Hydrogenophilus bacterium mutant having improved crotonyl-coa production capability

Abstract

Provided is a Hydrogenophilus bacterium mutant in which at least one of the below-mentioned enzyme genes (a) to (d) is disrupted, wherein the enzyme genes are located on the chromosome of a Hydrogenophilus bacterium and catalyze any one of reactions in the pathway for generating 3-hydroxybutyryl-CoA from crotonyl-CoA. The mutant is improved in terms of crotonyl-CoA production capability and can therefore be used for the efficient production of useful substances generated via crotonyl-CoA. (a) A gene that encodes a polypeptide having a 3-hydroxybutyryl-CoA dehydratase activity; (b) a gene that encodes a polypeptide having an enoyl-CoA hydratase activity; (c) a gene that encodes a polypeptide having a 3-hydroxyacyl-CoA dehydrogenase activity; and (d) a gene that encodes a polypeptide having a methylglutaconyl-CoA hydratase activity.

Description

Hydrogenophilus bacterium mutant with improved crotonyl-CoA production ability

[0001] The present invention relates to a mutant Hydrogenophilus bacterium that has an improved ability to produce crotonyl-CoA and, therefore, is able to improve its ability to produce various useful substances produced by metabolism of crotonyl-CoA.

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

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

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

[0005] Examples of useful chemical substances produced via crotonyl CoA include crotyl alcohol, butadiene, and butanol.

[0006] 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 chemicals such as adiponitrile, 1,4-butanediol, cyclododecatriene, chloroprene, and sulfolane.

[0007] Since most butadiene currently produced is produced using petrochemical technology, attempts have been made to develop technologies for producing butadiene using microorganisms in order to eliminate petroleum dependence and achieve sustainability. 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 not usually possible to produce butadiene at an industrial level. For this reason, various methods have been proposed for producing butadiene by culturing a transformant in which one or more foreign genes encoding the enzymes that make up this metabolic pathway have been introduced into the host.

[0008] Butanol is an organic chemical raw material with a wide range of applications in industrial sectors, including the chemical, pharmaceutical, and petroleum industries. It is used in the production of acrylic and methacrylic acid esters, which are used in the production of coatings, plastics, textiles, and adhesives; glycol ethers, which are used in the production of coatings and electronic products; butyl acetate, which is used in the production of paints, inks, coatings, and synthetic fruit fragrances; butylamine, which is used in the production of pesticides and pharmaceuticals; and amine resins. Butanol itself also has direct applications as a solvent in inks and dyes, an extraction solvent, an antifreeze fluid, a cosmetic ingredient, and a component of chromatography eluents. In particular, butanol, which has four carbon atoms, is attracting attention as a next-generation biofuel following bioethanol because of its many advantages, including higher fuel efficiency than ethanol, which has two carbon atoms, ease of blending with gasoline and diesel, low corrosivity, and low water content.

[0009] Microbial butanol production techniques using relatively fast-growing aerobic or facultative anaerobic bacteria have been attempted. Strictly anaerobic Clostridium bacteria have a metabolic pathway for producing butanol from glucose via pyruvate, acetyl-CoA, crotonyl-CoA, and butyryl-CoA, as shown in Figure 2. By referencing this metabolic pathway, it is possible to construct a metabolic pathway from pyruvate to butyryl-CoA and then to butyryl-CoA in bacteria that do not naturally produce butanol. According to Non-Patent Document 1, in aerobic and facultative anaerobic bacteria, the reduction reaction of enoyl-CoA via fatty acid synthesis or fatty acid beta-oxidation pathways is ubiquitous in the reaction from crotonyl-CoA to butyryl-CoA. Therefore, it is theoretically possible to produce butyryl-CoA from glucose. However, aerobic and facultative anaerobic bacteria are unable to actually produce butyryl-CoA from butyryl-CoA. For this reason, various methods have been proposed for producing butanol by culturing a transformant in which one or more foreign genes encoding enzymes that catalyze the metabolic pathway for producing butanol from butyryl-CoA are introduced into an aerobic bacterium or a facultative anaerobic bacterium host.

[0010] Advances in Biochemical Engineering / biotechnology 155:141-163

[0011] Hydrogen bacteria, which can grow using carbon dioxide as their sole carbon source, can utilize the chemical energy generated by the reaction of hydrogen and oxygen to produce chemical products using a mixture of oxygen, hydrogen, and carbon dioxide as a raw material, efficiently converting carbon dioxide into organic matter. Furthermore, they can be cultured in simple media that do not contain organic matter such as sugar. While hydrogen bacteria generally grow slowly, the growth rate of Hydrogenophilus bacteria is remarkably fast among hydrogen bacteria. The Mitsubishi Research Institute Bulletin No. 34, 1999, praised Hydrogenophilus bacteria, stating, "Their growth rate is so high that it cannot be compared to the carbon dioxide fixation ability of plants, clearly demonstrating the high carbon dioxide fixation ability of microorganisms." For this reason, there is a demand for using Hydrogenophilus bacteria to obtain valuable products from carbon dioxide as a resource.

[0012] The main objective of the present invention is to provide a mutant bacterium belonging to the genus Hydrogenophilus that has improved crotonyl-CoA production ability and can therefore be used for the efficient production of useful substances produced via crotonyl-CoA.

[0013] The present inventors conducted extensive research to solve the above problems and obtained the following findings: (1) Hydrogenophilus bacteria possess a metabolic pathway for producing crotonyl-CoA from pyruvate, as shown in Figure 1. However, Hydrogenophilus bacteria also possess a metabolic pathway for converting crotonyl-CoA to 3-hydroxybutyryl-CoA. When this metabolic pathway is active, the production of butadiene from crotonyl-CoA via crotyl alcohol or butanol from crotonyl-CoA via butyryl-CoA decreases the production amount of the target product.

[0014] (2) The present inventors predicted that 14 enzymes from Hydrogenophilus bacteria might catalyze side reactions that reduce the production of the target product. They expressed the genes for these 14 enzymes from Hydrogenophilus thermolluteolus in Escherichia coli and evaluated their side reaction activity. They found that the enzymes encoded by the genes HPTL_0565 (SEQ ID NO: 1), HPTL_1099 (SEQ ID NO: 3), HPTL_1333 (SEQ ID NO: 5), HPTL_0624 (SEQ ID NO: 7), HPTL_0998 (SEQ ID NO: 9), and HPTL_0272 (SEQ ID NO: 11) each have the activity to produce 3-hydroxybutyryl-CoA from crotonyl-CoA as a substrate. The enzyme encoded by HPTL_0565 (SEQ ID NO: 3) showed the strongest activity (Figures 3A and 3B). These six enzymes were found to catalyze side reactions.

[0015] (3) Furthermore, the present inventors evaluated the enzymatic activity of a strain in which HPTL_0565 (SEQ ID NO: 1) on the genome of Hydrogenophilus thermorteolus was disrupted and the wild-type strain before disruption, using crotonyl-CoA from the cell lysate as a substrate. The wild-type strain showed that crotonyl-CoA had almost completely disappeared 1 minute after the start of the enzymatic reaction, whereas the HPTL_0565-disrupted strain still contained crotonyl-CoA 30 minutes after the start of the enzymatic reaction (Figure 4). The present inventors also evaluated the enzymatic activity of a strain in which HPTL_0565 (SEQ ID NO: 1) on the genome of Hydrogenophilus thermorteolus was disrupted and the wild-type strain before disruption, using 3-hydroxybutyryl-CoA from the cell lysate as a substrate. The HPTL_0565-disrupted strain produced a higher amount of crotonyl-CoA than the wild-type strain (Figure 5). These results indicate that strains in which one or more of HPTL_0565, HPTL_1099, HPTL_1333, HPTL_0624, HPTL_0998, and HPTL_0272 on the genome of Hydrogenophilus thermorteolus have been disrupted have increased amounts of crotonyl-CoA, which increases the efficiency of production of useful substances such as crotyl alcohol, butadiene, and butanol, which are produced via crotonyl-CoA. Similar effects can be obtained in other Hydrogenophilus bacteria by disrupting the corresponding genes.

[0016] The present invention was completed based on the above findings and provides the following [1] to [4]: ​​[1] A mutant of a bacterium of the genus Hydrogenophilus, in which one or more (at least one) of the following enzyme genes (a) to (d) present on the chromosome of the bacterium of the genus Hydrogenophilus and catalyzing any reaction in a pathway for producing 3-hydroxybutyryl-CoA from crotonyl-CoA are disrupted: (a) a gene encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase activity, (b) a gene encoding a polypeptide having enoyl-CoA hydratase activity, (c) a gene encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity, and (d) a gene encoding a polypeptide having methylglutaconyl-CoA hydratase activity. [2] The mutant of [1], in which one or more of the genes (a) to (d) above are disrupted by deletion of all or a part of the gene, insertion or addition of nucleotides, or substitution of all or a part of the gene with other nucleotides. [3] A mutant of [1] or [2] having an exogenous gene for producing a target substance. [4] A method for producing a target substance, comprising the step of culturing the mutant of [3].

[0017] Measures to curb the increase in carbon dioxide 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, even these energy sources have not been able 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, and other raw materials for chemical 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, fuel, and raw materials for chemical products.

[0018] The mutant of the present invention is a Hydrogenophilus bacterium with an extremely high growth rate and therefore extremely high substance-producing capacity. Therefore, the gene encoding an enzyme that catalyzes a side reaction pathway in the pathway for producing useful substances from crotonyl-CoA on its genome has been disrupted. Therefore, when used to produce useful substances such as crotyl alcohol, butadiene, and butanol via crotonyl-CoA, the production efficiency can be improved. Because Hydrogenophilus bacteria do not have the enzyme genes that catalyze the reaction for producing crotyl alcohol, butadiene, or butanol from crotonyl-CoA, introducing these enzyme genes into the mutant of the present invention allows the resulting transformant to produce these useful substances with extremely high efficiency. Therefore, the Hydrogenophilus bacterium mutant of the present invention can address the global warming problem caused by increased carbon dioxide while meeting the demand for the production of chemical products such as plastics, rubber, fibers, polymers, and biofuels.

[0019] This figure shows the metabolic pathway in which Clostridium bacteria produce butanol from glucose via crotonyl-CoA. This figure shows the metabolic pathway in which many microorganisms produce butanol from pyruvate. This figure shows chromatograms showing the results of high-performance liquid chromatography (HPLC) detection of 3-hydroxybutyryl-CoA and crotonyl-CoA at 5 and 10 minutes after reacting cell lysates of 14 strains of Escherichia coli, each of which had been introduced with genes predicted to be side reaction enzymes of the Hydrogenophilus thermorteolus TH-1 strain, with crotonyl-CoA as a substrate for 10 minutes. This figure shows chromatograms showing the results of high-performance liquid chromatography (HPLC) detection of 3-hydroxybutyryl-CoA and crotonyl-CoA at 5 and 10 minutes after reacting cell lysates of 14 strains of Escherichia coli, each of which had been introduced with genes predicted to be side reaction enzymes of the Hydrogenophilus thermorteolus TH-1 strain, with crotonyl-CoA as a substrate for 10 minutes.

[0023] Figure 1 shows a chromatogram of crotonyl-CoA and 3-hydroxybutyryl-CoA detected by HPTL in the reaction solution after reacting a cell lysate of a Hydrogenophilus thermomorteolus HPTL_0565 gene-disrupted strain with crotonyl-CoA. Figure 2 shows a chromatogram of crotonyl-CoA and 3-hydroxybutyryl-CoA detected by HPTL in the reaction solution after reacting a cell lysate of a Hydrogenophilus thermomorteolus HPTL_0565 gene-disrupted strain with 3-hydroxybutyryl-CoA. Figure 3 shows an explanatory diagram of the procedure for disrupting the HPTL-0565 gene of Hydrogenophilus thermomorteolus. Figure 4 shows an explanatory diagram of the procedure for disrupting the HPTL-0565 gene of Hydrogenophilus thermomorteolus.

[0020] The present invention is described in detail below. (1) Hydrogenophilus bacterium mutant The Hydrogenophilus bacterium mutant of the present invention is a mutant in which one or more of the following genes (a) to (d) present on the chromosome of a Hydrogenophilus bacterium have been disrupted. The enzymes encoded by the following genes (a) to (d) produce 3-hydroxybutyryl-CoA from crotonyl-CoA, either alone or in combination with other enzymes. Two or more structurally different genes may be present for each of genes (a) to (d). Therefore, "one or more genes (a) to (d) have been disrupted" includes, for example, cases in which gene (a) and gene (b) have been disrupted, or cases in which multiple genes corresponding to gene (b) have been disrupted. (a) A gene encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase activity. In Hydrogenophilus thermorteolus, an example of gene (a) is HPTL_0565. (b) Genes encoding polypeptides with enoyl-CoA hydratase activity. In Hydrogenophilus thermorteolus, examples of genes in (b) include HPTL_1099 and HPTL_1333. (c) Genes encoding polypeptides with 3-hydroxyacyl-CoA dehydrogenase activity. In Hydrogenophilus thermorteolus, examples of genes in (c) include HPTL_0624 and HPTL_0998. (d) Genes encoding polypeptides with methylglutaconyl-CoA hydratase activity. In Hydrogenophilus thermorteolus, examples of genes in (d) include HPTL_0272.

[0021] Gene Disruption In the present invention, "gene disruption" refers to the reduction of the activity of each gene product to 50% or less of that of the parent strain before disruption. Preferably, it is 10% or less, particularly 1% or less. This reduces the production of 3-hydroxybutyryl-CoA from crotonyl-CoA compared to the parent strain. The activity of a gene product is evaluated by measuring the catalytic activity of the enzyme. A decrease in gene expression level reduces the activity of the gene product. Gene expression level can be measured by quantitative PCR (qPCR) or next-generation sequencing (NGS). Although a gene may be expressed normally even if part of its coding region or expression regulatory region is mutated, this also constitutes "gene disruption" as long as the activity of the gene product is 50% or less of that of the parent strain. Gene disruption can be achieved by mutating (by base deletion, substitution, insertion, addition, or a combination thereof) the coding region or gene expression regulatory region, such as the promoter region, of the gene on the chromosome of a Hydrogenophilus bacterium. Methods for gene disruption are well known, and include, for example, gene knockout methods using homologous recombination, and methods that involve the introduction of random gene mutations using mutagens and screening based on phenotypes.

[0022] The mutant of the present invention preferably has one or more mutations in the coding regions of the genes (a) to (d) above. Mutation of each coding region typically involves deleting the entire or partial region of each gene. Alternatively, a gene can be disrupted by inserting a nucleotide, oligonucleotide, or polynucleotide into the gene. Alternatively, the entire or partial region of each gene can be replaced with another nucleotide, oligonucleotide, or polynucleotide. When multiple nucleotide deletions, substitutions, and / or insertions are introduced, they may be introduced at a single site within the gene, or may be distributed across multiple sites.

[0023] The number of nucleotides to be deleted, substituted, or inserted is preferably 3 or more. It is also preferable to delete, replace, or insert 1% or more of the nucleotides of the entire gene length. This ensures that each gene is disrupted. It is also possible to delete or replace the entire length of each gene (i.e., 100% of the nucleotides constituting each gene). The number of nucleotides to be inserted can be 100,000 or less.

[0024] Examples of the above-mentioned (a) gene of a bacterium of the genus Hydrogenophilus include the following genes (a1) to (a5): (a1) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 1; (a2) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 1, and encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase activity; (a3) ​​DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 2; (a4) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 2, and having 3-hydroxybutyryl-CoA dehydratase activity; (a5) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence in which 1 to 10, particularly 1 to 5, particularly 1 to 3, particularly 1 amino acid has been deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 2, and having 3-hydroxybutyryl-CoA dehydratase activity. SEQ ID NO: 1 is the nucleotide sequence of HPTL_0565, a gene of the Hydrogenophilus thermorteolus wild strain, and SEQ ID NO: 2 is the amino acid sequence of a polypeptide encoded by HPTL_0565 of the Hydrogenophilus thermorteolus wild strain.

[0025] Examples of the above-mentioned (b) gene of bacteria of the genus Hydrogenophilus include the following genes (b1) to (b5): (b1) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 3. (b2) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 3, and encoding a polypeptide having enoyl-CoA hydratase activity. (b3) DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 4. (b4) DNA encoding a polypeptide having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 4, and having enoyl-CoA hydratase activity. (b5) DNA encoding a polypeptide having enoyl-CoA hydratase activity, comprising (particularly consisting of) an amino acid sequence in which 1 to 20, particularly 1 to 10, particularly 1 to 5, particularly 1 to 3, particularly 1 amino acid is deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 3 is a DNA encoding a polypeptide having enoyl-CoA hydratase activity. SEQ ID NO: 4 is the nucleotide sequence of HPTL_1099, a gene of the Hydrogenophilus thermorteolus wild strain, and SEQ ID NO: 5 is the amino acid sequence of the polypeptide encoded by HPTL_1099 of the Hydrogenophilus thermorteolus wild strain.

[0026] Examples of the above-mentioned (b) gene of bacteria of the genus Hydrogenophilus also include the following genes (b6) to (b10): (b6) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 5. (b7) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 5, and encoding a polypeptide having enoyl-CoA hydratase activity. (b8) DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 6. (b9) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 6, and having enoyl-CoA hydratase activity. (b10) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence in which 1 to 20, particularly 1 to 10, particularly 1 to 5, particularly 1 to 3, particularly 1 amino acid is deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 6, and having enoyl-CoA hydratase activity. SEQ ID NO: 5 is a DNA encoding a polypeptide of SEQ ID NO: 5 from Hydrogenophilus SEQ ID NO: 6 is the nucleotide sequence of HPTL_1333, a gene of the Hydrogenophilus thermorteolus wild strain, and SEQ ID NO: 7 is the amino acid sequence of the polypeptide encoded by HPTL_1333 of the Hydrogenophilus thermorteolus wild strain.

[0027] Examples of the above-mentioned (c) gene of a bacterium of the genus Hydrogenophilus include the following genes (c1) to (c5): (c1) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 7. (c2) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 7, and encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity. (c3) DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 8. (c4) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 8, and having 3-hydroxyacyl-CoA dehydrogenase activity. (c5) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence in which 1 to 20, particularly 1 to 10, particularly 1 to 5, particularly 1 to 3, particularly 1 amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 8, and having 3-hydroxyacyl-CoA dehydrogenase activity. SEQ ID NO: 7 is the nucleotide sequence of HPTL_0624, a gene of the Hydrogenophilus thermorteolus wild strain, and SEQ ID NO: 8 is the amino acid sequence of the polypeptide encoded by HPTL_0624 of the Hydrogenophilus thermorteolus wild strain.

[0028] Examples of the above-mentioned gene (c) of a bacterium of the genus Hydrogenophilus also include the following genes (c6) to (c10): (c6) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 9; (c7) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 9, and encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity; (c8) DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 10; (c9) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 10, and having 3-hydroxyacyl-CoA dehydrogenase activity; (c10) DNA encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity, comprising (particularly consisting of) an amino acid sequence in which 1 to 70, preferably 1 to 50, preferably 1 to 20, preferably 1 to 10, preferably 1 to 5, preferably 1 to 3, or preferably 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 9 is the base sequence of HPTL_0998, a gene of a Hydrogenophilus thermorteolus wild-type strain, and SEQ ID NO: 10 is the amino acid sequence of the polypeptide encoded by HPTL_0998 of a Hydrogenophilus thermorteolus wild-type strain.

[0029] Examples of the gene (d) of a bacterium belonging to the genus Hydrogenophilus include the following genes (d1) to (d5): (d1) DNA comprising (particularly consisting of) the nucleotide sequence of SEQ ID NO: 11; (d2) DNA comprising (particularly consisting of) a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to the nucleotide sequence of SEQ ID NO: 11, and encoding a polypeptide having methylglutaconyl-CoA hydratase activity; (d3) DNA encoding a polypeptide comprising (particularly consisting of) the amino acid sequence of SEQ ID NO: 12; (d4) DNA encoding a polypeptide comprising (particularly consisting of) an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 12, and having methylglutaconyl-CoA hydratase activity; (d5) DNA encoding a polypeptide having methylglutaconyl-CoA hydratase activity, comprising (particularly consisting of) an amino acid sequence in which 1 to 20, preferably 1 to 10, preferably 1 to 5, preferably 1 to 3, or preferably 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 12. SEQ ID NO: 11 is the base sequence of HPTL_0272, a gene of a Hydrogenophilus thermorteolus wild-type strain, and SEQ ID NO: 12 is the amino acid sequence of the polypeptide encoded by HPTL_0272 of a Hydrogenophilus thermorteolus wild-type strain.

[0030] In the present invention, the identity of the base sequence and amino acid sequence is a value calculated using GENETYX ver. 17 (manufactured by GENETYX Corporation).

[0031] Whether a test polypeptide has 3-hydroxybutyryl-CoA dehydratase activity, enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, or methylglutaconyl-CoA hydratase activity is confirmed by in vitro reaction of the test polypeptide with 1 mM crotonyl-CoA as a substrate at 52°C for 10 minutes, analyzing the reaction solution by high-performance liquid chromatography (HPLC), and observing the decrease in crotonyl-CoA.

[0032] Examples of Hydrogenophilus bacteria include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidans, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038. Among these, Hydrogenophilus thermoluteolus is preferred because it has the highest growth rate and carbon dioxide fixation ability of any carbon dioxide-fixing microorganism. Hydrogenophilus bacteria can be easily isolated from all over the world. A preferred strain of Hydrogenophilus thermorteolus is the TH-1 (NBRC 14978) strain. The TH-1 (NBRC 14978) strain exhibits an extremely high growth rate (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.

[0033] The Hydrogenophilus bacterium may be a bacterium isolated from nature, or may be a bacterium obtained by genetically modifying a bacterium isolated from nature. The modification can be carried out for the purpose of enabling high expression of an introduced gene. Such modification can be carried out, for example, by disrupting a gene in the genome that prevents production of a target substance, or by removing (curing) an endogenous plasmid in the Hydrogenophilus bacterium.

[0034] The Hydrogenophilus bacterium mutant of the present invention can produce a substance by expressing genes inherently present in the genome. Alternatively, metabolic enzyme genes encoding metabolic pathways necessary for the biosynthesis of a target substance can be introduced into the mutant to produce the target substance. That is, the mutant of the present invention can have exogenous metabolic enzyme genes encoding metabolic pathways necessary for the biosynthesis of the target substance. One or more metabolic enzyme genes can be introduced into the Hydrogenophilus bacterium mutant of the present invention, and therefore, the mutant can have one or more exogenous metabolic enzyme genes. In this way, the Hydrogenophilus bacterium mutant of the present invention can be used as a host for introducing an enzyme gene that catalyzes any step in the metabolic pathway that produces a target substance from crotonyl-CoA. Target substances or useful substances produced by the metabolism of crotonyl-CoA include crotyl alcohol, butadiene, and butanol. One or more enzyme genes catalyzing any step in the metabolic pathway that produces these substances from crotonyl-CoA can be introduced into the mutant. In the present invention, a gene introduced into a host is referred to as a "foreign gene," whether it is an endogenous gene present in the genome of the Hydrogenophilus bacterium or a heterologous gene not present in the genome of the host Hydrogenophilus bacterium.

[0035] (2) Method for Producing a Target Substance The present invention provides a method for producing a target substance, comprising the step of culturing the above-described Hydrogenophilus bacterium mutant of the present invention (both a mutant having an enzyme gene catalyzing any step in a metabolic pathway for producing a target substance from crotonyl-CoA introduced therein and a mutant having no such enzyme gene introduced therein). This method comprises the step of culturing the mutant of the present invention in an inorganic or organic medium while supplying a mixed gas containing hydrogen, oxygen, and carbon dioxide. The supplied gas is preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide, but other gases may be mixed in as long as the target substance can be efficiently produced.

[0036] 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 a target substance using substantially only carbon dioxide (especially only carbon dioxide) as a carbon source. 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 only carbon dioxide as a carbon source). In the present invention, "using carbon dioxide as the sole carbon source" includes cases where unavoidable amounts of other carbon sources are mixed in.

[0037] The pH of the culture medium used for cultivation 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 mixed gas solubility in the medium are high, allowing for highly efficient production of the target substance. When performing batch cultivation, the mixed gas can be sealed in a sealed culture vessel and cultured statically or with shaking, with shaking culture being preferred due to improved solubility of the mixed gas in the medium. When performing continuous cultivation, the mixed gas can be continuously supplied to a sealed culture vessel while being cultured with shaking, or the mutant can be cultured in a sealed culture vessel while introducing the mixed gas into the medium by bubbling. The volume ratio of hydrogen, oxygen, and carbon dioxide (hydrogen:oxygen:carbon dioxide) in the supply gas is preferably 1.75 to 7.5:1:0.25 to 3, more preferably 5 to 7.5:1:1 to 2, and even more preferably 6.25 to 7.5:1:1.5. Within this range, mutant growth is favorable and the target substance can be efficiently produced. The supply rate of the mixed gas or raw material gas should 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 mutant is favorable, the target substance 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 mutant is favorable and the target substance can be produced efficiently.

[0038] By culturing as described above, the target substance is produced in the culture solution. The target substance 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.

[0039] Next, the present invention will be described with reference to examples. The technical scope of the present invention is not limited by the following examples. Example 1: Functional verification of candidate side reaction enzymes expressed in Escherichia coli To identify the side reaction enzymes possessed by Hydrogenophilus bacteria, genes for 14 enzymes (SEQ ID NOs: 3, 5, 7, 9, 11, 12, 13-20) thought to be capable of the relevant reaction in Hydrogenophilus thermorteolus were cloned using the Nde I and EcoR I restriction enzyme sites of the plasmid pCAMO-6 (SEQ ID NO: 21). The gene sequences cloned on each plasmid were confirmed to be correct by DNA sequencing. The genes for the 14 enzymes thought to be capable of the side reaction are listed below. HPTL_1099 (SEQ ID NO: 3) HPTL_0565 (SEQ ID NO: 1) HPTL_0624 (SEQ ID NO: 7) HPTL_0998 (SEQ ID NO: 9) HPTL_1333 (SEQ ID NO: 5) HPTL_0272 (SEQ ID NO: 11) HPTL_1429 (SEQ ID NO: 13) HPTL_1650 (SEQ ID NO: 14) HPTL_1427 (SEQ ID NO: 15) HPTL_0993 (SEQ ID NO: 16) HPTL_0471 (SEQ ID NO: 17) HPTL_0239 (SEQ ID NO: 18) HPTL_0472 (SEQ ID NO: 19) HPTL_1001 (SEQ ID NO: 20)

[0040] E. coli JM109 strain was transformed with each of the plasmids containing the 14 enzyme genes. Transformant colonies grown on LB agar medium containing 50 μg / mL kanamycin were cultured overnight at 37°C with shaking in 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The target enzymes were expressed during culture under the control of the tac promoter in pCAMO-6, without the addition of an inducer. Two mL of the overnight culture was centrifuged at 4°C, 5,000 xg, for 10 minutes to harvest the cells. The harvested cells were suspended in 300 μL of 100 mM Tris-HCl (pH 7.5) buffer and sonicated. Crotonyl-CoA was added to each cell lysate to a final concentration of 1 mM, and the mixture was incubated at 52°C for 10 minutes. Reaction samples collected 5 and 10 minutes after the start of the reaction were analyzed by HPLC to detect crotonyl-CoA and 3-hydroxybutyryl-CoA. Crotonyl-CoA and 3-hydroxybutyryl-CoA were detected in the same manner for the E. coli JM109 strain into which the empty vector pCAMO-6 had been introduced.

[0041] HPLC chromatograms are shown in Figures 3A and 3B. Figures 3A and 3B include chromatograms taken after 5 and 10 minutes. Among the 14 candidates, HPTL_1099, HPTL_0565, HPTL_0624, HPTL_0998, HPTL_1333, and HPTL_0272 produced less crotonyl-CoA than the empty vector in the cell lysate, or the amount of crotonyl-CoA was reduced after 10 minutes compared to 5 minutes. Furthermore, HPTL_0565, HPTL_0998, HPTL_1333, and HPTL_0272 produced more 3-hydroxybutyryl-CoA than the empty vector. Therefore, HPTL_1099, HPTL_0565, HPTL_0624, HPTL_0998, HPTL_1333, and HPTL_0272 were considered to be enzyme genes catalyzing side reactions that interfere with the reaction producing useful substances from crotonyl-CoA. Among them, the strain expressing the HPTL_0565 gene showed the greatest reduction in crotonyl-CoA and had the strongest side reaction activity.

[0042] Example 2: Preparation of a gene-disrupted strain of HPTL_0565

[0123] A gene-disrupted strain of HPTL_0565, which gave the highest side reaction activity in Example 1, was obtained by homologous recombination using a wild-type strain of Hydrogenophilus thermorteolus TH-1 (NBRC 14978).

[0043] (1) Obtaining a streptomycin-resistant strain Hydrogenophilus thermorteolus TH-1 strain (NBRC 14978) (hereinafter referred to as "TH-1 strain") was cultured in liquid medium A [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, and CoCl2 6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0). The bacteria were inoculated into a glass vial containing 5 mL of TH-1000 (7.0) and incubated at 52°C under a gas mixture of H2:O2:CO2 (7.5:1:1.5) with a platinum loop. After 24 hours, the culture was inoculated onto LB solid medium containing 100 μg / mL streptomycin and incubated at 52°C for 72 hours. Three colonies were confirmed on the LB solid medium containing 100 μg / mL streptomycin. These colonies were then inoculated into a glass vial containing 5 mL of liquid medium A containing 100 μg / mL streptomycin with a platinum loop and incubated at 52°C under a gas mixture of H2:O2:CO2 (7.5:1:1.5). Since bacterial growth was observed after 24 hours, these strains were streptomycin-resistant variants of the TH-1 strain, and one of them was designated strain SR88. Streptomycin-resistant strains of hydrogen bacteria emerge at a certain rate when the culture solution of hydrogen bacteria is cultured on a solid medium containing 10 to 100 μg / mL of streptomycin.

[0044] (2) PCR, electrophoresis, DNA recovery, and seamless cloning in the construction of marker cassettes and expression plasmids. The plasmid vector pCAMO-6 for PCR, electrophoresis, DNA recovery, and seamless cloning in the construction of marker cassettes and expression plasmids was prepared as follows.

[0045] (2-1) PCR, electrophoresis, and DNA recovery PCR was performed using a Life Technologies DNA Thermal Cycler and KOD One PCR Master Mix (Toyobo Co., Ltd.) as the reaction reagent, according to standard methods. The resulting reaction mixture was then subjected to electrophoresis using a 1% agarose gel, and appropriate DNA fragments were recovered from the gel using a GEL / PCR Purification Mini Kit (FAVORGEN) as needed.

[0046] (2-2) Preparation of Plasmid Vector pCAMO-6 for Seamless Cloning To perform seamless cloning, the plasmid vector pCAMO-6 was amplified by PCR using the DNA of SEQ ID NO: 21 as a template. The following primers were used for PCR. Primers used for amplification of plasmid vector pCAMO-6: (a-1) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 22) (b-1) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 23) As a result of electrophoresis, a DNA fragment of approximately 5.2 kbp corresponding to the vector gene was detected, and the DNA fragment was recovered from the gel.

[0047] (2-3) Ligation of the DNA fragment of the vector pCAMO-6 with other DNA fragments Using recombinase extracted from Escherichia coli JM109, the DNA fragment of the vector pCAMO-6 synthesized above was ligated to the DNA fragment to be inserted. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the transformed cells were plated on LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours.

[0048] (2-4) Plasmid extraction and gene sequence confirmation 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, and plasmid DNA was extracted from the culture medium. The 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.

[0049] (3) Construction of Marker Cassette (3-1) Preparation of Counterselection Marker Genomic DNA was extracted from a wild-type TH-1 strain (a streptomycin-sensitive strain) according to standard methods. Using the extracted genomic DNA as a template, a DNA fragment containing the rpsL gene, which encodes the S12 ribosomal protein and is responsible for streptomycin sensitivity, was amplified by PCR. The following primers were used for PCR: Primers for amplifying the wild-type rpsL gene of the TH-1 strain: (a-2) 5'-CTGGAGGAGAAACGCATATGCCAACCATCAACCAGTTGGTG-3' (SEQ ID NO: 24) (b-2) 5'-CGACGGAGCTCGAATTCTTATTTCTTGCCCGCAGCGGC-3' (SEQ ID NO: 25). Primers (a-2) and (b-2) contained sequences homologous to those of the pCAMO-6 vector. As a result of electrophoresis, a DNA fragment of approximately 0.4 kbp corresponding to the rpsL gene of the wild-type TH-1 strain was detected, and the DNA fragment was recovered from the gel.

[0050] The DNA fragment of vector pCAMO-6 and the DNA fragment of rpsL were ligated to each other and used to transform Escherichia coli JM109 strain, after which the plasmid was extracted and the sequence of the rpsL gene was confirmed.

[0051] The rpsL gene containing the tac promoter region was amplified using pCAMO-6, into which the rpsL gene had been introduced, as a template. Primers for amplifying the rpsL gene containing the tac promoter region were: (a-3) 5'-CATAACGGTTCTGGCAAATATTC-3' (SEQ ID NO: 26) and (b-3) 5'-GCCATATGCGATACTCCTCCTCATTTCTTGCCCGCAGCGGCGCC-3' (SEQ ID NO: 27). Primer (b-3) contained the sequence required for binding to the bleomycin resistance gene. Electrophoresis detected a DNA fragment of approximately 0.4 kbp corresponding to the rpsL gene containing the tac promoter region, and the DNA fragment was recovered from the gel.

[0052] (3-2) Preparation of a Positive Selection Marker. A DNA fragment of the bleomycin resistance gene sequence was amplified by PCR using artificially synthesized (partially codon-replaced) DNA from Streptoalloteichus hindustanus as a template using techniques known to those skilled in the art. The following primers were used for PCR: (a-4) 5'-GAAATGAGGAGGAGTATCGCATATGGCTAAACTTACTTCTGCTG-3' (SEQ ID NO: 28) (b-4) 5'-TCAATCTTGTTCCTCTGCAACAAAATG-3' (SEQ ID NO: 29). Primer (a-4) contained the sequence required for binding to the rpsL gene. Electrophoresis detected a DNA fragment of approximately 0.4 kbp corresponding to the bleomycin resistance gene, and the DNA fragment was recovered from the gel.

[0053] (3-3) Preparation of Marker Cassette The rpsL gene fragment containing the tac promoter region and the bleomycin resistance gene fragment were mixed, ligated, and amplified by PCR. The following primers were used for PCR: (a-5) 5'-CCAAAGTAAAGGAGAGTAGCACATAACGGTTCTGGCAAATATTC-3' (SEQ ID NO: 30) (b-5) 5'-CGGTGAAGCTCGAAACTCAATCTTGTTCCTCTGCAACAAAATG-3' (SEQ ID NO: 31). Primer (a-5) contained the sequence required for binding to the 5' region of the HPTL_0565 gene, and primer (b-5) contained the sequence required for binding to the 3' region of the HPTL_0565 gene. Electrophoresis detected a DNA fragment of approximately 1.0 kbp corresponding to the ligated marker cassette gene, and the DNA fragment was recovered from the gel.

[0054] (4) Disruption of the HPTL_0565 gene in the host (4-1) Construction of DNA for disrupting the HPTL_0565 gene Using genomic DNA from the wild-type TH-1 strain as a template, a DNA fragment corresponding to the 5' region of the HPTL_0565 gene (including the 5'-terminal region and upstream region of the HPTL_0565 gene) and a DNA fragment corresponding to the 3' region of HPTL_0565 (including the 3'-terminal region and downstream region of the 0565 gene) were amplified by PCR. The following primers were used for PCR: Primers for amplifying the 5' region of the HPTL_0565 gene: (a-6) 5'-CTTCTCCCGAGCCCAATCGCGGG-3' (SEQ ID NO: 32) (b-6) 5'-CAGAATATTTGCCAGAACCGTTATGTTAAGCCAAGATATCGGCTTCCGAAACGG-3' (SEQ ID NO: 33) Primer (b-6) contained a sequence necessary for ligation with the marker cassette. Primers for amplifying the 3' region of the HPTL_0565 gene: (a-7) 5'-CATTTTGTTGCAGAGGAACAAGATTGAATCTCGACGCGGAGTTCGCG-3' (SEQ ID NO: 34) (b-7) 5'-GTGGTTGCTCTGCTTGCGCC-3' (SEQ ID NO: 35). Primer (a-7) contained the sequence necessary for binding to the marker cassette. Electrophoresis detected DNA fragments of approximately 1.7 kbp and approximately 1.3 kbp corresponding to the 5' and 3' regions of the HPTL_0565 gene, respectively, and the DNA fragments were recovered from the gel.

[0055] The DNA fragments from the 5' and 3' regions of the HPTL_0565 gene prepared in this manner and the marker cassette prepared in (3-3) were mixed and ligated by PCR for amplification. The following primers were used for PCR: (a-6) 5'-CTTCTCCCGAGCCCAATCGCGGG-3' (SEQ ID NO: 32) (b-7) 5'-GTGGTTGCTCTGCTTGCGCC-3' (SEQ ID NO: 35). Electrophoresis detected a DNA fragment of approximately 4.2 kbp corresponding to a DNA fragment containing the DNA fragments from the 5' and 3' regions and the marker cassette DNA fragment, and the DNA fragment was recovered from the gel. This DNA fragment was used as DNA for labeling the HPTL_0565 gene.

[0056] (4-2) Labeling of the HPTL_0565 Gene in Streptomycin-Resistant Strains (Positive Selection) The streptomycin-resistant SR88 strain of the TH-1 strain was transformed with the HPTL_0565 gene-labeling DNA prepared in (4-1) by electroporation. The transformants were plated onto LB medium containing 50 μg / mL of the bleomycin antibiotic Zeocin® and cultured at 52°C for 48 hours. Each strain grown on LB medium was restreaked onto LB medium containing 50 μg / mL of the bleomycin antibiotic Zeocin® (trade name) and cultured at 52°C for 24 hours. Using the resulting genomic DNA of the Zeocin-resistant strain as a template, the DNA region containing the HPTL_0565 gene was amplified by PCR. The following primers were used for PCR: PCR using the primer combination (a-6) and (b-7) amplifies a DNA region of approximately 4.2 kbp when the HPTL_0565 gene is replaced with the marker cassette. (a-6) 5'-CTTCTCCCGAGCCCAATCGCGGG-3' (SEQ ID NO: 32) (b-7) 5'-GTGGTTGCTCTGCTTGCGCC-3' (SEQ ID NO: 35) Electrophoresis detected a DNA fragment of approximately 4.2 kbp corresponding to the sequence of the HPTL_0565 gene replaced with the marker cassette. It was determined that the HPTL_0565 gene in this strain was disrupted by replacement with the HPTL_0565 gene labeling DNA. Counterselection to remove the marker cassette was not performed. The procedure from construction of the marker cassette to disruption of the HPTL_0565 gene is shown in Figures 6A and 6B.

[0057] Example 3: Confirmation of reduced by-product reaction capacity of the HPTL_0565 gene-disrupted strain. No. 1 The wild-type strain and the HPTL_0565 gene-disrupted strain were inoculated into test tubes containing 5 mL of liquid medium A using a platinum loop. 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 for 24 hours. Bacterial cells were collected from 2 mL of the culture medium by centrifugation (4°C, 5,000 g, 10 minutes). Each bacterial cell was suspended in 300 μL of 100 mM Tris-HCl (pH 7.5) and disrupted by sonication.

[0058] Crotonyl-CoA was added to each bacterial cell lysate as a reaction substrate to a final concentration of 1 mM (initial concentration in the reaction mixture), and the reaction was initiated at 52°C. 50 μL samples were taken at 1, 2, 3, 5, 10, 15, and 30 minutes after the start of the reaction. Crotonyl-CoA was detected by HPLC analysis. 3-Hydroxybutyryl-CoA was also detected. In the wild-type sample, 1 mM crotonyl-CoA was rapidly consumed, decreasing to below 0.1 mM within 1 minute of reaction. Crotonyl-CoA decreased to below the detection limit 10 minutes after the start of the reaction. Concomitantly, 3-hydroxybutyryl-CoA was produced as crotonyl-CoA decreased. The resulting 3-hydroxybutyryl-CoA was further metabolized (Figure 4, wild-type). These results indicate that the wild-type strain rapidly converted crotonyl-CoA to 3-hydroxybutyryl-CoA. In contrast, the HPTL_0565 disruptant sample showed a decrease in the 1 mM crotonyl-CoA used in the reaction over time. However, after 1 minute of reaction, the decrease was only about 40%, meaning that approximately 0.6 mM remained. Even after 30 minutes of reaction, approximately 0.2 mM crotonyl-CoA remained. Although the production of 3-hydroxybutyryl-CoA from crotonyl-CoA was detected, the metabolism of 3-hydroxybutyryl-CoA was slower than in the wild-type strain. The metabolism (decrease) of 3-hydroxybutyryl-CoA in the HPTL_0565 disruptant sample was as slow as the decrease of crotonyl-CoA (Figure 4, HPTL_0565 gene disruptant). The reaction of the cell lysate with crotonyl-CoA simulated the metabolism of crotonyl-CoA within the cells. The above results suggest that the wild-type strain rapidly reduces crotonyl-CoA, resulting in little or no production or accumulation of crotonyl-CoA. In contrast, the HPTL_0565 disruptant significantly attenuated the ability to reduce crotonyl-CoA, promoting its production and accumulation. This suggests that crotonyl-CoA can be used to produce useful substances.

[0059] Example 4: Confirmation of Reduction in By-Product Reaction Ability of the HPTL_0565 Gene-Disrupted Strain No. 2 Using the same sample as in Example 3, crotonyl-CoA and 3-hydroxybutyryl-CoA were detected over time by HPLC in the same manner as in Example 3, except that the reaction substrate was changed to 3-hydroxybutyryl-CoA. The purpose of this example was to verify the effect of HPTL_0565 gene disruption on the improvement of crotonyl-CoA production (supply) by generating hydroxybutyryl-CoA and crotonyl-CoA from 1 mM 3-hydroxybutyryl-CoA added to the reaction system using endogenous enzymes present in the cell lysate of Hydrogenophilus thermorteolus. The detected amounts were compared between the wild-type strain and the HPTL_0565 gene-disrupted strain. Because the substrate was 3-hydroxybutyryl-CoA, this example was evaluated under conditions closer to the natural state than in Example 3. As a result, 3-hydroxybutyryl-CoA almost disappeared after 30 minutes from the reaction mixtures using cell lysates of both the wild-type strain and the HPTL_0565 gene-disrupted strain. Crotonyl-CoA production by endogenous enzymes was also detected in both strains. The amount of crotonyl-CoA detected 1 minute after the start of the reaction was compared by HPLC chromatographic peak area between the wild-type strain and the HPTL_0565-disrupted strain. The amount of crotonyl-CoA detected in the HPTL_0565-disrupted strain was approximately four times higher than that in the wild-type strain, confirming that HPTL_0565 gene disruption can increase the amount of crotonyl-CoA produced (Figure 5).

[0060] SEQ ID NOs: 1 to 21 are shown below.

[0061] The Hydrogenophilus bacterium mutant of the present invention can be used to efficiently produce useful substances such as crotyl alcohol, butadiene, and butanol via crotonyl-CoA, while solving the problem of global warming caused by increased carbon dioxide, thereby meeting the demand for the production of chemical products such as plastics, rubber, fibers, polymers, and biofuels.

Claims

1. A mutant of a bacterium belonging to the genus Hydrogenophilus in which one or more of the following enzyme genes (a) to (d) present on the chromosome of the bacterium belonging to the genus Hydrogenophilus that catalyze any reaction in the pathway for producing 3-hydroxybutyryl-CoA from crotonyl-CoA are disrupted: (a) a gene encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase activity; (b) a gene encoding a polypeptide having enoyl-CoA hydratase activity; (c) a gene encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity; (d) a gene encoding a polypeptide having methylglucoconyl-CoA hydratase activity.

2. The mutant according to claim 1, wherein the gene (a) above comprises any one of the following genes (a1) to (a5): (a1) DNA comprising the nucleotide sequence of SEQ ID NO: 1; (a2) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 1 and encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase activity; (a3) ​​DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (a4) DNA encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase activity and having an amino acid sequence having 90% or more identity with SEQ ID NO: 2; (a5) DNA encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase activity and comprising an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO:

2.

3. The mutant according to claim 1 or 2, wherein the gene (b) above comprises any one of the following genes (b1) to (b5): (b1) DNA comprising the nucleotide sequence of SEQ ID NO: 3; (b2) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 3 and encoding a polypeptide having enoyl-CoA hydratase activity; (b3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4; (b4) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 4 and having enoyl-CoA hydratase activity; (b5) DNA encoding a polypeptide having an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4 and having enoyl-CoA hydratase activity.

4. The mutant according to any one of claims 1 to 3, wherein the gene (b) above comprises any one of the genes (b6) to (b10) below: (b6) DNA comprising the nucleotide sequence of SEQ ID NO: 5; (b7) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 5 and encoding a polypeptide having enoyl-CoA hydratase activity; (b8) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; (b9) DNA encoding a polypeptide having enoyl-CoA hydratase activity and comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 6; (b10) DNA encoding a polypeptide having enoyl-CoA hydratase activity and comprising an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO:

6.

5. The mutant according to any one of claims 1 to 4, wherein the gene (c) above comprises any one of the following genes (c1) to (c5): (c1) DNA comprising the nucleotide sequence of SEQ ID NO: 7; (c2) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 7 and encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity; (c3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8; (c4) DNA encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity and comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 8; (c5) DNA encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity and comprising an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO:

8.

6. The mutant according to any one of claims 1 to 5, wherein the gene (c) above comprises any one of the genes (c6) to (c10) below: (c6) DNA comprising the nucleotide sequence of SEQ ID NO: 9; (c7) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 9 and encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity; (c8) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10; (c9) DNA encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity and comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 10; (c10) DNA encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity and comprising an amino acid sequence in which 1 to 70 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO:

10.

7. The mutant according to any one of claims 1 to 6, wherein the gene (d) above comprises any one of the following genes (d1) to (d5): (d1) DNA comprising the nucleotide sequence of SEQ ID NO: 11; (d2) DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 11 and encoding a polypeptide having methylglucosaconyl-CoA hydratase activity; (d3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 12; (d4) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 12 and having methylglucosaconyl-CoA hydratase activity; (d5) DNA encoding a polypeptide having an amino acid sequence in which 1 to 20 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 12 and having methylglucosaconyl-CoA hydratase activity.

8. A mutant described in any one of claims 1 to 7, in which one or more of the genes (a) to (d) above have been disrupted by deletion of all or part of the gene, insertion or addition of nucleotides, or substitution of all or part of the gene with other nucleotides.

9. A mutant according to any one of claims 1 to 8, which has an exogenous gene for producing a target substance.

10. The mutant according to any one of claims 1 to 9, wherein the bacterium of the genus Hydrogenophilus is Hydrogenophilus thermorteolus.

11. A method for producing a target substance, comprising the step of culturing the mutant described in claim 9.

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