Crotonaldehyde-crotyl alcohol dehydrogenase mutant

Abstract

In the present invention, bacteria into which has been introduced a gene encoding a crotonaldehyde-crotyl alcohol dehydrogenase mutant of (1) or (2) below can efficiently produce crotyl alcohol, which serves as a raw material for butadiene production. (1) DNA having the base sequence of SEQ ID NO: 1 comprising a base sequence in which the isoleucine at amino acid position 403 of SEQ ID NO: 2, which is encoded by the base sequence of SEQ ID NO: 1, is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid. (2) DNA encoding the polypeptide of (2A) or (2B) below (where, in the amino acid sequence thereof, the amino acid corresponding to amino acid position 403 of SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid). (2A) A polypeptide: comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2, where the isoleucine at amino acid position 403 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid; and having crotonaldehyde-crotyl alcohol dehydrogenase activity. (2B) A polypeptide: comprising an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 2 where the isoleucine at amino acid position 403 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid, 1-80 amino acids are deleted, substituted, inserted, or added; and having crotonaldehyde-crotyl alcohol dehydrogenase activity.

Description

Crotonaldehyde-crotyl alcohol dehydrogenase mutants

[0001] The present invention relates to a crotonaldehyde-crotyl alcohol dehydrogenase mutant having an improved ability to produce crotyl alcohol from crotonyl-CoA, a gene encoding the mutant, a transformant into which the gene has been introduced, 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] Currently, most butadiene is produced using petrochemical technology, necessitating a shift away from petroleum and toward sustainability. Biobutadiene, a type of butadiene produced from biologically derived materials, has been attracting attention. The synthesis of butadiene from ethanol has been known since the 1940s. However, with the development of butadiene extractive distillation technology for C4 fractions obtained by steam cracking naphtha, this method lost its competitiveness and fell into widespread use. However, in recent years, the production of butadiene from biologically derived bioethanol has once again attracted attention. Recently, a four-stage continuous biobutadiene production technology has been reported (Non-Patent Document 1). The four-stage continuous process involves a first-stage reaction in which ethanol is dehydrogenated to produce acetaldehyde; a second-stage reaction in which acetaldehyde is condensed to produce crotonaldehyde; a third-stage reaction in which crotonaldehyde is selectively hydrogenated to produce crotyl alcohol; and a fourth-stage reaction in which crotyl alcohol is dehydrated to produce butadiene. Although bioethanol is also used as the starting material for the four-stage continuous process, this method does not sufficiently reduce the environmental impact because the conversion of ethanol to butadiene is carried out through a chemical reaction.

[0007] Therefore, attempts have been made to develop technologies for producing butadiene using microorganisms. As shown in Figure 1, many microorganisms have a metabolic pathway that produces butadiene from pyruvate via crotonyl-CoA and crotyl alcohol (crotonyl alcohol). However, it is usually not possible to produce butadiene on an industrial scale. 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 a transformant of Escherichia coli and a step of chemically converting this crotyl alcohol into butadiene (Claim 1). The transformant used here has introduced therein exogenous genes encoding the following enzymes (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 (Figure 1, sections 2 to 3), acetoacetyl-CoA reductase (Figure 1, sections 3 to 4), and 3-hydroxybutyryl-CoA dehydratase (Figure 1, sections 4 to 5). 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 a complex process to convert biomass into sugars, which makes it expensive, 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 contacting a transformant obtained by introducing an exogenous gene encoding the following enzyme (A) or (B) into bacteria of the genus Burkholderia, Propionibacterium, Propionispira, Clostridium, Bacillus, Escherichia, Pelobacter, or Lactobacillus 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 by using a transformant obtained by introducing into a host, which is a methylotrophic strain such as a methanol-assimilating yeast, a gene encoding an enzyme that converts methanol and / or formic acid to formaldehyde and a gene that imparts formaldehyde fixation ability, as well as a gene encoding at least one enzyme selected from a gene encoding acetoacetyl-CoA thiolase (see FIGS. 2 and 3 in FIG. 1), a gene encoding 3-hydroxybutyryl-CoA dehydrogenase (see FIGS. 3 and 4 in FIG. 1), a gene encoding 3-hydroxybutyryl-CoA dehydratase (see FIGS. 4 and 5 in FIG. 1), a gene encoding crotonyl-CoA hydrolase (see FIGS. 5 and 8 in FIG. 1), a crotonate reductase (see FIGS. 8 and 7 in FIG. 1), and an alcohol-forming crotonaldehyde reductase (see FIGS. 7 and 6 in FIG. 1) (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] Journal of the Japan Petroleum Institute, 63, (2), 70-78(2020)

[0013] An objective of the present invention is to provide a crotonaldehyde-crotyl alcohol dehydrogenase mutant capable of efficiently producing crotyl alcohol, which is a raw material for butadiene production, a gene encoding the mutant, a transformant, and a method for producing crotyl alcohol.

[0014] The present inventors conducted extensive research to solve the above problems and discovered the following: (i) As mentioned above, many bacteria are inherently unable to produce crotyl alcohol on an industrial scale. By introducing 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, into bacteria (hereinafter, sometimes referred to as "crotonaldehyde-crotyl alcohol dehydrogenase." This enzyme is a type of aldehyde-alcohol dehydrogenase suitable for producing crotyl alcohol), the ability of the bacteria to produce crotyl alcohol can be improved.

[0015] (ii) The polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 derived from Fonticella tunisiensis is an enzyme with weak crotonaldehyde-crotyl alcohol dehydrogenase activity. The function of this enzyme was discovered for the first time in the present invention, and its function has not been reported. Because the activity of this enzyme is weak, improvement of its activity is necessary for its use in industrial crotyl alcohol production. Mutation is often used as a method for improving enzyme activity, but information necessary for determining the site of mutation introduction for this enzyme, such as the substrate or coenzyme binding site and binding mode, is not known.

[0016] (iii) To analyze information regarding substrate binding of this enzyme, the present inventors compared the amino acid sequences of this enzyme with those of aldehyde-alcohol dehydrogenase from Escherichia coli, which does not use crotonyl-CoA as a substrate but for which information on the active center is available based on the protein's three-dimensional structure analysis, and crotonaldehyde-crotyl alcohol dehydrogenase from Clostridium acetobutylicum ATCC 824, which has been reported to use crotonyl-CoA as a substrate (Microb Cell Fact (2018) 17:194). Specifically, based on information regarding the active center of E. coli aldehyde-alcohol dehydrogenase, the amino acids constituting the substrate binding site of each enzyme were predicted and the differences were confirmed. In addition, analysis taking into account the differences in activity of these three enzymes when crotonyl-CoA is used as a substrate revealed that the 403rd isoleucine (hereinafter referred to as "I403") of the aldehyde-alcohol dehydrogenase of Fonticella tunisiensis plays an important role in the crotonaldehyde-crotyl alcohol dehydrogenase activity when crotonyl-CoA is used as a substrate.

[0017] (iv) To verify this prediction, we constructed mutants of Fonticella tunisiensis crotonaldehyde-crotyl alcohol dehydrogenase in which I403 was replaced with 19 other amino acids. The wild-type crotonaldehyde-crotyl alcohol dehydrogenase from Fonticella tunisiensis expressed in Escherichia coli was compared with 19 mutant enzymes to compare their activity in producing crotyl alcohol from crotonyl-CoA. The crotyl alcohol production activity of 12 mutants, namely, serine mutant (I403S), glycine mutant (I403G), methionine mutant (I403M), alanine mutant (I403A), proline mutant (I403P), glutamine mutant (I403Q), phenylalanine mutant (I403F), leucine mutant (I403L), tryptophan mutant (I403W), lysine mutant (I403K), cysteine ​​mutant (I403C), and glutamic acid mutant (I403E), ranged from 3- to over 20-fold higher than that of the wild-type. This demonstrated that I403 plays an important role in crotonaldehyde-crotyl alcohol dehydrogenase activity.

[0018] (v) Genes encoding the eight most active mutants (serine mutant (I403S), glycine mutant (I403G), methionine mutant (I403M), alanine mutant (I403A), proline mutant (I403P), glutamine mutant (I403Q), phenylalanine mutant (I403F), and leucine mutant (I403L)) were introduced into Hydrogenophilus bacteria, along with a gene encoding wild-type crotonaldehyde-crotyl alcohol dehydrogenase from Fonticella tunisiensis. When these transformants were cultured using carbon dioxide as the sole carbon source, the mutant enzymes were expressed at levels comparable to those of the wild-type enzyme.

[0019] (vii) The crotonaldehyde-crotyl alcohol dehydrogenase activity of the cell lysates of a transformant producing wild-type crotonaldehyde-crotyl alcohol dehydrogenase from Fonticella tunisiensis and the eight transformants producing mutant crotonaldehyde-crotyl alcohol dehydrogenases was evaluated. Activity was evaluated by quantifying crotyl alcohol produced from the substrate crotonyl-CoA by gas chromatography. Crotyl alcohol production was below the detection limit with the wild-type enzyme, whereas it was detected with the eight mutant enzymes. Although it is difficult to induce the introduction of foreign genes into Hydrogenophilus bacteria to produce functional proteins, these mutant enzyme genes expressed functional proteins within Hydrogenophilus bacteria.

[0020] The present invention was completed based on the above findings and provides the following [1] to [7]. [1] A gene encoding a crotonaldehyde-crotyl alcohol dehydrogenase mutant of the following (1) or (2): (1) A DNA comprising a nucleotide sequence of SEQ ID NO: 1 having a nucleotide substitution in which isoleucine at amino acid number 403 of SEQ ID NO: 2, which it encodes, is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid. (2) A DNA encoding a polypeptide of the following (2A) or (2B): (2A) A polypeptide having crotonaldehyde-crotyl alcohol dehydrogenase activity, comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 in which isoleucine at amino acid number 403 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid (provided that in this amino acid sequence, the amino acid corresponding to amino acid number 403 of SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid). (2B) 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, in which isoleucine at amino acid number 403 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid, and which has crotonaldehyde-crotyl alcohol dehydrogenase activity (provided that in this amino acid sequence, the amino acid corresponding to amino acid number 403 in SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid).) [2] (1a) The above (1) is a DNA containing the following nucleotide sequences: a nucleotide sequence in which the ATA at nucleotide numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with TCT, TCC, TCA, TCG, AGT, or AGC; a nucleotide sequence in which the ATA at nucleotide numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with GGT, GGC, GGA, or GGG; a nucleotide sequence in which the ATA at nucleotide numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with ATG; a nucleotide sequence in which the ATA at nucleotide numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with GCT, GCC, GCA, or GCG; a nucleotide sequence in which the ATA at nucleotide numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with CCT, CCC, CCA, or CCG; a nucleotide sequence in which the ATA at nucleotide numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with CAA or CAG; A base sequence in which the ATA at bases 1207 to 1209 of SEQ ID NO: 1 is substituted with TTT or TTC, a base sequence in which the ATA at bases 1207 to 1209 of SEQ ID NO: 1 is substituted with TTA, TTG, CTT, CTC, CTA, or CTG, a base sequence in which the ATA at bases 1207 to 1209 of SEQ ID NO: 1 is substituted with TGG, a base sequence in which the ATA at bases 1207 to 1209 of SEQ ID NO: 1 is substituted with AAA or AAG, a base sequence in which the ATA at bases 1207 to 1209 of SEQ ID NO: 1 is substituted with TGT or TGC, or a base sequence in which the ATA at bases 1207 to 1209 of SEQ ID NO: 1 is substituted with GAA or GAG. The gene according to claim 1, wherein the (2) is a DNA comprising a nucleotide sequence having 90% or more identity with any of the nucleotide sequences described in the (1a) above and encoding a polypeptide having crotonaldehyde-crotyl alcohol dehydrogenase activity (provided that this nucleotide sequence is a nucleotide sequence in which bases other than 1207 to 1209 of the nucleotide sequence of the (1a) above have been deleted, substituted, inserted, or added). [3] The gene according to [1] or [2], which is the DNA of the following (A) or (B):(A) DNA comprising any of the nucleotide sequences of SEQ ID NOs: 3 to 14. (B) DNA comprising a DNA having a nucleotide sequence with 90% or more identity to SEQ ID NOs: 3 to 14 and encoding a polypeptide having crotonaldehyde-crotyl alcohol dehydrogenase activity (however, this nucleotide sequence is a nucleotide sequence in which bases other than 1207 to 1209 of SEQ ID NOs: 3 to 10 have been deleted, substituted, inserted, or added). [4] A crotonaldehyde-crotyl alcohol dehydrogenase mutant of (3) or (4) below. (3) A polypeptide comprising the amino acid sequence of SEQ ID NO: 2 in which isoleucine at amino acid number 403 has been substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid. (4) A polypeptide of (4A) or (4B) below. (4A) A polypeptide comprising an amino acid sequence having 90% or more identity to an amino acid sequence in which the isoleucine at amino acid number 403 in SEQ ID NO: 2 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid, and having crotonaldehyde-crotyl alcohol dehydrogenase activity (provided that in this amino acid sequence, the amino acid corresponding to amino acid number 403 in SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid).) (4B) 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, in which amino acid 403, isoleucine, is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine ​​or glutamic acid, and having crotonaldehyde-crotyl alcohol dehydrogenase activity (provided that in this amino acid sequence, the amino acid corresponding to amino acid 403 in SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine ​​or glutamic acid). [5] A transformant obtainable by introducing a gene according to any one of [1] to [3] into a bacterium. [6] The transformant according to [5], wherein the bacterium is a hydrogen bacterium. [7] A method for producing crotyl alcohol, comprising a step of culturing the transformant according to [5] or [6].

[0021] A four-stage continuous process for butadiene production that has been reported in recent years is a method for producing butadiene from ethanol via acetaldehyde, crotonaldehyde, and crotyl alcohol. Even if bioethanol is used, each step from ethanol to butadiene is performed by chemical synthesis, so the environmental burden is not sufficiently reduced. Therefore, there is a demand for all or part of this process to be performed by bioprocesses.

[0022] The crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention produces crotyl alcohol with high efficiency. Although bacteria generally do not produce crotyl alcohol on an industrial scale, by introducing a gene encoding the crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention into bacteria and culturing the resulting transformant, crotyl alcohol can be produced efficiently with little environmental impact. Converting the biocrotyl alcohol obtained in this way into butadiene in a single-step reaction reduces the use of chemical raw materials, thereby achieving sustainable materials development. Furthermore, it is also possible to produce biobutadiene from biocrotyl alcohol using microorganisms. Crotyl alcohol can also be produced efficiently with little environmental impact by reacting the crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention with crotonyl-CoA in vitro.

[0023] 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.

[0024] 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."

[0025] Hydrogenophilus bacteria naturally possess the gene for the enzyme that catalyzes the reaction that produces crotonyl-CoA from pyruvate, but do not possess the gene for the enzyme that catalyzes the reaction that produces crotonyl-CoA 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 Hydrogenophilus bacteria. However, the gene for the crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention is expressed in Hydrogenophilus bacteria and produces an enzyme that functions as crotonaldehyde-crotyl alcohol dehydrogenase. Therefore, transformants of Hydrogenophilus bacteria into which the mutant gene of the present invention has been introduced grow rapidly using carbon dioxide as the sole carbon source and can produce biocrotyl alcohol with high efficiency.

[0026] The mutant of the present invention can produce crotyl alcohol more efficiently than the wild-type enzyme. In particular, when a Hydrogenophilus bacterium is used as a host, the production rate of crotyl alcohol is dramatically improved and crotyl alcohol can be produced by fixing carbon dioxide, enabling industrial production of crotyl alcohol while reducing the environmental load.

[0027] This figure shows the pathway for producing butadiene from pyruvate via crotonyl-CoA, which is found in many microorganisms. This figure shows the results of comparing the activity of wild-type Fonticella tunisiensis-derived crotonaldehyde-crotyl alcohol dehydrogenase expressed in Escherichia coli with 19 mutant enzymes for producing crotyl alcohol from crotonyl-CoA. The peak area of ​​crotyl alcohol obtained using the wild-type enzyme is set to 1, and the peak area of ​​crotyl alcohol obtained using each mutant enzyme is expressed as a relative value. (A) is a gel photograph showing the results of polyacrylamide gel electrophoresis of proteins contained in a culture of a transformant obtained by introducing the wild-type Fonticella tunisiensis-derived crotonaldehyde-crotyl alcohol dehydrogenase gene and each of the eight mutant enzyme genes into Hydrogenophilus thermorteolus. (B) is a gas chromatography chromatogram showing that the crotonaldehyde-crotyl alcohol dehydrogenase mutant produced by Hydrogenophilus thermorteolus produced crotyl alcohol using crotonyl-CoA as a substrate.

[0028] The present invention is described in detail below. (1) Crotonaldehyde-crotyl alcohol dehydrogenase mutant The crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention is a polypeptide or a homolog thereof, which comprises an amino acid sequence in which isoleucine (Ile) at amino acid number 403 in the amino acid sequence of SEQ ID NO: 2 is substituted with serine (Ser), glycine (Gly), methionine (Met), alanine (Ala), proline (Pro), glutamine (Gln), phenylalanine (Phe), leucine (Leu), tryptophan (Trp), lysine (Lys), cysteine ​​(Cys), or glutamic acid (Glu) (particularly, the amino acid sequence in which Ile at amino acid number 403 in the amino acid sequence of SEQ ID NO: 2 is substituted with Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu). However, in the homolog, the amino acid corresponding to or equivalent to amino acid number 403 of SEQ ID NO: 2 is Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu. SEQ ID NO: 2 is the amino acid sequence of wild-type crotonaldehyde-crotyl alcohol dehydrogenase from Fonticella tunisiensis.

[0029] Examples of homologs include amino acid sequences that have 90% or more, preferably 95% or more, preferably 98% or more, and particularly 99% or more identity to the amino acid sequence of SEQ ID NO: 2 in which Ile at amino acid 403 is substituted with Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu (particularly, amino acid sequences that have 90% or more, preferably 95% or more, preferably 98% or more, and particularly 99% or more identity to the amino acid sequence of SEQ ID NO: 2 in which Ile at amino acid 403 is substituted with Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu), and polypeptides that have crotonaldehyde-crotyl alcohol dehydrogenase activity.

[0030] Furthermore, as a homolog, in the amino acid sequence of SEQ ID NO: 2, Ile at amino acid number 403 is replaced with Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu. and a polypeptide having crotonaldehyde-crotyl alcohol dehydrogenase activity.

[0031] However, in these homologs, the amino acid corresponding to or equivalent to amino acid number 403 in SEQ ID NO: 2 is Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu, respectively. That is, these homologs have an amino acid sequence in which Ile at amino acid number 403 in SEQ ID NO: 2 is substituted with Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu, and have been modified (deleted, substituted, inserted, or added) with respect to amino acids other than the amino acid resulting from the substitution.

[0032] 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.

[0033] (2) Crotonaldehyde-crotyl alcohol dehydrogenase gene mutant The gene encoding the crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention is a DNA comprising a base sequence of SEQ ID NO: 1 having a base substitution in which Ile at amino acid number 403 of SEQ ID NO: 2, which it encodes, is substituted with Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu (particularly, consisting of a base sequence of SEQ ID NO: 1 having a base substitution in which Ile at amino acid number 403 of SEQ ID NO: 2, which it encodes, is substituted with Ser, Gly, Met, Ala, Pro, Gln, Phe, Leu, Trp, Lys, Cys, or Glu), or a homologous DNA thereof. However, in these homolog DNAs, the trinucleotide encoding the amino acid corresponding to or equivalent to amino acid 403 of SEQ ID NO: 2 in the amino acid sequence encoded by the homolog DNA is a trinucleotide encoding Met, Ser, Gly, Ala, Pro, Glu, Phe, Leu, Trp, Lys, Cys, or Glu. SEQ ID NO: 1 is a DNA encoding a wild-type crotonaldehyde-crotyl alcohol dehydrogenase derived from Fonticella tunisiensis.

[0034] In the nucleotide sequence of SEQ ID NO: 1, an example of a nucleotide sequence having a substitution of Ile with Ser at amino acid 403 in the nucleotide sequence of SEQ ID NO: 2 that it encodes includes a nucleotide sequence in which ATA at nucleotides 1207 to 1209 in SEQ ID NO: 1 is substituted with TCT, TCC, TCA, TCG, AGT, or AGC (these may be referred to as "nucleotide sequence I403S"). Of these, a preferred nucleotide sequence is SEQ ID NO: 3, in which ATA at nucleotides 1207 to 1209 in SEQ ID NO: 1 is substituted with TCG. In the nucleotide sequence of SEQ ID NO: 1, an example of a nucleotide sequence having a substitution of Ile with Gly at amino acid 403 in the nucleotide sequence of SEQ ID NO: 2 that it encodes includes a nucleotide sequence in which ATA at nucleotides 1207 to 1209 in SEQ ID NO: 1 is substituted with GGT, GGC, GGA, or GGG (these may be referred to as "nucleotide sequence I403G"). Of these, a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with GGC (SEQ ID NO: 4) is preferred. An example of a nucleotide sequence in SEQ ID NO: 1 that has a substitution of Ile at amino acid 403 of SEQ ID NO: 2, which it encodes, with Met is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with ATG (these may be referred to as "nucleotide sequence I403M"). Of these, a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with ATG (SEQ ID NO: 5) is preferred. An example of a nucleotide sequence in which Ile at amino acid 403 of SEQ ID NO: 2, which it encodes, is substituted with Ala is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with GCT, GCC, GCA, or GCG (these may be referred to as "nucleotide sequence I403A"). Of these, a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with GCG (SEQ ID NO: 6) is preferred. Examples of the nucleotide sequence in SEQ ID NO: 1 having a nucleotide substitution in which Ile at amino acid 403 of SEQ ID NO: 2, which it encodes, is substituted with Pro include nucleotide sequences in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with CCT, CCC, CCA, or CCG (these may be referred to as "nucleotide sequence I403P").Of these, a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with CCG (SEQ ID NO: 7) is preferred. An example of a nucleotide sequence in which Ile at amino acid 403 of SEQ ID NO: 2, which it encodes, is substituted with Gln, is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with CAA or CAG (these may be referred to as "nucleotide sequence I403Q"). Of these, a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with CAG (SEQ ID NO: 8) is preferred. An example of a nucleotide sequence in which Ile at amino acid 403 of SEQ ID NO: 2, which it encodes, is substituted with Phe, is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with TTT or TTC (these may be referred to as "nucleotide sequence I403F"). Of these, a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with TTC (SEQ ID NO: 9) is preferred. An example of a nucleotide sequence in which Ile at amino acid 403 of SEQ ID NO: 2, which it encodes, is substituted with Leu, is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with TTA, TTG, CTT, CTC, CTA, or CTG (these may be referred to as "nucleotide sequence I403L"). Of these, a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with CTC (SEQ ID NO: 10) is preferred. An example of a nucleotide sequence in which Ile at amino acid 403 of SEQ ID NO: 2, which it encodes, is substituted with Trp, is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 of SEQ ID NO: 1 is substituted with TGG (these may be referred to as "nucleotide sequence I403W"). Among these, the base sequence (SEQ ID NO: 11) in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with TGG is preferred.An example of a nucleotide sequence of SEQ ID NO: 1 having a nucleotide substitution in which Ile at amino acid 403 in the encoded SEQ ID NO: 2 is substituted with Lys is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 in SEQ ID NO: 1 is substituted with AAA or AAG (these may be referred to as "nucleotide sequence I403K"). Of these, a preferred nucleotide sequence is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 in SEQ ID NO: 1 is substituted with AAA (SEQ ID NO: 12). An example of a nucleotide sequence of SEQ ID NO: 1 having a nucleotide substitution in which Ile at amino acid 403 in the encoded SEQ ID NO: 2 is substituted with Cys is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 in SEQ ID NO: 1 is substituted with TGT or TGC (these may be referred to as "nucleotide sequence I403C"). Of these, a preferred nucleotide sequence is a nucleotide sequence in which ATA at nucleotides 1207 to 1209 in SEQ ID NO: 1 is substituted with TGC (SEQ ID NO: 13). An example of the nucleotide sequence of SEQ ID NO: 1 having a nucleotide substitution in which Ile at amino acid number 403 in SEQ ID NO: 2, which it encodes, is a nucleotide sequence in which ATA at nucleotide numbers 1207 to 1209 in SEQ ID NO: 1 is substituted with GAA or GAG (these may be referred to as "nucleotide sequence I403E"). Of these, the nucleotide sequence in which ATA at nucleotide numbers 1207 to 1209 in SEQ ID NO: 1 is substituted with GAA (SEQ ID NO: 14) is preferred.

[0035] The homologous DNA is a DNA encoding a homolog of the crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention described above. Examples of the homologous DNA include a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity with the nucleotide sequence I403S, the nucleotide sequence I403G, the nucleotide sequence I403M, the nucleotide sequence I403A, the nucleotide sequence I403P, the nucleotide sequence I403Q, the nucleotide sequence I403F, the nucleotide sequence I403L, the nucleotide sequence I403W, the nucleotide sequence I403K, the nucleotide sequence I403C, or the nucleotide sequence I403E (particularly, the nucleotide sequence I403S, the nucleotide sequence I403G, the nucleotide sequence I403M, the nucleotide sequence I403A, the nucleotide sequence I403P, the nucleotide sequence I403Q, the nucleotide sequence I403F, the nucleotide sequence I403L, the nucleotide sequence I403W, the nucleotide sequence I403K, the nucleotide sequence I403C, or the nucleotide sequence I403E). and a DNA encoding a polypeptide having crotonaldehyde-crotyl alcohol dehydrogenase activity, the DNA comprising a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence I403M, nucleotide sequence I403A, nucleotide sequence I403P, nucleotide sequence I403Q, nucleotide sequence I403F, nucleotide sequence I403L, nucleotide sequence I403W, nucleotide sequence I403K, nucleotide sequence I403C, or nucleotide sequence I403E. However, homologous DNA includes a nucleotide sequence in which a change (deletion, substitution, insertion, or addition) has been made to a base other than base numbers 1207 to 1209 of the above nucleotide sequence I403S, nucleotide sequence I403G, nucleotide sequence I403M, nucleotide sequence I403A, nucleotide sequence I403P, nucleotide sequence I403Q, nucleotide sequence I403F, nucleotide sequence I403L, nucleotide sequence I403W, nucleotide sequence I403K, nucleotide sequence I403C, or nucleotide sequence I403E.

[0036] In the present invention, the identities of base sequences and amino acid sequences are values ​​calculated using GENETYX ver. 17 (GENETYX).

[0037] The crotonaldehyde-crotyl alcohol dehydrogenase mutants of the present invention and the genes encoding the crotonaldehyde-crotyl alcohol dehydrogenase mutants may be naturally occurring mutants that have arisen, for example, through spontaneous mutation, or may be artificially synthesized using techniques known to those skilled in the art, or may be obtained by site-specific mutation of natural DNA using PCR technology, or by treatment with a mutagen.

[0038] (2) Transformant Ability to Produce Crotyl Alcohol The transformant (transformed cell) of the present invention is a transformant obtained by introducing into a host bacterium a gene encoding the above-described crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention. In other words, the transformant of the present invention has an exogenous gene encoding the crotonaldehyde-crotyl alcohol dehydrogenase mutant of the present invention. The transformant of the present invention may be one into which one or more mutants of the gene encoding the crotonaldehyde-crotyl alcohol dehydrogenase of the present invention have been introduced. Furthermore, the transformant may be one into which other genes have been introduced in addition to the mutant of the gene encoding the crotonaldehyde-crotyl alcohol dehydrogenase of the present invention.

[0039] Host bacteria include, but are not limited to, Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Clostridium thermocellum, etc. Furthermore, when hydrogen bacteria are used as a host, crotyl alcohol can be produced using carbon dioxide as the sole carbon source. Examples of hydrogen bacteria include Pseudonocardia bacteria such as Pseudonocardia autotrophica; Hydrogenobacter bacteria such as Hydrogenobacter hydrogenophilus; Hydrogenophilus thermoluteolus, Hydrogenophilus thiooxidans, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038.Hydrogenophilus bacteria such as Paracoccus denitrificans and Paracoccus pantotrophus; Xanthobacter bacteria such as Xanthobacter autotrophicus; Cupriavidus bacteria such as Cupriavidus basilensis, Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus pauculus, and Cupriavidus taiwanensis; Hydrogenophaga pseudoflava Examples include Hydrogenophaga bacteria such as Hydrogenophaga pseudoflava; and Variovorax bacteria such as Variovorax paradoxus.

[0040] Among these, hydrogenophilus bacteria are preferred, with hydrogenophilus thermorteolus being more preferred, due to their top-level growth rate and carbon dioxide fixation ability among carbon dioxide fixating microorganisms. 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 hydrogenophilus thermorteolus TH-1 (NBRC 14978) strain exhibits the highest growth rate among carbon dioxide fixating microorganisms [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.

[0041] The host bacterium may be a bacterium isolated from nature, or a bacterium that has been genetically modified from a bacterium isolated from nature. Modifications can be performed for purposes such as enabling high expression of introduced genes. Such modifications can be achieved, for example, by removing (curing) endogenous plasmids in the bacterium or by disrupting genes on the bacterial genome. 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.

[0042] Method for Preparing Transformants A method for obtaining transformants by introducing the mutant genes of the present invention into host bacteria is described below. Introduction of the mutant genes into host bacteria can be carried out using standard methods for introducing foreign genes into bacteria. These genes may be introduced directly into the host bacterium, or a vector incorporating these genes into a transformation vector (e.g., a plasmid vector, a viral vector, a cosmid, a fosmid, a BAC, etc.) may be introduced. The vector for transformation may contain DNA capable of autonomous replication in the host bacterium. 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 based on its nucleotide sequence (SEQ ID NO: 15). 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 host bacteria using conventional methods, such as the calcium chloride method, the rubidium chloride method, and the electric pulse method (electroporation).

[0043] (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, particularly a step of culturing the transformant of the present invention in an inorganic or organic medium while supplying a gas containing carbon dioxide, 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.

[0044] 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" includes cases where unavoidable amounts of other carbon sources are mixed in.

[0045] 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 the culture vessel while being cultured by shaking, or the mixed gas can be introduced into the culture medium by bubbling, and the transformant can be cultured with stirring. The volume ratio of hydrogen, oxygen, and carbon dioxide (hydrogen:oxygen:carbon dioxide) in the supply gas is preferably 1.75 to 7.5:1:0.25 to 3, more preferably 5 to 7.5:1:1 to 2, and even more preferably 6.25 to 7.5:1:1.5. Within this range, bacterial growth is favorable and 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.

[0046] 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.

[0047] 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.

[0048] (1) Construction of Gene Expression Plasmids Gene expression plasmids were constructed using Gibson Assembly. First, a DNA fragment of the crotonaldehyde-crotyl alcohol dehydrogenase gene consisting of the base sequence of SEQ ID NO: 1 was amplified by PCR using the genomic DNA of Fonticella tunisiensis as a template. The following primers were used for PCR: (a-1) 5'-GATCTGGAGGAGAAACGCATATGAAAGTCACCAACGCGGAAG-3' (SEQ ID NO: 16) (a-2) 5'-TGTCGACGGAGCTCGAATTCTCATTTGTTGTTTTCTTCGGTTTC-3' (SEQ ID NO: 17). Primers (a-1) and (a-2) contained sequences homologous to the vector pCAMO-6. The resulting reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of approximately 2.7 kbp was detected. The agarose gel containing this DNA fragment was excised, and the DNA fragment was recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0049] To perform DNA ligation by Gibson Assembly, the plasmid vector pCAMO-6 was amplified by PCR using the DNA consisting of the base sequence of SEQ ID NO: 15 as a template. The following primers were used for PCR: Primers for amplification of plasmid vector pCAMO-6: (a-20) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 18) (b-20) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 19). 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).

[0050] The DNA fragment of the vector pCAMO-6 synthesized above and the DNA fragment of the crotonaldehyde-crotyl alcohol dehydrogenase gene were ligated using Gibson Assembly Master Mix (New England Biolabs). The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the resulting mixture was plated on LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C with shaking. Plasmid DNA was extracted from the culture (pCAMO-6-Fonti). The nucleotide sequence of the inserted gene was analyzed by Sanger analysis at Eurofins Genomics, and confirmed to match the sequence in the database. The constructed plasmid can replicate not only in Escherichia coli but also in the Hydrogenophilus thermorteolus (TH-1) NBRC 14978 strain.

[0051] The constructed plasmid (pCAMO-6-Fonti) containing the gene for the wild-type crotonaldehyde-crotyl alcohol dehydrogenase of Fonticella tunisiensis was used as a template. The primers used to mutate the 403rd amino acid, isoleucine (I403), of the enzyme to one of 19 other amino acids are shown in Tables 1A and 1B. *In Tables 1A and 1B, the "amino acid" indicates the amino acid generated by site-directed mutagenesis using the primer set to replace isoleucine at position 403 in the amino acid sequence of wild-type crotyl alcohol dehydrogenase.

[0052] Each amino acid mutation was performed using PCR-based site-directed mutagenesis. The template DNA was a plasmid (pCAMO-6-Fonti) carrying the wild-type crotyl alcohol dehydrogenase gene from Fonticella tunisiensis. PCR reactions were performed using each of the 19 primer combinations listed in Table 1 to amplify the full-length plasmid DNA. The PCR reaction mixture was digested with the restriction enzyme DpnI to remove the template plasmid DNA (37°C, 1 hour). A portion of the DpnI-digested plasmid solution was transformed into Escherichia coli JM109 by heat shock, and the resulting transformants were plated on LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into 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 then extracted from the culture medium. The base sequence of the gene inserted into each plasmid was analyzed by the Sanger method, which was entrusted to Eurofins Genomics, Inc., and it was confirmed that the desired amino acid mutations had occurred.

[0053] The resulting plasmid was used to transform Escherichia coli JM109 strain by electroporation, and the transformed strain was inoculated onto LB solid medium containing 50 μg / mL of kanamycin and cultured at 37° C. for 24 hours.

[0054] (2) Measurement of crotonaldehyde-crotyl alcohol dehydrogenase activity of the enzyme produced by E. coli transformants. The Escherichia coli transformants grown on LB solid medium were inoculated into LB solid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C for 24 hours with shaking. Bacterial cells were harvested from 2 mL of culture medium of each transformant by centrifugation (4°C, 5,000 g, 10 minutes). The cells were suspended in 300 μL of reaction buffer (100 mM Tris-HCl (pH 7.5), 5 μM FeSO4, 0.2 mM DTT), disrupted by sonication, and then centrifuged (4°C, 20,000 g, 10 minutes) to obtain the cell disruption supernatant. The cell lysate supernatant was also obtained from Escherichia coli JM109 strain transformed with pCAMO-6-Fonti, which contains the wild-type crotonaldehyde-crotyl alcohol dehydrogenase gene from Fonticella tunisiensis. The cell lysate supernatant was used as a crude enzyme solution to measure crotonaldehyde-crotyl alcohol dehydrogenase activity. The crude enzyme solution was mixed with reaction buffer, 10 mM NADH as coenzyme, and 4 mM crotonyl-CoA as substrate, and the reaction was carried out at 52°C for 30 minutes. The reaction solution was analyzed by gas chromatography (Shimadzu Corporation: GC-2014, Polar-WAX column). The crotyl alcohol produced was identified by detecting a peak with the same column retention time as the authentic sample. Figure 2 shows the relative crotyl alcohol peak area obtained with each mutant enzyme, with the crotyl alcohol peak area obtained with the wild-type enzyme set to 1. The crotyl alcohol-producing activity of 12 mutants, namely, serine mutant (I403S), glycine mutant (I403G), methionine mutant (I403M), alanine mutant (I403A), proline mutant (I403P), glutamine mutant (I403Q), phenylalanine mutant (I403F), leucine mutant (I403L), tryptophan mutant (I403W), lysine mutant (I403K), cysteine ​​mutant (I403C), and glutamic acid mutant (I403E), was improved by 3- to more than 20-fold compared to the wild type (Ile:WT).This demonstrated that I403 plays an important role in crotonaldehyde-crotyl alcohol dehydrogenase activity.

[0055] (3) Hydrogenophilus thermorteolus Transformants (3-1) Gene Introduction: Hydrogenophilus thermorteolus TH-1 strain was transformed by electroporation with the empty vector plasmid pCAMO-6, a plasmid expressing wild-type crotonaldehyde-crotyl alcohol dehydrogenase from Fonticella tunisiensis, or a mutant enzyme I403S, I403G, I403M, I403A, I403P, I403Q, I403F, or I403L. The transformed strains were then plated on 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 for each strain grown on LB solid medium by PCR. As a result, amplification of DNA fragments of lengths corresponding to each gene was observed.

[0056] (3-2) Evaluation of the crotyl alcohol-producing enzyme produced by the transformant. The transgenic strain of Hydrogenophilus thermorteolus prepared as described above was inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. 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 300 μL of reaction buffer (100 mM Tris-HCl (pH 8.5), 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.

[0057] The supernatant of the cell lysate was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). The gel photograph is shown in Figure 3(A). The approximately 95 kDa band indicated by the arrow in the figure is crotonaldehyde-crotyl alcohol dehydrogenase. The eight transformants into which the mutant gene had been introduced produced crotonaldehyde-crotyl alcohol dehydrogenase at levels comparable to those of the transformant into which the wild-type crotonaldehyde-crotyl alcohol dehydrogenase gene had been introduced.

[0058] The cell lysate supernatant was used as a crude enzyme solution to evaluate the crotyl alcohol production ability. 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). The produced crotyl alcohol was identified by detecting a peak with the same column retention time as the authentic sample.

[0059] Figure 3(B) shows the chromatograms obtained for the reaction products of crotonyl-CoA and the crude enzyme solution produced by each transformant. Here, NC denotes the negative control strain transformed with the empty vector pCAMO-6, and Std denotes 1 mM crotyl alcohol. The crude enzyme produced by the transformant, Hydrogenophilus thermorteolus TH-1, carrying the wild-type crotonaldehyde-crotyl alcohol dehydrogenase gene, did not produce crotyl alcohol using crotonyl-CoA as a substrate (below the detection limit). In contrast, the crude enzyme produced by the transformants carrying the eight mutant enzyme genes (I403S, I403G, I403M, I403A, I403P, I403Q, I403F, and I403L) produced crotyl alcohol using crotonyl-CoA as a substrate.

[0060] (3-3) Production of Crotyl Alcohol by Transformants The transformants producing the wild-type enzyme, the eight transformant strains producing the mutant enzymes, and the NC strain containing the empty vector were each inoculated into a medium containing 50 μg / mL of kanamycin and cultured with shaking while supplying a mixture of H2, O2, and CO2. After cultivation, the culture supernatant was obtained by centrifugation (4°C, 5,000 g, 10 minutes). Unlike the culture supernatants of the empty vector-introduced strain and the transformant strain producing wild-type crotonaldehyde-crotyl alcohol dehydrogenase, the culture supernatant of the transformant strain expressing the mutant enzyme contained crotyl alcohol.

[0061] 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.

[0062] SEQ ID NOs: 1 to 15 are shown below.

[0063] The mutant crotonaldehyde-crotyl alcohol dehydrogenase 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, and therefore contributes to the sustainability of chemical production.

Claims

1. A gene encoding a crotonaldehyde - crotyl alcohol dehydrogenase variant of the following (1) or (2): (1) DNA containing a base sequence having a base substitution in which isoleucine at amino acid number 403 of SEQ ID NO: 2 encoded by it is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid in the base sequence of SEQ ID NO: 1 (2) DNA encoding a polypeptide of the following (2A) or (2B): (2A) A polypeptide having an amino acid sequence having 90% or more identity with an amino acid sequence in which isoleucine at amino acid number 403 of SEQ ID NO: 2 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid, and having crotonaldehyde - crotyl alcohol dehydrogenase activity (however, in this amino acid sequence, the amino acid corresponding to amino acid number 403 of SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid.) (2B) A polypeptide having an amino acid sequence in which 1 to 80 amino acids are deleted, substituted, inserted, or added in an amino acid sequence in which isoleucine at amino acid number 403 of SEQ ID NO: 2 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid, and having crotonaldehyde - crotyl alcohol dehydrogenase activity (however, in this amino acid sequence, the amino acid corresponding to amino acid number 403 of SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid.) 2. (1a) The DNA described in (1) above contains the following base sequence, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with TCT, TCC, TCA, TCG, AGT, or AGC, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with GGT, GGC, GGA, or GGG, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with ATG, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with GCT, GCC, GCA, or GCG, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with CCT, CCC, CCA, or CCG, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with CAA or CAG, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with TTT or TTC, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with TTA, TTG, CTT, CTC, CTA, or CTG, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with TGG, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with AAA or AAG, a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with TGT or TGC, or a base sequence in which ATA at base numbers 1207 to 1209 of SEQ ID NO: 1 is substituted with GAA or GAG. (2) above contains a base sequence having 90% or more identity with any of the base sequences described in (1a) above and encodes a polypeptide having crotonaldehyde-crotyl alcohol dehydrogenase activity (provided that this base sequence is a base sequence in which deletions, substitutions, insertions, or additions have been made to bases other than base numbers 1207 to 1209 of the base sequence of (1a) above). The gene according to claim 1.

3. The gene according to claim 1 or 2, which is the DNA of the following (A) or (B). (A) DNA containing any of the base sequences of SEQ ID NOs: 3 to 14. (B) DNA containing a DNA having a base sequence having 90% or more identity with SEQ ID NOs: 3 to 14 and encoding a polypeptide having crotonaldehyde-crotyl alcohol dehydrogenase activity (however, this base sequence is a base sequence obtained by deletion, substitution, insertion, or addition to bases other than base numbers 1207 to 1209 of SEQ ID NOs: 3 to 10).

4. The crotonaldehyde - crotyl alcohol dehydrogenase variant of the following (3) or (4). (3) A polypeptide comprising an amino acid sequence in which isoleucine at amino acid number 403 in the amino acid sequence of SEQ ID NO: 2 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid. (4) The polypeptide of the following (4A) or (4B). (4A) A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence in which isoleucine at amino acid number 403 in the amino acid sequence of SEQ ID NO: 2 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid, and having crotonaldehyde - crotyl alcohol dehydrogenase activity (however, in this amino acid sequence, the amino acid corresponding to amino acid number 403 of SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid). (4B) 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 in which isoleucine at amino acid number 403 in the amino acid sequence of SEQ ID NO: 2 is substituted with serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid, and having crotonaldehyde - crotyl alcohol dehydrogenase activity (however, in this amino acid sequence, the amino acid corresponding to amino acid number 403 of SEQ ID NO: 2 is serine, glycine, methionine, alanine, proline, glutamine, phenylalanine, leucine, tryptophan, lysine, cysteine, or glutamic acid).

5. A transformant obtained by introducing the gene according to any one of claims 1 to 3 into a bacterium.

6. The transformant according to claim 5, wherein the bacterium is a hydrogen bacterium.

7. A method for producing crotyl alcohol, comprising the step of culturing the transformant according to claim 5 or 6.

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