were equivalent to the protruding ends produced 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 designated 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) (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. , ren To amplify the ligated DNA fragment, PCR was carried out using primers (a-9) and (b-10). The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 0.7 kbp corresponding to the DNA formed by ligating the mazF and mazE DNA fragments was detected and recovered from the gel.

[0066] The prepared DNA fragment of about 0.7 kbp and the plasmid pCAMO-5 were each digested with the restriction enzyme SfiI and ligated to each other using T4 DNA ligase. The constructed plasmid was designated pCAMO-6.

[0067] (2) Construction of gene expression plasmids Gene expression plasmids were constructed by Gibson Assembly. DNA fragments of the genes 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) contain 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 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 crotyl alcohol dehydrogenase gene (SEQ ID NO: 17) of Fonticella tunisiensis (a-19) 5'-GATCTGGAGGAGAAACGCATATGAGGGTTGCAGAACCTATAGGA-3' (SEQ ID NO: 55) (b-19) 5'-TGTCGACGGAGCTCGAATTCTTATTTATTATTTTCTTCAGTTTC-3' (SEQ ID NO: 56) Primers (a-19) and (b-19) contain sequences homologous to the vector pCAMO-6. The resulting reaction mixture was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 1.0 kbp was detected for the crotyl alcohol dehydrogenase gene, and a DNA fragment of approximately 2.7 kbp was detected 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 by Gibson Assembly, the plasmid vector pCAMO-6 was amplified by PCR using the following primers: Primers for amplifying the 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 recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0070] Using Gibson Assembly Master Mix (New England Biolabs), the DNA fragments of the vector pCAMO-6 synthesized above and the DNA fragments of each dehydrogenase gene were ligated together. The resulting reaction mixture was used to transform Escherichia coli JM109 strain by the heat shock method, and the transformed transformant was plated on LB medium containing 50 μg / mL of kanamycin and cultured at 37° C. for 24 hours. Each strain growing on LB medium was inoculated using a platinum loop into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL of kanamycin, and cultured with shaking at 37°C. Plasmid DNA was extracted from the culture medium. The base sequence of the gene inserted into each plasmid was analyzed by the Sanger method at Eurofins Genomics, and it was confirmed to match the sequence in the database.

[0071] (3) Transformants of Hydrogenophilus thermorteolus (3-1) Gene transfer The resulting plasmid was used to transform the Hydrogenophilus thermorteolus TH-1 strain by electroporation, and the transformed strain was seeded 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 fragment of each plasmid was confirmed by PCR for each strain grown on LB solid medium. As a result, amplification of DNA fragments of lengths corresponding to the respective genes was confirmed. The Hydrogenophilus thermorteolus TH-1 transformants transformed with the plasmids containing the genes derived from each bacterium were named as shown in Table 2.

[0072] [Table 2]

[0073] (3-2) Measurement of crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity of the enzymes produced by the transformants The CA-1 transformant of Hydrogenophilus thermorteolus 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 culture was incubated with shaking for 24 hours. Bacteria were harvested from 2 mL of the culture 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. Reaction buffer was added to the crude enzyme solution, and 10 mM NADH as a coenzyme and 1 mM crotonyl-CoA as a substrate were mixed, followed by a reaction 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] The chromatogram obtained for the reaction product of crotonyl-CoA with the crude enzyme solution produced by the transformant strain CA-1 is shown in Figure 2. Here, NC represents the negative control strain transformed with an empty vector, and Std represents the respective standards (1 mM) of crotyl alcohol and crotonaldehyde. 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 that a bifunctional enzyme with crotonaldehyde dehydrogenase activity and crotyl alcohol dehydrogenase activity was produced. Thus, the Hydrogenophilus thermorteolus CA-1 strain produced functional crotonaldehyde-crotyl alcohol dehydrogenase by growing using carbon dioxide as the sole carbon source, demonstrating that it can 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 transformant The cell lysates 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 coenzyme, and 10 mM crotonaldehyde as substrate, and the reaction was carried out at 52°C for 15 minutes. The reaction mixture was analyzed by gas chromatography (Shimadzu 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] The 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 standards (1 mM) of crotyl alcohol and crotonaldehyde. The crude enzyme produced by the transformant in which the DNA of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17 was introduced into the Hydrogenophilus thermoruteolus 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) Crotyl alcohol production 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 culture was incubated with shaking at 52°C for 24 hours. After incubation, the culture supernatant was collected 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. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [Industrial Applicability]

[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 with crotonaldehyde dehydrogenase and crotyl alcohol dehydrogenase activities (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 selected from the group consisting of the following DNAs (a), (b), (c), (d), and (e): (a) DNA containing the nucleotide sequence of SEQ ID NO: 1 (b) DNA containing 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) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (d) DNA encoding a polypeptide comprising 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) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 80 amino acids are 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. 3. The transformant according to claim 1, wherein the gene encoding the enzyme having crotyl alcohol dehydrogenase activity is a DNA selected from the group consisting of (f), (g), (h), (i), and (j): (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 comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18 and having crotyl alcohol dehydrogenase activity. (j) DNA encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18 in which 1 to 30 amino acids have been deleted, substituted, inserted, or added, and having crotyl alcohol dehydrogenase activity.

4. 3. The transformant according to claim 1, wherein the bacterium of the genus Hydrogenophilus is Hydrogenophilus thermorteolus.

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

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