WO2025220580A1 - Hydrogenophilus bacterium mutant having improved crotonyl-coa production capability - Google Patents

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

The present invention relates to a mutant Hydrogenophilus bacterium that has improved crotonyl-CoA production ability, thereby improving its ability to produce various useful substances produced by its metabolism.

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.

Globally, the majority of chemical production relies on petroleum as raw materials, resulting in problems such as rising 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 carried out in various countries. However, converting biomass into sugars to use as a feedstock for microbial fermentation requires complex processes, posing a high cost issue. Furthermore, using biomass that 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.

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 fixing and effectively utilizing carbon dioxide, which contributes significantly to global warming.

Examples of useful chemicals produced via crotonyl CoA include crotyl alcohol, butadiene, and butanol.

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. It is also used as a raw material for other chemical products such as adiponitrile, 1,4-butanediol, cyclododecatriene, chloroprene, and sulfolane.

As most butadiene currently produced is produced using petrochemical technology, attempts are being made to develop technologies to produce butadiene using microorganisms in an effort to move away from 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), but it is not usually 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 the host.

Butanol is an organic chemical raw material with a wide range of applications in industrial sectors such as the chemical, pharmaceutical, and petroleum industries. It is used to manufacture 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 also has direct uses as a solvent in inks and dyes, extraction solvent, antifreeze fluid, cosmetic ingredient, and chromatography eluent component.
In particular, butanol, which has four carbon atoms, has many advantages over ethanol, which has two carbon atoms: it is more fuel efficient, it can be easily mixed with gasoline or diesel oil, and it has low corrosiveness and low water content. Therefore, it is attracting attention as a next-generation biofuel to follow bioethanol.

As a method for producing butanol using microorganisms, techniques have been attempted in which butanol is produced using aerobic bacteria or facultative anaerobic bacteria, which grow relatively quickly.
Clostridium bacteria, which are obligate anaerobes, have a metabolic pathway shown in Figure 2 that produces butanol from glucose via pyruvate, acetyl-CoA, crotonyl-CoA, and butyryl-CoA. By referring to this metabolic pathway, it is possible to construct a metabolic pathway from pyruvate to butyryl-CoA and then to butanol in bacteria that do not naturally produce butanol.
According to Non-Patent Document 1, in aerobic bacteria and facultative anaerobic bacteria, the reduction reaction of enoyl-CoA via fatty acid synthesis, fatty acid beta-oxidation pathways, etc., is ubiquitous in the reaction of crotonyl-CoA to butyryl-CoA, and therefore it is theoretically possible to produce butyryl-CoA from glucose. However, aerobic bacteria and facultative anaerobic bacteria cannot actually produce butyryl-CoA from butyryl-CoA. For this reason, various methods for producing butanol have been proposed, in which a transformant is cultured in which one or more foreign genes encoding enzymes that catalyze the metabolic pathway for producing butyryl-CoA from butyryl-CoA are introduced into an aerobic or facultative anaerobic host.

Advances in Biochemical Engineering/biotechnology 155:141-163

Hydrogen bacteria can grow using carbon dioxide as the sole carbon source, utilizing the chemical energy generated by the reaction of hydrogen and oxygen. This allows them to produce chemical products using a mixture of oxygen, hydrogen, and carbon dioxide as raw materials, enabling them to efficiently organicize carbon dioxide. They can also be cultured in simple media that do not contain organic substances such as sugars.
Generally, hydrogen bacteria grow slowly, but among hydrogen bacteria, the Hydrogenophilus bacteria grow at an exceptionally fast rate. The Mitsubishi Research Institute Report No. 34, 1999, praised the Hydrogenophilus bacteria, saying, "Their growth rate is so high that it cannot be compared to the carbon dioxide fixation ability of plants, and it clearly demonstrates the high carbon dioxide fixation ability of microorganisms." For this reason, there is a demand to use Hydrogenophilus bacteria to obtain valuable products from carbon dioxide as a resource.

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

The present inventors have conducted extensive research to solve the above problems and have 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 the target product decreases when butadiene is produced from crotonyl-CoA via crotyl alcohol, or when butanol is produced from crotonyl-CoA via butyryl-CoA.

(2) The inventors predicted that 14 enzymes from Hydrogenophilus bacteria may 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 possess the activity to produce 3-hydroxybutyryl-CoA from crotonyl-CoA as a substrate. The enzyme encoded by HPTL_0565 (SEQ ID NO: 3) exhibited the strongest activity (Figures 3A and 3B). These six enzymes were found to catalyze side reactions.

(3) Furthermore, the present inventors evaluated the enzyme activity of a strain in which HPTL_0565 (SEQ ID NO: 1) on the genome of Hydrogenophilus thermorteolus was disrupted and a wild-type strain before disruption, using crotonyl-CoA as a substrate in the cell lysate. As a result, in the wild-type strain, crotonyl-CoA had almost completely disappeared 1 minute after the start of the enzymatic reaction, whereas in the HPTL_0565-disrupted strain, crotonyl-CoA remained even 30 minutes after the start of the enzymatic reaction (Figure 4).
The present inventors also evaluated the enzyme activity of a strain in which HPTL_0565 (SEQ ID NO: 1) on the genome of Hydrogenophilus thermorteolus was disrupted and a wild-type strain before disruption, using 3-hydroxybutyryl-CoA from the cell lysate as a substrate. As a result, the strain in which HPTL_0565 was disrupted 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 Hydrogenophilus thermorteolus genome 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.

The present invention was completed based on the above findings and provides the following [1] to [4].
[1] A Hydrogenophilus bacterium mutant in which one or more (at least one) of the enzyme genes present on the chromosome of the Hydrogenophilus bacterium, which catalyze any reaction in the pathway that produces 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 methylglutaconyl-CoA hydratase activity [2] A mutant of [1] in which one or more of the genes (a) to (d) above have been disrupted by deletion of the entire region or a portion thereof, insertion or addition of nucleotides, or substitution of the entire region or a portion thereof 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].

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

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

FIG. 1 shows a metabolic pathway in which Clostridium bacteria produce butanol from glucose via crotonyl-CoA. FIG. 1 shows a metabolic pathway for producing butanol from pyruvate that many microorganisms have. This is a chromatogram showing the results of reacting cell lysates of 14 strains of Escherichia coli, each of which had been introduced with genes presumed to be side reaction enzymes of the Hydrogenophilus thermorteolus TH-1 strain, with the substrate crotonyl-CoA for 10 minutes, and detecting 3-hydroxybutyryl-CoA and crotonyl-CoA at 5 and 10 minutes using high-performance liquid chromatography (HPLC). This is a chromatogram showing the results of reacting cell lysates of 14 strains of Escherichia coli, each of which had been introduced with genes presumed to be side reaction enzymes of the Hydrogenophilus thermorteolus TH-1 strain, with the substrate crotonyl-CoA for 10 minutes, and detecting 3-hydroxybutyryl-CoA and crotonyl-CoA at 5 and 10 minutes using high-performance liquid chromatography (HPLC). This is a chromatogram showing crotonyl-CoA and 3-hydroxybutyryl-CoA detected by HPTL in the reaction solution after reacting the cell lysate of the HPTL_0565 gene-disrupted strain of Hydrogenophilus thermorteolus with crotonyl-CoA. This is a chromatogram showing crotonyl-CoA and 3-hydroxybutyryl-CoA detected by HPTL in the reaction solution after reacting the cell lysate of the HPTL_0565 gene-disrupted strain of Hydrogenophilus thermorteolus with 3-hydroxybutyryl-CoA. This is a diagram illustrating the procedure for disrupting the HPTL-0565 gene in Hydrogenophilus thermorteolus. This is a diagram illustrating the procedure for disrupting the HPTL-0565 gene in Hydrogenophilus thermorteolus.

The present invention will be 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:
For each of genes (a) to (d), two or more genes with different structures may exist. Therefore, "one or more genes (a) to (d) are disrupted" includes, for example, cases where gene (a) and gene (b) are disrupted, and cases where multiple genes corresponding to gene (b) are disrupted.

(a) A gene encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase activity. In Hydrogenophilus thermorteolus, an example of the gene (a) is HPTL_0565.
(b) A gene encoding a polypeptide having enoyl-CoA hydratase activity. In Hydrogenophilus thermorteolus, examples of the genes of (b) include HPTL_1099 and HPTL_1333.
(c) A gene encoding a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity. In Hydrogenophilus thermorteolus, examples of the genes in (c) include HPTL_0624 and HPTL_0998.
(d) A gene encoding a polypeptide having methylglutaconyl-CoA hydratase activity. In Hydrogenophilus thermorteolus, an example of the gene (d) is HPTL_0272.

Gene Disruption In the present invention, "gene disruption" refers to reducing the activity of the product of each gene to 50% or less of that of the parent strain before disruption. Preferably, it is 10% or less, and particularly 1% or less. This results in less production of 3-hydroxybutyryl-CoA from crotonyl-CoA than in the parent strain. The activity of the gene product is evaluated by measuring the activity of the enzyme it catalyzes.
A decrease in gene expression leads to a decrease in the activity of the gene product. Gene expression levels can be measured using quantitative PCR (qPCR) or next-generation sequencing (NGS). Even if a gene's coding region or expression regulatory region is partially mutated, the gene may still be expressed normally. However, even in this case, if the activity of the gene product is 50% or less of that of the parent strain, this constitutes "gene disruption."
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, including gene knockout using homologous recombination and phenotype-based screening using random gene mutations introduced using mutagens.

The mutant of the present invention preferably has a mutation in one or more of the above-mentioned genes (a) to (d), which are coding regions. Mutation of each coding region typically involves deleting all or part of each gene. Alternatively, a gene can be disrupted by inserting a nucleotide, oligonucleotide, or polynucleotide into the gene. Alternatively, all or part 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 location within the gene, or may be distributed across multiple locations.

The number of nucleotides to be deleted, substituted, or inserted is preferably 3 or more. It is also preferable to delete, replace, or insert nucleotides that account for 1% or more of the entire length of the gene. This ensures that each gene is disrupted reliably. 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.

Examples of the above-mentioned (a) gene of a bacterium of the genus Hydrogenophilus include the following genes (a1) to (a5):
(a1) DNA containing (particularly consisting of) the base sequence of SEQ ID NO: 1
(a2) DNA containing (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, preferably 1 to 5, preferably 1 to 3, preferably 1 amino acid in the amino acid sequence of SEQ ID NO: 2 have been deleted, substituted, inserted, or added, and which has 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.

Examples of the above-mentioned (b) gene of bacteria of the genus Hydrogenophilus include the following genes (b1) to (b5):
(b1) DNA containing (particularly consisting of) the base sequence of SEQ ID NO: 3
(b2) DNA containing (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 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: 4 and having enoyl-CoA hydratase activity.
(b5) DNA encoding a polypeptide 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, preferably 1 amino acid in the amino acid sequence of SEQ ID NO: 4 have been deleted, substituted, inserted, or added, and which has enoyl-CoA hydratase activity.
SEQ ID NO: 3 is the nucleotide sequence of HPTL_1099, a gene of the Hydrogenophilus thermorteolus wild strain, and SEQ ID NO: 4 is the amino acid sequence of a polypeptide encoded by HPTL_1099 of the Hydrogenophilus thermorteolus wild strain.

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 base 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, preferably 1 to 10, preferably 1 to 5, preferably 1 to 3, preferably 1 amino acid in the amino acid sequence of SEQ ID NO: 6 have been deleted, substituted, inserted, or added, and which has enoyl-CoA hydratase activity.
SEQ ID NO: 5 is the nucleotide sequence of HPTL_1333, a gene of the Hydrogenophilus thermorteolus wild strain, and SEQ ID NO: 6 is the amino acid sequence of a polypeptide encoded by HPTL_1333 of the Hydrogenophilus thermorteolus wild strain.

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 base sequence of SEQ ID NO: 7
(c2) DNA containing (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, preferably 1 to 10, preferably 1 to 5, preferably 1 to 3, preferably 1 amino acid in the amino acid sequence of SEQ ID NO: 8 have been deleted, substituted, inserted, or added, and which has 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.

Examples of the above-mentioned (c) gene of bacteria of the genus Hydrogenophilus also include the following genes (c6) to (c10):
(c6) DNA comprising (particularly consisting of) the base sequence of SEQ ID NO: 9
(c7) DNA containing (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 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, preferably 1 amino acid in the amino acid sequence of SEQ ID NO: 10 have been deleted, substituted, inserted, or added, and which has 3-hydroxyacyl-CoA dehydrogenase activity.
SEQ ID NO: 9 is the nucleotide sequence of HPTL_0998, which is a gene of the Hydrogenophilus thermorteolus wild strain, and SEQ ID NO: 10 is the amino acid sequence of the polypeptide encoded by HPTL_0998 of the Hydrogenophilus thermorteolus wild strain.

Examples of the above-mentioned (d) gene of a bacterium of the genus Hydrogenophilus include the following genes (d1) to (d5):
(d1) DNA comprising (particularly consisting of) the base sequence of SEQ ID NO: 11
(d2) DNA containing (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 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, preferably 1 amino acid in the amino acid sequence of SEQ ID NO: 12 have been deleted, substituted, inserted, or added, and which has methylglutaconyl-CoA hydratase activity.
SEQ ID NO: 11 is the nucleotide sequence of HPTL_0272, a gene of the Hydrogenophilus thermorteolus wild strain, and SEQ ID NO: 12 is the amino acid sequence of a polypeptide encoded by HPTL_0272 of the Hydrogenophilus thermorteolus wild strain.

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

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.

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 among carbon dioxide fixation 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 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.

Hydrogenophilus bacteria can be bacteria isolated from nature, or bacteria that have been genetically modified from bacteria isolated from nature. Modifications can be carried out for purposes such as enabling high expression of introduced genes. Such modifications can be carried out, for example, by disrupting genes in the genome that prevent the production of target substances, or by removing (curing) endogenous plasmids in Hydrogenophilus bacteria.

The Hydrogenophilus bacterium mutant of the present invention can produce a substance by expressing genes inherently contained in its genome, but for the production of a target substance, metabolic enzyme genes encoding a metabolic pathway necessary for the biosynthesis of the target substance can also be introduced. That is, the mutant of the present invention can have an exogenous metabolic enzyme gene encoding a metabolic pathway 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 or useful substances produced by the metabolism of crotonyl-CoA include crotyl alcohol, butadiene, butanol, etc., and one or more enzyme genes that catalyze any step in the metabolic pathway that produces these substances from crotonyl-CoA can be introduced.
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.

(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 the mutant and the mutant having no enzyme gene introduced therein, which catalyzes any step in the metabolic pathway for producing the target substance from crotonyl-CoA). This method includes 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.

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 essentially only carbon dioxide (especially using 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 essentially only carbon dioxide as a carbon source (especially using carbon dioxide as the only 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.

The pH of the medium used for the culture is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, the growth of the bacteria and the solubility of the mixed gas in the medium are high, and the target substance can be produced with high efficiency.
In the case of batch culture, the mixed gas can be sealed in a sealed culture vessel and cultured by static culture or shaking, with shaking culture being preferred since it improves the dissolution of the mixed gas into the medium. In the case of continuous culture, the mixed gas can be continuously supplied to a sealed culture vessel while the culture is shaken, 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 in the feed gas (hydrogen:oxygen:carbon dioxide) is preferably 1.75-7.5:1:0.25-3, more preferably 5-7.5:1:1-2, and even more preferably 6.25-7.5:1:1.5. Within these ranges, the growth of the mutant is favorable and the target substance can be efficiently produced.
The supply rate of the mixed gas or raw material gas may be 10 to 60 L/hour, preferably 10 to 40 L/hour, and more preferably 10 to 20 L/hour per L of medium. Within this range, the growth of the 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 mutant grows well and the target substance can be produced efficiently.

By culturing as described above, the target substance is produced in the culture medium. The target substance can be recovered by collecting the culture medium, but it can also be separated from the reaction solution using known methods. Such known methods include membrane separation and distillation.

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.
Example 1: Functional verification of candidate side reaction enzymes expressed in E. 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 into the plasmid pCAMO-6 (SEQ ID NO: 21) using the Nde I and EcoR I restriction enzyme sites. The gene sequences cloned into each plasmid were confirmed to be correct by DNA sequencing.
The genes for 14 enzymes that are thought to be potential side effects 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)

E. coli JM109 strain was transformed with each of the plasmids containing the 14 enzyme genes listed above. Each transformant colony grown on LB agar medium containing 50 μg/mL kanamycin was cultured overnight at 37°C with shaking in a test tube containing 5 mL of LB liquid medium containing 50 μg/mL kanamycin. During culture, each target enzyme is expressed without the addition of an inducer by the tac promoter contained in pCAMO-6. 2 mL of the overnight culture was centrifuged (4°C, 5,000 g, 10 minutes) to recover the bacterial cells. The recovered bacterial cells were suspended in 300 μL of 100 mM Tris-HCl (pH 7.5) buffer and disrupted by ultrasound. Crotonyl-CoA was added to each bacterial cell lysate to a final concentration of 1 mM, mixed, and 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 also detected in the E. coli JM109 strain containing the empty vector pCAMO-6.

The 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, the results showed that the bacterial cell lysates expressing HPTL_1099, HPTL_0565, HPTL_0624, HPTL_0998, HPTL_1333, and HPTL_0272 either produced less crotonyl-CoA than the empty vector, or the amount of crotonyl-CoA was lower after 10 minutes than after 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 thought to be enzyme genes that catalyze side reactions that interfere with the reaction that produces useful substances from crotonyl-CoA. Among them, the strain expressing the HPTL_0565 gene had the greatest reduction in crotonyl-CoA and possessed the strongest side reaction activity.

Example 2: Preparation of a gene-disrupted strain of HPTL_0565 A gene-disrupted strain of HPTL_0565, which had 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).

(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, _CoCl2 · _6H2O 4.0 _{mg ]} . The bacteria were inoculated into a glass vial containing 5 mL of [100 mg dissolved in 1 L of distilled water (pH 7.0)] using a platinum loop, and cultured with shaking at 52°C under a gas mixture of H ₂ :O ₂ :CO ₂ = 7.5:1:1.5. After 24 hours, the culture was inoculated onto LB solid medium containing 100 μg/mL streptomycin and cultured at 52°C for 72 hours.
As a result, the formation of three colonies was confirmed on LB solid medium containing 100 μg/mL streptomycin. These colonies were inoculated using a platinum loop into a glass vial containing 5 mL of liquid medium A containing 100 μg/mL streptomycin, and the mixture was cultured at 52°C under a gas mixture of H ₂ :O ₂ :CO ₂ = 7.5:1:1.5. Since bacterial growth was observed after 24 hours, these strains were streptomycin-resistant strains 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.

(2) PCR, electrophoresis, DNA recovery, and seamless cloning in the construction of marker cassettes and expression plasmids
In constructing the marker cassette and expression plasmid, PCR, electrophoresis, DNA recovery, and preparation of the plasmid vector pCAMO-6 for seamless cloning were carried out as follows.

(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.

(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.

Primer (a-1) used for amplification of the plasmid vector pCAMO-6: 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.

(2-3) Ligation of the DNA fragment of the vector pCAMO-6 with another DNA fragment 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 used to transform Escherichia coli JM109 strain by the heat shock method, and the transformed transformant was plated on LB medium containing 50 μg/mL of kanamycin and cultured at 37° C. for 24 hours.

(2-4) Plasmid extraction and gene sequence confirmation Each strain grown 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, Inc., and confirmed to match the sequence in the database.

(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)
Primer (a-2) and primer (b-2) contain sequences homologous to the vector pCAMO-6.

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.

The DNA fragment of vector pCAMO-6 and the DNA fragment of rpsL were ligated together and used to transform Escherichia coli strain JM109. Plasmid was then extracted and the sequence of the rpsL gene was confirmed.

The rpsL gene containing the tac promoter region was amplified using pCAMO-6, into which the rpsL gene had been introduced, as a template.

Primer (a-3) for amplifying the rpsL gene containing the tac promoter region: 5'-CATAACGGTTCTGGCAAATATTC-3' (SEQ ID NO: 26)
(b-3) 5'-GCCATATGCGATACTCCTCCTCATTTCTTGCCCGCAGCGGCGCC-3' (SEQ ID NO: 27)
The primer (b-3) contains a sequence required for binding to the bleomycin resistance gene.

As a result of electrophoresis, a DNA fragment of about 0.4 kbp corresponding to the rpsL gene containing the tac promoter region was detected, and the DNA fragment was recovered from the gel.

(3-2) Preparation of a positive selection marker: Using artificially synthesized (partially codon-substituted) DNA from Streptoalloteichus hindustanus as a template, a DNA fragment of the bleomycin resistance gene sequence was amplified by PCR using the following primers:

(a-4) 5'-GAAATGAGGAGGAGTATCGCATATGGCTAAACTTACTTCTGCTG-3' (SEQ ID NO: 28)
(b-4) 5'-TCAATCTTGTTCCTCTGCAACAAAATG-3' (SEQ ID NO: 29)
The primer (a-4) contains a sequence necessary for binding to the rpsL gene.

As a result of electrophoresis, a DNA fragment of approximately 0.4 kbp corresponding to the bleomycin resistance gene was detected, and the DNA fragment was recovered from the gel.

(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) contains a sequence required for binding to the 5' region of the HPTL_0565 gene, and primer (b-5) contains a sequence required for binding to the 3' region of the HPTL_0565 gene.

As a result of electrophoresis, a DNA fragment of about 1.0 kbp corresponding to the ligated marker cassette gene was detected, and the DNA fragment was recovered from the gel.

(4) Disruption of the host HPTL_0565 gene
(4-1) Construction of DNA for disrupting the HPTL_0565 gene Using the genomic DNA of 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:

Primer (a-6) for amplifying the 5' region of the HPTL_0565 gene: 5'-CTTCTCCCGAGCCCAATCGCGGG-3' (SEQ ID NO: 32)
(b-6) 5'-CAGAATATTTGCCAGAACCGTTATGTTAAGCCAAGATATCGGCTTCCGAAACGG-3' (SEQ ID NO: 33)
The primer (b-6) contains a sequence required for binding to the marker cassette.

Primer (a-7) for amplifying the 3' region of the HPTL_0565 gene: 5'-CATTTTGTTGCAGAGGAACAAGATTGAATCTCGACGCGGAGTTCGCG-3' (SEQ ID NO: 34)
(b-7) 5'-GTGGTTGCTCTGCTTGCGCC-3' (SEQ ID NO: 35)
The primer (a-7) contains a sequence required for binding to the marker cassette.

As a result of electrophoresis, DNA fragments of approximately 1.7 kbp and approximately 1.3 kbp corresponding to the 5' and 3' regions of the HPTL_0565 gene were detected, respectively, and the DNA fragments were recovered from the gel.

The DNA fragments from the 5' and 3' regions of the HPTL_0565 gene prepared in this way and the marker cassette prepared in (3-3) were mixed and ligated by PCR, followed by 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 revealed a DNA fragment of approximately 4.2 kbp, corresponding to a DNA fragment containing the 5'-region DNA fragment, the 3'-region DNA fragment, and the marker cassette DNA fragment. This DNA fragment was recovered from the gel and used to label the HPTL_0565 gene.

(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 DNA for labeling the HPTL_0565 gene prepared in (4-1) by electroporation, and the transformed strain was plated on LB medium containing 50 μg/mL of Zeocin, a bleomycin antibiotic, and cultured at 52°C for 48 hours.
Each strain grown on the LB medium was restreaked on an 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. When PCR was performed using a combination of primers (a-6) and (b-7), a DNA region of approximately 4.2 kbp was amplified if the HPTL_0565 gene was 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 4.2-kbp DNA fragment corresponding to the sequence in which the HPTL_0565 gene had been replaced with the marker cassette, indicating that the HPTL_0565 gene in this strain had been disrupted by replacement with the HPTL_0565 gene labeling DNA.
Here, counter-selection 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.

Example 3: Confirmation of the reduction in by-product reaction ability of the HPTL_0565 gene-disrupted strain No. 1
The wild-type strain and the HPTL_0565 gene disruptant were inoculated into 5 mL of liquid medium A in a test tube using a platinum loop. The tube was then filled with a gas mixture of H ₂ :O ₂ :CO ₂ = 7.5:1:1.5 and cultured with shaking at 52°C for 24 hours. Bacterial cells were harvested from 2 mL of the culture by centrifugation (4°C, 5,000 g, 10 minutes). Each cell was suspended in 300 μL of 100 mM Tris-HCl (pH 7.5) and disrupted by sonication.

Crotonyl-CoA was added as a reaction substrate to each bacterial cell lysate to a final concentration of 1 mM (the initial concentration in the reaction solution), and the reaction was initiated at 52°C. 50 μL of the reaction solution was sampled at 1, 2, 3, 5, 10, 15, and 30 minutes after the start of the reaction. The samples taken at each time point were analyzed by HPLC to detect crotonyl-CoA. 3-Hydroxybutyryl-CoA was also detected.
As a result, in the wild-type strain sample, 1 mM crotonyl-CoA used in the reaction was rapidly consumed, and the detected crotonyl-CoA decreased to less than 0.1 mM after 1 minute of reaction. 10 minutes after the start of the reaction, crotonyl-CoA decreased to below the detection limit. Simultaneously, 3-hydroxybutyryl-CoA was produced in association with the decrease in crotonyl-CoA. The produced 3-hydroxybutyryl-CoA was further metabolized (Figure 4, wild-type strain). These results indicate that the reaction from crotonyl-CoA to 3-hydroxybutyryl-CoA occurred rapidly in the wild-type strain.
In contrast, in the HPTL_0565 disruptant sample, the 1 mM crotonyl-CoA used in the reaction decreased over time, but the decrease was only about 40% after 1 minute of reaction, leaving approximately 0.6 mM. Even after 30 minutes of reaction, approximately 0.2 mM crotonyl-CoA remained. The production of 3-hydroxybutyryl-CoA from crotonyl-CoA was detected, but 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 in crotonyl-CoA (Figure 4, HPTL_0565 gene disruptant).
The reaction of the cell lysate with crotonyl-CoA simulates the metabolism of crotonyl-CoA within the cell. The above results suggest that the wild-type strain rapidly reduces crotonyl-CoA, resulting in little 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. Therefore, it was demonstrated that crotonyl-CoA can be used to produce useful substances.

Example 4: Confirmation of the 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 produce 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, and to compare the detected amounts between the wild-type strain and the HPTL_0565 gene-disrupted strain to verify the effect of HPTL_0565 gene disruption on improving the production (supply) of crotonyl-CoA. 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).

SEQ ID NOs: 1 to 21 are shown below.

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

Claims (11)

A Hydrogenophilus bacterium mutant in which one or more of the following enzyme genes (a) to (d) present on the chromosome of the Hydrogenophilus bacterium, which 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 The mutant according to claim 1, wherein the gene (a) includes any one of the following genes (a1) to (a5):
(a1) DNA containing the base sequence of SEQ ID NO: 1
(a2) DNA containing 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 comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 2 and having 3-hydroxybutyryl-CoA dehydratase activity.
(a5) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2 and having 3-hydroxybutyryl-CoA dehydratase activity. The mutant according to claim 1 or 2, wherein the gene (b) includes any one of the following genes (b1) to (b5):
(b1) DNA containing the base sequence of SEQ ID NO: 3
(b2) DNA containing 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 comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 4 and having enoyl-CoA hydratase activity.
(b5) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4 and having enoyl-CoA hydratase activity. The mutant according to any one of claims 1 to 3, wherein the gene (b) includes any one of the genes (b6) to (b10) below.
(b6) DNA containing the base sequence of SEQ ID NO: 5
(b7) DNA encoding a polypeptide having enoyl-CoA hydratase activity and containing a nucleotide sequence having 90% or more identity with the nucleotide sequence of SEQ ID NO: 5.
(b8) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6
(b9) DNA encoding a polypeptide comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 6 and having enoyl-CoA hydratase activity.
(b10) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6 and having enoyl-CoA hydratase activity. The mutant according to any one of claims 1 to 4, wherein the gene (c) includes any one of the genes (c1) to (c5) below.
(c1) DNA containing the base sequence of SEQ ID NO: 7
(c2) DNA containing 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 comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 8 and having 3-hydroxyacyl-CoA dehydrogenase activity.
(c5) DNA encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 8 in which 1 to 20 amino acids are deleted, substituted, inserted, or added, and having 3-hydroxyacyl-CoA dehydrogenase activity. The mutant according to any one of claims 1 to 5, wherein the gene (c) includes any one of the genes (c6) to (c10) below.
(c6) DNA containing the base sequence of SEQ ID NO: 9
(c7) DNA containing 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 comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 10 and having 3-hydroxyacyl-CoA dehydrogenase activity.
(c10) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 70 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 10, and having 3-hydroxyacyl-CoA dehydrogenase activity. The mutant according to any one of claims 1 to 6, wherein the gene (d) includes any one of the genes (d1) to (d5) below.
(d1) DNA containing the base sequence of SEQ ID NO: 11
(d2) DNA containing 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 comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 12 and having methylglucosaconyl-CoA hydratase activity.
(d5) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 12 and having methylglucosaconyl-CoA hydratase activity. The mutant of any one of claims 1 to 7, wherein one or more of the genes (a) to (d) above are 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. A mutant described in any one of claims 1 to 8, having an exogenous gene for producing a target substance. The mutant described in any one of claims 1 to 9, wherein the bacterium of the genus Hydrogenophilus is Hydrogenophilus thermorteolus. A method for producing a target substance, comprising the step of culturing the mutant according to claim 9.