Aliphatic alcohol-producing transformant of bacterium belonging to genus hydrogenophilus

Abstract

Bacteria belonging to the genus Hydrogenophilus do not have a gene encoding an enzyme that catalyzes an aliphatic alcohol production reaction and do not inherently produce an aliphatic alcohol. However, by introducing a gene encoding a thioesterase that produces a fatty acid from acyl-ACP and a gene encoding an aliphatic acyl-CoA reductase that produces an aliphatic alcohol from an aliphatic acyl-CoA into a bacterium belonging to the genus Hydrogenophilus, said bacterium belonging to genus Hydrogenophilus produces an aliphatic alcohol. Furthermore, the production efficiency of a fatty acid alcohol is improved by disrupting the acyl-CoA dehydrogenase gene on the chromosome of the bacterium belonging to the genus Hydrogenophilus.

Description

Transformants of Hydrogenophilus bacteria producing fatty alcohols

[0001] The present invention relates to a transformant of a bacterium belonging to the genus Hydrogenophilus having the ability to produce an aliphatic alcohol, and a method for producing an aliphatic alcohol using the transformant.

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

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

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

[0005] Fatty alcohols are widely used as raw materials for detergents, cosmetics, pharmaceuticals, food additives, diesel fuel, and other applications. Fatty alcohols are primarily derived from natural plant oils. Palm oil, extracted from the fruit of the oil palm tree, is the most widely consumed vegetable oil in the world. In recent years, major palm oil-producing countries like Indonesia and Malaysia have been developing large-scale oil palm plantations to meet the rapidly increasing demand for oil. However, large-scale development of oil palm plantations has led to social issues such as deforestation, biodiversity loss, and forced labor. In 2004, the Roundtable on Sustainable Palm Oil (RSPO) was established to promote sustainable palm oil production and use.

[0006] Recently, attempts to use microorganisms to produce fatty alcohols independently of agricultural products have been reported. Many microorganisms synthesize fatty acyl-acyl carrier proteins (acyl-ACPs), the final product of fatty acid synthesis, but lack the genes required for fatty alcohol synthesis. Therefore, various methods for producing fatty alcohols have been proposed, including culturing transformants of Escherichia coli or other hosts that have been transformed with exogenous genes encoding the enzymes that make up this metabolic pathway.

[0007] For example, Non-Patent Document 1 reports an attempt to induce high levels of fatty alcohol production in Escherichia coli by introducing a thioesterase gene derived from Escherichia coli lacking a DNA region encoding a periplasmic transport signal sequence and a fatty acyl-CoA reductase gene derived from Acinetobacter calcoaceticus into Escherichia coli and disrupting the fatty acid degrading enzyme gene of the host Escherichia coli. Non-Patent Document 2, similar to Non-Patent Document 1, also reports an attempt to induce high levels of fatty alcohol production in Escherichia coli by introducing a thioesterase gene derived from Escherichia coli lacking a DNA region encoding a signal sequence and a fatty acyl-CoA reductase gene derived from Marinobacter aquaeolei into Escherichia coli and disrupting the fatty acid degrading enzyme gene of the host Escherichia coli. However, the methods in Non-Patent Documents 1 and 2 use glucose or starch derived from biomass as a carbon source. As mentioned above, the use of biomass requires complex processes to convert biomass into sugars, which results in high costs, and the industrial use of biomass has the drawback of placing a burden on the environment.

[0008] Furthermore, Patent Document 1 teaches a method for producing aliphatic alcohols using a transformant obtained by introducing a gene encoding aliphatic alcohol synthase into Synechocystis sp. PCC6803. This method involves producing aliphatic alcohols using a cyanobacterium, a photosynthetic bacterium, as a host. Although cyanobacteria have a higher carbon dioxide fixation capacity than plants, this capacity is not sufficient, and therefore the method using cyanobacteria as a host has not been put into practical use as an industrial method for producing aliphatic alcohols.

[0009] Patent Publication No. 2018-143192

[0010] Stee EJ, et al. 2010. Microbial production of fatty-acid derived fuels and chemicals from plant biomass. Nature. 463: 559-562. Liu A, et al.,2013. Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases. Appl microbiol Biotechnol. 97: 7061-7071.

[0011] An object of the present invention is to provide a transformant of a bacterium belonging to the genus Hydrogenophilus that can efficiently produce aliphatic alcohols using carbon dioxide as a sole carbon source, and a method for efficiently producing aliphatic alcohols using the transformant.

[0012] The present inventors conducted extensive research to solve the above problems and discovered the following: (i) Hydrogenophilus bacteria do not inherently produce fatty alcohols because they do not possess a gene encoding an enzyme that catalyzes a fatty alcohol synthesis reaction. However, introducing a gene encoding an enzyme that catalyzes a fatty alcohol synthesis reaction allows them to produce fatty alcohols. Specifically, by introducing a gene encoding a thioesterase that synthesizes fatty acids from acyl-ACP and a gene encoding a fatty acyl-CoA reductase that synthesizes fatty alcohols from fatty acyl-CoA into Hydrogenophilus bacteria, these genes are expressed in the Hydrogenophilus bacteria, imparting the ability to produce fatty alcohols. (ii) Because a gene encoding acyl-CoA dehydrogenase is present on the chromosome of Hydrogenophilus bacteria, fatty alcohol precursors are decomposed, resulting in a decrease in fatty alcohol production efficiency. Disruption of the gene encoding acyl-CoA dehydrogenase in Hydrogenophilus bacteria suppresses the decomposition of fatty alcohol precursors, improving fatty alcohol production efficiency. This allows the Hydrogenophilus bacterium to produce aliphatic alcohols very efficiently.

[0013] The present invention was completed based on the above findings and provides the following [1] to [7]: [1] A transformant in which a gene encoding a thioesterase that produces a fatty acid from acyl-ACP and a gene encoding a fatty acyl-CoA reductase that produces a fatty alcohol from fatty acyl-CoA have been introduced into a bacterium of the genus Hydrogenophilus. [2] The transformant according to [1], in which the thioesterase gene that produces a fatty acid from acyl-ACP is the DNA of (a1), (a2), (a3), (a4), or (a5) below. (a1) DNA comprising the base sequence of SEQ ID NO: 1; (a2) DNA comprising a base sequence having 90% or more identity with SEQ ID NO: 1 and encoding a polypeptide having thioesterase activity to synthesize fatty acids from acyl-ACP; (a3) ​​DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (a4) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 2 and having thioesterase activity to synthesize fatty acids from acyl-ACP; (a5) 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: 2 and having thioesterase activity to synthesize fatty acids from acyl-ACP. [3] The transformant according to [1] or [2], wherein the fatty acyl-CoA reductase gene that synthesizes fatty alcohols from fatty acyl-CoA is DNA of the following (b1), (b2), (b3), (b4), or (b5).(b1) DNA comprising the base sequence of SEQ ID NO: 3. (b2) DNA comprising a base sequence having 90% or more identity with SEQ ID NO: 3 and encoding a polypeptide having fatty acyl-CoA reductase activity that produces fatty alcohols from fatty acyl-CoA. (b3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. (b4) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 4 and having fatty acyl-CoA reductase activity that produces fatty alcohols from fatty acyl-CoA. (b5) DNA encoding a polypeptide having an amino acid sequence in which 1 to 50 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4 and having fatty acyl-CoA reductase activity that produces fatty alcohols from fatty acyl-CoA. [4] A transformant according to any of [1]-[3], in which the acyl-CoA dehydrogenase gene on the chromosome of a bacterium of the genus Hydrogenophilus is disrupted. [5] The transformant according to [4], wherein the acyl-CoA dehydrogenase gene is the DNA of the following (c1), (c2), (c3), (c4), or (c5): (c1) DNA comprising the nucleotide sequence of SEQ ID NO: 5; (c2) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 5, and encoding a polypeptide having acyl-CoA dehydrogenase activity; (c3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; (c4) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 6, and having acyl-CoA dehydrogenase activity; (c5) DNA encoding a polypeptide having acyl-CoA dehydrogenase activity, comprising an amino acid sequence in which 1 to 80 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6. [6] The transformant according to any of [1] to [5], wherein the Hydrogenophilus bacterium is Hydrogenophilus thermorteolus. [7] A method for producing an aliphatic alcohol, comprising a step of culturing the transformant according to any one of [1] to [6].

[0014] Measures to curb the increase in carbon dioxide emissions include reducing carbon dioxide emissions and fixing emitted carbon dioxide. To reduce carbon dioxide emissions, solar, wind, geothermal, and other energy sources are being used in place of fossil fuels. However, in reality, the use of these energy sources has not sufficiently curbed the increase in carbon dioxide. Therefore, it is necessary to promote the fixation or resource recovery of emitted carbon dioxide. Carbon dioxide can be fixed physically or chemically, but if it is fixed using living organisms, it can be used to produce organic matter that can be used as food, feed, fuel, and other products. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve both the two problems of global warming caused by increased carbon dioxide and the difficulty in securing food, feed, and fuel. Furthermore, it can produce in-demand chemical raw materials while curbing global warming caused by increased carbon dioxide.

[0015] Hydrogen bacteria, which can grow using carbon dioxide as their sole carbon source, can use the chemical energy generated by the reaction between hydrogen and oxygen to produce chemical products using a mixture of oxygen, hydrogen, and carbon dioxide as raw materials, making it possible to efficiently organicize carbon dioxide and to cultivate them in simple culture media. While hydrogen bacteria generally grow slowly, Hydrogenophilus bacteria have a significantly faster growth rate. The Mitsubishi Research Institute Bulletin No. 34 1999 evaluated Hydrogenophilus bacteria, saying, "Their growth rate is so high that it cannot be compared to the carbon dioxide fixation ability of plants, and clearly demonstrates the high carbon dioxide fixation ability of microorganisms."

[0016] Like many microorganisms, Hydrogenophilus bacteria possess a set of enzyme genes that catalyze the production of acyl-ACP, the final product of fatty acid synthesis, but do not possess a set of enzyme genes that catalyze the reaction of producing fatty alcohols from acyl-ACP. The present inventors have found that it is difficult to express functional proteins even when foreign genes are introduced into Hydrogenophilus bacteria. However, according to the present invention, by introducing into Hydrogenophilus bacteria a gene encoding a thioesterase that produces fatty acids from acyl-ACP and a gene encoding a fatty acyl-CoA reductase that produces fatty alcohols from fatty acyl-CoA, these genes are expressed in the Hydrogenophilus bacteria, enabling the Hydrogenophilus bacteria to produce fatty alcohols using carbon dioxide as the sole carbon source.

[0017] Wild-type Hydrogenophilus bacteria have the gene for acyl-CoA dehydrogenase, a fatty acid-degrading enzyme, on their genome, and have the ability to decompose fatty acids by producing active acyl-CoA dehydrogenase. Disrupting the gene encoding acyl-CoA dehydrogenase, a fatty acid-degrading enzyme, suppresses the breakdown of fatty acids, which are precursors of fatty alcohols, and improves fatty alcohol productivity.

[0018] As described above, among organisms that have the ability to fix carbon dioxide, bacteria of the genus Hydrogenophilus have particularly excellent carbon dioxide fixation ability. Therefore, by using the transformant of the present invention, it is possible to fix carbon dioxide and produce aliphatic alcohols industrially.

[0019] Fatty alcohols are widely used as raw materials for detergents, additives for cosmetics, pharmaceuticals, foods, etc., diesel fuel, etc. The transformant of the present invention enables fatty alcohols to be produced on an industrial scale, thereby resolving various problems associated with the mass production of palm oil, which is a raw material for fatty alcohols.

[0020] This is an SDS-PAGE gel photograph showing that the cell lysate of a strain of Hydrogenophilus thermorteolus transformed with a thioesterase gene decomposed C12:0-acyl-ACP. This is a gas chromatography chromatogram showing that the cell lysate of a strain of Hydrogenophilus thermorteolus transformed with a fatty acyl-CoA reductase gene produced 1-dodecanol from lauroyl-CoA and 1-tetradecanol from myristoyl-CoA.

[0021] The present invention is described in detail below. (1) Transformant Capable of Producing Fatty Alcohols The transformant (transformed cell) of the present invention is a host Hydrogenophilus bacterium, into which a gene encoding a thioesterase that synthesizes fatty acids from acyl-ACPs and a gene encoding a fatty acyl-CoA reductase that synthesizes fatty alcohols from fatty acyl-CoAs have been introduced. In other words, the transformant of the present invention is a Hydrogenophilus bacterium harboring an exogenous thioesterase gene that synthesizes fatty acids from acyl-ACPs and an exogenous fatty acyl-CoA reductase gene that synthesizes fatty alcohols from fatty acyl-CoA. In the thioesterase of the present invention that synthesizes fatty acids from acyl-ACPs, the range of acyl groups in the acyl-ACP that can serve as substrates varies depending on the amino acid sequence of the thioesterase. For example, a linear or branched, saturated or unsaturated acyl-ACP having an acyl group with 8 to 18 carbon atoms (particularly, any of 8 to 18 carbon atoms) can be used as a substrate. Furthermore, in the fatty acyl-CoA reductase of the present invention that produces fatty alcohols from fatty acyl-CoA, the range of fatty acyl groups in fatty acyl-CoA that can serve as substrates varies depending on the amino acid sequence of the fatty acyl-CoA reductase, but for example, fatty acyl-CoA having a linear or branched, saturated or unsaturated fatty acyl group having 12 to 18 carbon atoms (particularly, any of 8 to 18 carbon atoms) can be used as a substrate. The thioesterase gene and fatty acyl-CoA reductase gene may be DNA of a thioesterase gene and DNA of a fatty acyl-CoA reductase gene isolated from a naturally occurring bacterium, respectively, or may be DNA artificially synthesized using techniques known to those skilled in the art.

[0022] Thioesterase Gene The gene for a thioesterase that produces fatty acids from acyl-ACP does not need to have been identified as the gene for this thioesterase; it is sufficient if it is a DNA that encodes a polypeptide having this thioesterase activity.

[0023] In the present invention, whether a polypeptide has thioesterase activity using acyl-ACP as a substrate can be determined by reacting the test polypeptide with RCO-ACP (R represents a linear saturated alkyl group having 11 carbon atoms), and confirming the degradation and consumption of RCO-ACP by SDS-PAGE. Specifically, a cell lysate prepared by the method described in the Examples section is used as a crude enzyme solution, which is reacted with RCO-ACP (R represents a linear saturated alkyl group having 11 carbon atoms) from Escherichia coli, and the reaction product is subjected to SDS-PAGE to confirm the disappearance of the RCO-ACP band. Using this method, consumption of RCO-ACP indicates that the test polypeptide has the activity of degrading acyl-ACP to produce fatty acids.

[0024] A preferred example of the thioesterase gene is that of Tepidiphilus thermophilus, particularly a thioesterase gene from Tepidiphilus lacking a DNA region encoding an N-terminal peptide presumed to be a periplasmic transport signal sequence. The thioesterase gene from Tepidiphilus lacking a presumed signal sequence is a DNA consisting of the nucleotide sequence of SEQ ID NO: 1. Alternatively, a DNA containing a nucleotide sequence having at least 90%, preferably at least 95%, preferably at least 98%, and preferably at least 99% identity to SEQ ID NO: 1 (particularly, a DNA having at least 90%, preferably at least 95%, preferably at least 98%, and preferably at least 99% identity to SEQ ID NO: 1) and encoding a polypeptide having thioesterase activity that synthesizes fatty acids from acyl-ACP can also be used.

[0025] Preferred examples of the thioesterase gene include DNA encoding a Tepidophilus thermophilus thioesterase, particularly DNA encoding a Tepidophilus thermophilus thioesterase lacking an N-terminal peptide presumed to be a periplasmic transport signal sequence. The Tepidophilus thermophilus thioesterase lacking a presumed signal sequence is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. DNA encoding a polypeptide containing an amino acid sequence having at least 90%, preferably at least 95%, preferably at least 98%, and preferably at least 99% identity to SEQ ID NO: 2 (particularly, an amino acid sequence having at least 90%, preferably at least 95%, preferably at least 98%, and preferably at least 99% identity to SEQ ID NO: 2) and having thioesterase activity that synthesizes fatty acids from acyl-ACP can also be used. Furthermore, DNAs that encode a polypeptide comprising an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2 (particularly, an amino acid sequence in which 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2) and that has thioesterase activity for synthesizing fatty acids from acyl-ACP can also be used.

[0026] Fatty acyl-CoA reductase gene The fatty acyl-CoA reductase gene does not need to be known to have been identified as a fatty acyl-CoA reductase gene, as long as it is a DNA encoding a polypeptide having fatty acyl-CoA reductase activity.

[0027] Whether a test polypeptide has fatty acyl-CoA reductase activity using fatty acyl-CoA as a substrate can be confirmed by reacting the test polypeptide with lauroyl-CoA in the presence of NADPH and detecting the produced 1-dodecanol using gas chromatography or the like.

[0028] A preferred example of a fatty acyl-CoA reductase gene is that of Marinobacter lutaoensis. The Marinobacter lutaoensis fatty acyl-CoA reductase gene is a DNA consisting of the nucleotide sequence of SEQ ID NO: 3. Alternatively, a DNA encoding a polypeptide having fatty acyl-CoA reductase activity that synthesizes fatty alcohols from fatty acyl-CoA may be used, which includes a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 3 (particularly, a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 3).

[0029] A preferred example of a fatty acyl-CoA reductase gene is DNA encoding the fatty acyl-CoA reductase of Marinobacter lutaoensis. The fatty acyl-CoA reductase of Marinobacter lutaoensis is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4. DNA encoding a polypeptide having an amino acid sequence with 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 4 (particularly, an amino acid sequence with 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 4) and having fatty acyl-CoA reductase activity that produces a fatty alcohol from fatty acyl-CoA can also be used. Furthermore, DNAs that encode a polypeptide comprising an amino acid sequence in which 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4 (particularly, consisting of an amino acid sequence in which 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4) and that has fatty acyl-CoA reductase activity that produces fatty alcohols from fatty acyl-CoA can also be used.

[0030] The DNA sequence encoding the protein containing the amino acid sequence of SEQ ID NO: 2 or 4 may have various base substitutions in the coding region, taking into account codon degeneracy or preferred codons in Hydrogenophilus bacteria, as long as the amino acid sequence of the protein expressed from the coding region is not changed.

[0031] In the present invention, the identities of base sequences and amino acid sequences are values ​​calculated using GENETYX ver. 17 (GENETYX).

[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. Hydrogenophilus thermorteolus TH-1 (NBRC 14978) exhibits the highest growth rate of any carbon-fixing microorganism (Agricultural and Biological Chemistry, 41, 685-690 (1977)) (doubling in one hour). The Hydrogenophilus thermorteolus NBRC 14978 strain has been internationally deposited under the Budapest Treaty and is publicly available.

[0033] The host Hydrogenophilus bacterium may be a bacterium isolated from nature, or a bacterium that has been genetically modified from a bacterium isolated from nature. Modifications can be performed for purposes such as enabling high expression of the introduced thioesterase gene and fatty acyl-CoA reductase gene. Such modifications can be achieved by, for example, removing (curing) the endogenous plasmid in the Hydrogenophilus bacterium. Methods for removing endogenous plasmids are well known, including methods that utilize plasmid incompatibility, such as the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide; destabilization by introducing a plasmid with the same replication origin as the endogenous plasmid; and destabilization by disrupting factors involved in the plasmid partition system.

[0034] Method for Preparing Transformants A method for obtaining transformants by introducing a thioesterase gene and a fatty acyl-CoA reductase gene into a bacterium belonging to the genus Hydrogenophilus is described below. These genes can be introduced into a bacterium belonging to the genus Hydrogenophilus using a standard method for introducing foreign genes into bacteria. These genes may be introduced directly into a bacterium belonging to the genus Hydrogenophilus. Alternatively, a vector containing the thioesterase gene and the fatty acyl-CoA reductase gene integrated into a transformation vector (e.g., a plasmid vector, a viral vector, a cosmid, a fosmid, a BAC, etc.) may be introduced into a bacterium belonging to the genus Hydrogenophilus. The vector for transformation may contain DNA capable of autonomous replication in Hydrogenophilus bacteria. Examples include broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI Reference Sequence: NC_004444.1), pC194 (NCBI Reference Sequence: NC_002013.1), pK18mobsacB (GenBank: FJ437239.1), and pUB110 (NCBI Reference Sequence: NC_001384.1), as well as genetically modified versions of these vectors (e.g., pCAMO-6). Among these, pCAMO-6 (SEQ ID NO: 7) is preferred. pCAMO-6 can be prepared from its DNA sequence by those skilled in the art using gene synthesis services, etc. Examples of promoters contained in the vector include the tac promoter, lac promoter, trc promoter, and each of the OXB1, OXB11 to OXB20 promoters from Oxford Genetics. Examples of terminators contained in the vector include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the bacteriophage λt0 transcription terminator, and the T7 terminator.The thioesterase gene and the fatty acyl-CoA reductase gene can be introduced (transformed) into a bacterium belonging to the genus Hydrogenophilus by a general method, such as the calcium chloride method or the electric pulse method (electroporation).

[0035] Disruption of Acyl-CoA Dehydrogenase Gene The transformant of the present invention preferably has an exogenous thioesterase gene and an exogenous fatty acyl-CoA reductase gene, and has the acyl-CoA dehydrogenase gene on the chromosome of a Hydrogenophilus bacterium disrupted. The acyl-CoA dehydrogenase gene on the genome of a Hydrogenophilus bacterium may be disrupted before the thioesterase gene and fatty acyl-CoA reductase gene are introduced, or the thioesterase gene and fatty acyl-CoA reductase gene may be introduced into the Hydrogenophilus bacterium, and then the acyl-CoA dehydrogenase gene on the genome may be disrupted.

[0036] In the present invention, "disruption of the acyl-CoA dehydrogenase gene" refers to the activity of the gene product, acyl-CoA dehydrogenase, being lower than that of a parent strain in which the acyl-CoA dehydrogenase gene is not disrupted. Preferably, the activity of the gene product is 50% or less, preferably 10% or less, and more preferably 1% or less, of that of the parent strain. A decrease in gene expression level leads to a decrease in the activity of the gene product. Gene expression level can be measured by quantitative PCR (qPCR) or next-generation sequencing (NGS). Although normal gene expression may occur even when a portion of the gene's coding region or expression regulatory region is mutated, this also constitutes "gene disruption" if the activity of the gene product is lower than that of a wild-type 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.

[0037] For example, a disrupted acyl-CoA dehydrogenase gene can be prepared by PCR or other methods, and then introduced into a parent strain to induce homologous recombination between the disrupted gene and a gene on the genome, thereby replacing the acyl-CoA dehydrogenase gene on the chromosome with the disrupted gene. Gene disruption methods using homologous recombination are well known. The present inventors have discovered the rpsL gene as a gene responsible for streptomycin sensitivity that functions in Hydrogenophilus bacteria (particularly in streptomycin-resistant strains) and can be used as a counterselection marker. Using this gene, the acyl-CoA dehydrogenase gene of Hydrogenophilus bacteria can be disrupted by homologous recombination.

[0038] The mutant of the present invention is preferably one in which the coding region of the acyl-CoA dehydrogenase gene is mutated. Mutation of the 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 a 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 in the gene or at multiple dispersed sites.

[0039] The number of nucleotides to be deleted, substituted, or inserted is preferably 3 or more, more preferably 5 or more, more preferably 10 or more, more preferably 20 or more, and even more preferably 50 or more. It is also preferable to delete, replace, or insert 1% or more, more preferably 5% or more, more preferably 10% or more, and even more preferably 50% or more of the nucleotides of the entire gene. This ensures that each gene is disrupted. The entire gene may be deleted or replaced (i.e., 100% of the nucleotides constituting the gene). The number of nucleotides to be inserted may be 100,000 or less, 1,000 or less, or 100 or less.

[0040] To explain the method for disrupting the acyl-CoA dehydrogenase gene in detail, we use the example of disrupting the gene by deleting the entire coding region. First, a DNA fragment containing the DNA encoding the acyl-CoA dehydrogenase gene of a Hydrogenophilus bacterium, the DNA 5' upstream of the acyl-CoA dehydrogenase gene coding region, and the DNA 3' downstream of the acyl-CoA dehydrogenase gene coding region is amplified by PCR using the genomic DNA of the Hydrogenophilus bacterium as a template. The amplified DNA fragment is then ligated to a marker cassette containing a streptomycin sensitivity gene (counterselection marker) functional in Hydrogenophilus bacteria and a kanamycin resistance gene (positive selection marker) functional in Hydrogenophilus bacteria to produce a plasmid. To improve the efficiency of homologous recombination, the DNA in the 5' upstream region and the DNA in the 3' downstream region are each preferably 10 nucleotides or longer, preferably 50 nucleotides or longer, and more preferably 100 nucleotides or longer. Alternatively, it is not necessary to ligate DNA consisting of a completely identical base sequence to the 5' upstream region of the region to be deleted and the 3' downstream region of the region to be deleted, respectively; the longer the region used for homologous recombination, the lower the identity between the 5' upstream region or the 3' downstream region of the region to be deleted, even if homologous recombination can occur.

[0041] Next, a gene for disruption lacking the acyl-CoA dehydrogenase gene is prepared by PCR amplification of the DNA fragment lacking the acyl-CoA dehydrogenase gene using the above-mentioned plasmid DNA containing the acyl-CoA dehydrogenase gene as a template, followed by circularization using T4 polynucleotide kinase and T4 DNA ligase.

[0042] Next, homologous recombination is performed using this disruption gene. The disruption gene is introduced into a streptomycin-resistant strain of a Hydrogenophilus bacterium, and a strain that is resistant to streptomycin but has lost its kanamycin resistance is selected. This results in a genetic recombinant in which the marker cassette inserted into the acyl-CoA dehydrogenase gene has been lost, i.e., a genetic recombinant in which the acyl-CoA dehydrogenase gene has been deleted.

[0043] Examples of wild-type acyl-CoA dehydrogenase genes include the following DNAs (c1) to (c5): (c1) DNA comprising the nucleotide sequence of SEQ ID NO: 5; (c2) DNA comprising a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 5 (particularly, consisting of a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity to SEQ ID NO: 5), and encoding a polypeptide having acyl-CoA dehydrogenase activity; (c3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; (c4) DNA comprising 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 (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 encoding a polypeptide having acyl-CoA dehydrogenase activity; (c5) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, in which 1 to 80, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been deleted, substituted, inserted, or added (particularly, consisting of the amino acid sequence of SEQ ID NO: 6, in which 1 to 80, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been deleted, substituted, inserted, or added), and having acyl-CoA dehydrogenation activity. SEQ ID NO: 5 is the nucleotide sequence of the HPTL_0940 gene, which is an acyl-CoA dehydrogenase gene in a Hydrogenophilus thermorteolus wild-type strain, and SEQ ID NO: 6 is the amino acid sequence of the polypeptide encoded by the HPTL_0940 gene of a Hydrogenophilus thermorteolus wild-type strain.

[0044] The acyl-CoA dehydrogenase activity of a test polypeptide is confirmed by spectrophotometric analysis of a redox reaction using ferrocenium hexafluorophosphate. Spectrophotometric analysis is performed by observing the change in the absorption spectrum at 300 nm when the substrate octanoyl-CoA is added to a mixed solution of ferrocenium hexafluorophosphate and a cell lysate of Escherichia coli JM109 strain in which the test polypeptide has been overexpressed. If the test polypeptide has acyl-CoA dehydrogenase activity, a reduction reaction of ferrocenium hexafluorophosphate occurs, and a decrease in the absorption spectrum at 300 nm is measured. Specific reaction conditions can be appropriately determined by those skilled in the art by referring to "Lodewijk I, et al., 1993. A simple spectrophotometric assay for long-chain acyl-CoA dehydrogenase activity measurements in human skin fibroblasts. Ann Clin Biochem. 30: 293-297."

[0045] (2) Method for Producing Fatty Alcohol The present invention provides a method for producing aliphatic alcohol using the transformant of the present invention described above. This method includes a step of culturing the transformant of the present invention, particularly a step of culturing the transformant of the present invention using an inorganic or organic medium while supplying a mixed gas containing hydrogen, oxygen, and carbon dioxide. The gas supplied is preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide, but other gases may be mixed in as long as aliphatic alcohol can be efficiently produced.

[0046] 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 aliphatic alcohols using substantially only carbon dioxide as a carbon source (especially using only carbon dioxide). Therefore, in the method of the present invention, it is preferable to use an inorganic medium that does not contain carbon sources such as organic matter or carbonates, i.e., to culture using substantially only carbon dioxide as a carbon source (especially using carbon dioxide as the only carbon source). In the present invention, "using carbon dioxide as the sole carbon source" includes cases where unavoidable amounts of other carbon sources are mixed in.

[0047] The pH of the culture medium used for culture is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, bacterial growth and mixed gas solubility in the medium are high, enabling highly efficient production of aliphatic alcohols. When performing batch culture, the mixed gas can be sealed in a sealed culture vessel and cultured statically or with shaking. Shaking culture is preferred because it improves the solubility of the mixed gas in the medium. When performing continuous culture, the mixed gas can be continuously supplied to a sealed culture vessel while being cultured with shaking, or the transformant 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, the transformant grows well and aliphatic alcohols can be produced efficiently. The supply rate of the mixed gas or raw material gas is 10 to 60 L / hour, preferably 10 to 40 L / hour, and more preferably 10 to 20 L / hour per L of medium. Within this range, the growth of the transformant is favorable, aliphatic alcohols can be produced efficiently, and waste of the mixed gas is reduced. The culture temperature is preferably 35 to 55°C, more preferably 37 to 52°C, and even more preferably 50 to 52°C. Within this range, the growth of the transformant is favorable, and aliphatic alcohols can be produced efficiently.

[0048] By culturing as described above, aliphatic alcohols are produced in the culture solution. The aliphatic alcohols can be recovered by recovering the culture solution, but they can also be separated from the reaction solution by known methods. Such known methods include separation and distillation.

[0049] 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.

[0050] (1) Construction of Gene Expression Plasmids. Gene expression plasmids were constructed using Gibson Assembly. Using Tepidophilus thermophilus genomic DNA as a template, a DNA fragment of SEQ ID NO: 1, a thioesterase gene lacking the DNA region encoding the N-terminal peptide presumed to be the periplasmic transport signal sequence (hereinafter sometimes referred to as a mutant thioesterase gene), was amplified by PCR. The periplasmic transport signal sequence was deduced using SignalP 6.0 (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ). Furthermore, using Marinobacter lutaoensis genomic DNA as a template, a DNA fragment of SEQ ID NO: 3, a fatty acyl-CoA reductase gene, was amplified by PCR. PCR was performed according to standard procedures using a Thermo Fisher Scientific 2720 Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as a reaction reagent. The resulting reaction solution was then subjected to electrophoresis using a 1% agarose gel, and the DNA fragment was recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0051] The following primers were used for PCR: Primers for amplifying the mutant thioesterase gene of Tepidophilus thermophilus (SEQ ID NO: 1): (a-1) 5'-GATCTGGAGGAGAAACGCATATGGCCACGATTCTCGTCTTCGGC-3' (SEQ ID NO: 8) (b-1) 5'-ACGCGGCGCCGCCTCCGGGCGCTGAGAATTCGAGCTCCGTCGAC-3' (SEQ ID NO: 9) Primers (a-1) and (b-1) contained sequences homologous to the vector pCAMO-6. Primers for amplifying the fatty acyl-CoA reductase gene of Marinobacter lutaoensis (SEQ ID NO: 3) were: (a-2) 5'-GATCTGGAGGAGAAACGCATATGGCAACACCGCATGTATCCACC-3' (SEQ ID NO: 10) and (b-2) 5'-AGCGAGAAAGAAACAGGCCGCCTGAGAATTCGAGCTCCGTCGAC-3' (SEQ ID NO: 11). Primers (a-2) and (b-2) contained sequences homologous to the vector pCAMO-6. Electrophoresis detected DNA fragments of approximately 0.6 kbp for the mutant thioesterase gene and approximately 1.5 kbp for the fatty acyl-CoA reductase gene, which were recovered from agarose gel.

[0052] To perform DNA ligation by Gibson Assembly, the plasmid vector pCAMO-6 was amplified by PCR using the DNA fragment of SEQ ID NO: 7 as a template. The following primers were used for PCR: Primers for amplifying the plasmid vector pCAMO-6: (a-3) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 12) (b-3) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 13) Electrophoresis detected a DNA fragment of approximately 5.2 kbp corresponding to the vector gene, which was recovered from an agarose gel.

[0053] The DNA fragment of the vector pCAMO-6 was ligated with either the DNA fragment of the thioesterase gene or the DNA fragment of the fatty acyl-CoA reductase gene using Gibson Assembly Master Mix (New England Biolabs). The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the resulting transformants were plated onto LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C with shaking. Plasmid DNA was extracted from the culture medium. The nucleotide sequence of the gene inserted into each plasmid was analyzed by Sanger analysis at Eurofins Genomics, and confirmed to match the sequence in the database.

[0054] (2) Construction of strains carrying mutant thioesterase genes or fatty acyl-CoA reductase genes (2-1) Gene introduction. Hydrogenophilus thermorteolus strain TH-1 (NBRC 14978) (hereinafter referred to as "TH-1 strain") was transformed with each of the plasmids obtained in "(1) Construction of gene expression plasmids" by electroporation. The strains were then plated on LB solid medium containing 50 μg / mL kanamycin and cultured at 52°C for 24 hours. Each strain grown on LB solid medium was inoculated into LB solid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 52°C for 24 hours. Amplification of the insert fragments of each plasmid was confirmed for each strain grown on LB solid medium by PCR. As a result, amplification of DNA fragments of lengths corresponding to the respective genes was confirmed. The TH-1 transformant transformed with a plasmid containing the mutant thioesterase gene of Tepidophilus thermophilus was designated as the TE strain, and the TH-1 transformant transformed with a plasmid containing the fatty acyl-CoA reductase gene of Marinobacter lutaoensis was designated as the FAR strain.

[0055] (2-2) Thioesterase Activity Measurement. The mutant thioesterase gene-transfected strain (TE strain) of Hydrogenophilus thermorteolus prepared as described above was inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. A gas mixture of H2:O2:CO2 (7.5:1:1.5) was supplied during the culture, and the culture was incubated at 52°C for 24 hours with shaking. Bacterial cells were harvested from 2 mL of the culture by centrifugation (4°C, 5,000 g, 10 minutes). The cells were suspended in 1 mL of reaction buffer (200 mM Tris-HCl (pH 7.5)) and disrupted by sonication to obtain a cell lysate. This was used as the crude enzyme solution. For the thioesterase activity assay, C12:0-acyl-ACP from Escherichia coli, which has a structure similar to that of C12:0-acyl-ACP from Hydrogenophilus thermorteolus (C12:0-acyl refers to a linear, saturated acyl group with 12 carbon atoms), was prepared as a substrate. Crude C12:0-acyl-ACP was prepared by conjugating lauroyl-CoA to ACP from Escherichia coli using Escherichia coli holo-ACP synthase as a catalyst. Specific preparation conditions can be appropriately determined by those skilled in the art by referring to "Leonardo L, et al., 2016. A high-yield optimized method for the production of acylated ACPs enabling the analysis of enzymes involved in P. falciparum fatty acid biosynthesis. Biochem Biophysics Reports. 8: 310-317." The crude enzyme solution was mixed with the same volume of crude C12:0-acyl-ACP solution and incubated at 52°C for 15 minutes. The reaction mixture was analyzed by SDS-PAGE, and thioesterase activity was assessed by confirming the disappearance of the C12:0-acyl-ACP band due to hydrolysis. A negative control (NC) strain, obtained by introducing the empty vector pCAMO-6 into Hydrogenophilus thermoruteolus TH-1, was also cultured and lysed in the same manner, and thioesterase activity was assessed.

[0056] Figure 1 shows the results of SDS-PAGE analysis of Tepidophilus thermophilus transformants with the mutant thioesterase gene. Here, NC is the negative control strain transformed with an empty vector, and Std is the crude C12:0-acyl-ACP solution used in the reaction. A band of approximately 10 kDa for C12:0-acyl-ACP was present in the NC strain but disappeared in the TE strain. It can be seen that when the DNA of SEQ ID NO: 1 was introduced into the TH-1 strain, an enzyme functioning as a thioesterase was produced, and C12:0-acyl-ACP was degraded.

[0057] (2-3) Measurement of Fatty Acyl-CoA Reductase Activity. A cell lysate was prepared from the Marinobacter lutaoensis (FAR strain) transfected with the fatty acyl-CoA reductase gene prepared as described above, as described in "(2-2) Measurement of Thioesterase Activity." The supernatant of this cell lysate was used as a crude enzyme solution to measure fatty acyl-CoA reductase activity. The crude enzyme solution was mixed with reaction buffer, 5 mM NADPH as a coenzyme, and 1 mM lauroyl-CoA or myristoyl-CoA as a substrate, and the reaction was carried out at 52°C for 60 minutes. The reaction solution was analyzed by gas chromatography (Shimadzu Corporation: GC-2014, Polar-WAX column). The produced 1-dodecanol or 1-tetradodecanol was identified by detecting peaks with the same column retention time as the authentic samples.

[0058] The chromatogram obtained for the transformant with the fatty acyl-CoA reductase gene of Marinobacter lutaoensis is shown in Figure 2. Here, NC is the negative control strain transformed with an empty vector, and Std is the 1-dodecanol and 1-tetradecanol standard (100 μM). Unlike the NC strain, the FAR strain, in which the DNA of SEQ ID NO: 3 was introduced into the TH-1 strain, produced 1-dodecanol and 1-tetradecanol, indicating that an enzyme functioning as a fatty acyl-CoA reductase was produced.

[0059] (2-4) Production of fatty alcohols. The FA-1 strain, in which both the mutant thioesterase gene of Tepidophilus thermophilus and the fatty acyl-CoA reductase gene of Marinobacter lutaoensis were introduced into the wild-type TH-1 strain, and the NC strain, in which an empty vector was introduced, were grown in liquid medium A containing 50 μg / mL of kanamycin [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4·7H2O 0.014 g, MgSO4·7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4·7H2O 28 mg, CuSO4·5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4·5H2O 4.0 mg, CoCl2·6H2O 4.0 mg]. Using a platinum loop, inoculate 1 L of distilled water (pH 7.0) containing 1 mg of FA-1 strain. A gas mixture of H2:O2:CO2 = 7.5:1:1.5 is supplied during cultivation, and the culture is incubated with shaking at 52°C for 24 hours. After cultivation, the culture supernatant is obtained by centrifugation (4°C, 5,000 g, 10 minutes). Unlike the culture supernatant of the NC strain, which contains an empty vector, the culture supernatant of the FA-1 strain contains 1-dodecanol and 1-tetradecanol.

[0060] (3) Construction of Acyl-CoA Dehydrogenase Gene Disruption Strains (3-1) Isolation of Streptomycin-Resistant Strains. Hydrogenophilus thermorteolus TH-1 strain was inoculated into a test tube containing 5 mL of liquid medium A using a platinum loop. The test tube was then cultured at 52°C under a gas mixture of H2:O2:CO2 = 7.5:1:1.5 with shaking. After 24 hours, the culture was spread onto LB solid medium containing 100 μg / ml streptomycin and cultured at 52°C for 72 hours. Three colonies were confirmed to form on the LB solid medium containing 100 μg / ml streptomycin. These colonies were then inoculated into a test tube containing 5 mL of liquid medium A containing 100 μg / ml streptomycin using a platinum loop. The test tube was then cultured at 52°C under a gas mixture of H2:O2:CO2 = 7.5:1:1.5 with shaking. 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.

[0061] (3-2) Construction of a plasmid (pCAMO-7) for gene disruption. 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, a streptomycin-sensitive gene encoding the S12 ribosomal protein, was amplified by PCR. The following primers were used for PCR:

[0062] Primers for amplifying the wild-type rpsL gene of the TH-1 strain: (a-4) 5'-CTGGAGGAGAAACGCATATGCCAACCATCAACCAGTTGGTG-3' (SEQ ID NO: 14) (b-4) 5'-CGACGGAGCTCGAATTCTTATTTCTTGCCCGCAGCGGC-3' (SEQ ID NO: 15) Primers (a-4) and (b-4) contain sequences homologous to the vector pCAMO-6. Electrophoresis detected a DNA fragment of approximately 0.4 kbp for the rpsL gene derived from the TH-1 strain, which was recovered from an agarose gel.

[0063] To perform DNA ligation by Gibson Assembly, the plasmid vector pCAMO-6 was amplified by PCR using the DNA fragment of SEQ ID NO: 7 as a template. Primers (a-3) and (b-3) were used for PCR. A DNA fragment of approximately 5.2 kbp corresponding to the vector gene was detected and recovered from an agarose gel.

[0064] The DNA fragment of the pCAMO-6 vector synthesized above and the DNA fragment of the rpsL gene were ligated using Gibson Assembly. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method. The resulting mixture was then plated onto LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C with shaking. Plasmid DNA was extracted from the culture medium. The sequence of the rpsL gene inserted into the plasmid was analyzed by Sanger analysis and confirmed to match the sequence in the database. The resulting plasmid was named pCAMO-7. pCAMO-7 contains the streptomycin sensitivity gene (rpsL gene) and the kanamycin resistance gene derived from pCAMO-6.

[0065] (3-3) Construction of DNA (pCAMO-8) for Disrupting the HPTL_0940 Gene Using genomic DNA from the wild-type TH-1 strain as a template, a DNA fragment containing the HPTL_0940 gene, the 5' upstream region of the HPTL_0940 gene, and the 3' downstream region of the HPTL_0940 gene was amplified by PCR. The following primers were used for PCR: Primers for amplifying a DNA fragment containing the HPTL_0940 gene and surrounding DNA: (a-5) 5'-GAATCGCGAAATAAGGCCCTTGGTCACGCCACTTGGCACGC-3' (SEQ ID NO: 16) (b-5) 5'-CTATCAACAGGAGTCCCACCTTTCATCAACCGGCAAGGGGAG-3' (SEQ ID NO: 17) Primers (a-5) and (b-5) contained sequences homologous to those of the vector pCAMO-7. As a result of electrophoresis, a DNA fragment of approximately 5.9 kbp corresponding to the HPTL_0940 gene and DNA of the upstream and downstream regions of the HPTL_0940 gene was detected and recovered from the agarose gel.

[0066] To perform DNA ligation using Gibson Assembly, the plasmid vector pCAMO-7 was amplified by PCR. The following primers were used for PCR: Primers for amplifying the plasmid vector pCAMO-7: (a-6) 5'-GGACTCCTGTTGATAGATCCAGTAATGACC-3' (SEQ ID NO: 18) (b-6) 5'-GGCCTTATTTCGCGATTCCCAAGAAGACAG-3' (SEQ ID NO: 19) Electrophoresis detected a DNA fragment of approximately 3.4 kbp, which was recovered from an agarose gel.

[0067] The DNA fragment of the pCAMO-7 vector synthesized above was ligated to a DNA fragment of the disruption gene containing the HPTL_0940 gene and its flanking regions using Gibson Assembly. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the transformation mixture was plated onto LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated using a platinum loop into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C. Plasmid DNA was extracted from the culture medium. The nucleotide sequence of the gene inserted into the plasmid was analyzed by Sanger analysis and confirmed to match the sequence in the database.

[0068] Using the thus prepared plasmid as a template, a DNA fragment of the HPTL_0940-deleted gene was amplified by PCR. The following primers were used for PCR: (a-7) 5'-GCGCACACCGGGAGCATAGCGTGGCGGATCG-3' (SEQ ID NO: 20) (b-7) 5'-CCATCTCCTATCCGAATGGATCGAACGTTTGTTTGAATGATACG-3' (SEQ ID NO: 21). Electrophoresis detected a DNA fragment of approximately 6.3 kbp. This DNA fragment was ligated using T4 DNA ligase and T4 DNA kinase (Takara Bio Inc.) to generate a circular plasmid lacking the HPTL_0940 gene. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, plated on LB medium containing 50 μg / mL kanamycin, and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured with shaking at 37°C. Plasmid DNA was extracted from the culture medium. The nucleotide sequence of the gene inserted into the plasmid was analyzed by Sanger analysis and confirmed to match the sequence in the database. The resulting plasmid was named pCAMO-8. pCAMO-8 contains both flanking regions of the HPTL_0940 gene and a marker cassette containing a streptomycin sensitivity gene (counterselection marker) and a kanamycin resistance gene (positive selection marker).

[0069] (3-4) Disruption of the HPTL_0940 gene (positive selection and counterselection) Using the HPTL_0940 gene disruption plasmid pCAMO-8 obtained in (3-3) as a template, a DNA fragment lacking the E. coli replication origin was amplified by PCR using the following primers: (a-8) 5'-GGCGGAGCCTATGGAAAAACGCC-3' (SEQ ID NO: 22) (b-8) 5'-CGGGGTCTGACGCTCAGTGGAAC-3' (SEQ ID NO: 23) Electrophoresis detected a DNA fragment of approximately 5.7 kbp. These DNA fragments were ligated to prepare a circular plasmid lacking the E. coli replication origin. The resulting reaction mixture was transformed into the streptomycin-resistant SR88 strain of Hydrogenophilus thermorteolus by electroporation. The transformants were then plated onto LB solid medium containing 50 μg / mL kanamycin and cultured at 52°C for 48 hours. The grown transgenic strain was then inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. A gas mixture of H2:O2:CO2 (7.5:1:1.5) was supplied during the culture, and the resulting culture was shaken at 52°C for 24 hours. Because the introduced circular plasmid lacked the origin of replication, the strains that grew on the kanamycin-containing medium were not those that grew due to the kanamycin resistance gene of the circular plasmid, but rather those that grew due to the integration of the kanamycin resistance gene into the genome of Hydrogenophilus thermorteolus through the first round of homologous recombination. In the first round of homologous recombination, the entire circular plasmid was integrated into the genome of H. thermorteolus by a single crossover. The resulting culture was plated on LB solid medium containing 100 μg / mL streptomycin and cultured at 52°C for 24 hours.Strains grown on streptomycin-containing medium were either wild-type strains or strains in which a marker cassette consisting of a kanamycin resistance gene and a streptomycin sensitivity gene had been lost due to a second homologous recombination between the 5' upstream region or the 3' downstream region of the Hydrogenophilus thermorteolus genome, resulting in the 5' upstream region and the 3' downstream region of the HPTL_0940 gene being adjacent to each other, i.e., the HPTL_0940 gene that was previously present between them. Strains grown on LB solid medium were screened for strains with a disrupted HPTL_0940 gene by colony PCR. Colony PCR was performed using primers (a-5) and (b-5) according to standard methods. A DNA fragment of approximately 5.9 kbp was amplified in wild-type strains, while a DNA fragment of approximately 3.3 kbp was amplified in strains lacking the HPTL_0940 gene. The strain in which a DNA fragment of approximately 3.3 kbp lacking the HPTL_0940 gene was amplified was designated Δ0940 strain.

[0070] (4) Introduction of the mutant thioesterase gene and fatty acyl-CoA reductase gene into the acyl-CoA dehydrogenase gene-disrupted strain (Δ0940 strain). (4-1) Gene Introduction. The resulting Δ0940 strain was transformed with a plasmid containing the mutant thioesterase gene from Tepidophilus thermophilus and the fatty acyl-CoA reductase gene from Marinobacter lutaoensis inserted into pCAMO-6. The resulting transformants were plated onto LB solid medium containing 50 μg / mL kanamycin and cultured at 52°C for 48 hours. Each strain growing on LB solid medium was inoculated onto LB solid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 52°C for 24 hours. PCR was used to confirm amplification of the plasmid insert fragments for the strains growing on LB solid medium, confirming amplification of DNA fragments of the length corresponding to each gene. The resulting transformant was designated FA-2.

[0071] (4-2) Production of Aliphatic Alcohols by Transformants. The prepared FA-2 strain was inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. A gas mixture of H2:O2:CO2 (7.5:1:1.5) was supplied during the culture, and the strain was cultured at 52°C for 24 hours with shaking. After the culture, the culture was centrifuged (4°C, 5,000 g, 10 minutes) to obtain the culture supernatant. The culture supernatant of the FA-2 strain produced greater amounts of 1-dodecanol and 1-tetradecanol than the culture supernatant of the FA-1 strain, a wild-type Hydrogenophilus thermorteolus TH-1 strain into which a mutant thioesterase gene from Tepidophilus thermophilus and a fatty acyl-CoA reductase gene from Marinobacter lutaoensis had been introduced.

[0072] The transformants of the Hydrogenophilus bacteria of the present invention can be prepared by referring to the description in the Examples. In addition, the other strains described herein are either internationally deposited under the Budapest Treaty, are held by an institution from which they can be obtained without conditions, are commercially available, or can be prepared by those skilled in the art based on the present specification and are publicly available.

[0073] SEQ ID NOs: 1 to 7 are shown below.

[0074] The transformant of the present invention can highly efficiently produce aliphatic alcohols, which are raw materials in high demand in the cosmetics, detergent, and other oleochemical product industries, using carbon dioxide as the sole carbon source. This contributes to the highly efficient industrial production of chemical products while resolving global warming caused by increased carbon dioxide levels.

Claims

1. A transformant of a Hydrogenophilus bacterium into which a gene encoding a thioesterase that produces fatty acids from acyl-ACP and a gene encoding a fatty acyl-CoA reductase that produces fatty alcohols from fatty acyl-CoA have been introduced.

2. The transformant according to claim 1, wherein the thioesterase gene for synthesizing fatty acids from acyl-ACP is the DNA of (a1), (a2), (a3), (a4), or (a5) below: (a1) DNA comprising the nucleotide sequence of SEQ ID NO: 1 (a2) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 1 and encoding a polypeptide having thioesterase activity for synthesizing fatty acids from acyl-ACP (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 with SEQ ID NO: 2 and having thioesterase activity for synthesizing fatty acids from acyl-ACP (a5) DNA encoding a polypeptide 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: 2 and having thioesterase activity for synthesizing fatty acids from acyl-ACP 3. The transformant according to claim 1 or 2, wherein the fatty acyl-CoA reductase gene that produces a fatty alcohol from fatty acyl-CoA is the DNA of (b1), (b2), (b3), (b4), or (b5) below. (b1) DNA comprising the base sequence of SEQ ID NO: 3; (b2) DNA encoding a polypeptide comprising a base sequence having 90% or more identity with SEQ ID NO: 3 and having fatty acyl-CoA reductase activity to produce fatty alcohol from fatty acyl-CoA; (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 with SEQ ID NO: 4 and having fatty acyl-CoA reductase activity to produce fatty alcohol from fatty acyl-CoA; (b5) DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 50 amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 4 and having fatty acyl-CoA reductase activity to produce fatty alcohol from fatty acyl-CoA.

4. The transformant according to any one of claims 1 to 3, in which the acyl-CoA dehydrogenase gene on the chromosome of the Hydrogenophilus bacterium is disrupted.

5. The transformant according to claim 4, wherein the acyl-CoA dehydrogenase gene is the DNA of (c1), (c2), (c3), (c4), or (c5) below: (c1) DNA comprising the nucleotide sequence of SEQ ID NO: 5; (c2) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 5, and encoding a polypeptide having acyl-CoA dehydrogenase activity; (c3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6; (c4) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 6 and having acyl-CoA dehydrogenase activity; (c5) DNA encoding a polypeptide having an amino acid sequence in which 1 to 80 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6, and having acyl-CoA dehydrogenase activity.

6. The transformant according to any one of claims 1 to 5, wherein the Hydrogenophilus bacterium is Hydrogenophilus thermorteolus.

7. A method for producing an aliphatic alcohol, comprising a step of culturing the transformant according to any one of claims 1 to 6.

Images