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WO2025134868A1 - Transformant producteur d'alcools aliphatiques d'une bactérie appartenant au genre hydrogenophilus - Google Patents

Transformant producteur d'alcools aliphatiques d'une bactérie appartenant au genre hydrogenophilus Download PDF

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WO2025134868A1
WO2025134868A1 PCT/JP2024/043666 JP2024043666W WO2025134868A1 WO 2025134868 A1 WO2025134868 A1 WO 2025134868A1 JP 2024043666 W JP2024043666 W JP 2024043666W WO 2025134868 A1 WO2025134868 A1 WO 2025134868A1
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fatty
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英明 湯川
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Utilization Of Carbon Dioxide Institute Co Ltd
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group

Definitions

  • the present invention relates to a Hydrogenophilus bacterial transformant capable of producing aliphatic alcohols, and a method for producing aliphatic alcohols using this transformant.
  • Japan aims to reduce its emissions of greenhouse gases such as carbon dioxide and methane by 46% by 2030 compared to 2013 levels.
  • gases such as carbon dioxide, methane, and carbon monoxide are attracting attention as carbon raw materials with a higher degree of sustainability, 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 fixing and effectively utilizing carbon dioxide, which contributes greatly to global warming.
  • aliphatic alcohols are widely used as raw materials for detergents, additives for cosmetics, medicines, foods, etc., diesel fuel, etc.
  • the main raw material for fatty alcohols is natural fats and oils contained in plants.
  • palm oil extracted from the fruit of the oil palm, is the most widely consumed in the world.
  • large-scale development of oil palm plantations has been carried out to meet the rapidly increasing demand for oil and fats.
  • large-scale development of oil palm plantations brings with it social problems such as deforestation, reduction of biodiversity, and forced labor.
  • RSPO Roundtable on Sustainable Palm Oil
  • microorganisms synthesize fatty acyl-acyl carrier protein (acyl-ACP), the final product, through fatty acid synthesis, but do not possess the genes involved in fatty alcohol synthesis. For this reason, various methods have been proposed for producing fatty alcohols by culturing transformants in which foreign genes encoding the enzymes that make up this metabolic pathway have been introduced into a host such as Escherichia coli.
  • acyl-ACP fatty acyl-acyl carrier protein
  • 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 decomposition enzyme gene of the host Escherichia coli.
  • Non-Patent Document 2 also reports an attempt similar to that of Non-Patent Document 1 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 decomposition enzyme gene of the host Escherichia coli.
  • 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 a complicated process 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.
  • 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 is a method for producing aliphatic alcohols using a cyanobacterium, a photosynthetic bacterium, as a host.
  • cyanobacteria have a higher carbon dioxide fixation capacity than plants, this capacity is not sufficient, and therefore the method of using cyanobacteria as a host has not yet been put to practical use as an industrial method for producing aliphatic alcohols.
  • the objective of the present invention is to provide a Hydrogenophilus bacterial transformant capable of efficiently producing aliphatic alcohols using carbon dioxide as the sole carbon source, and a method for efficiently producing aliphatic alcohols using this transformant.
  • Hydrogenophilus bacteria do not inherently produce fatty alcohols because they do not have a gene encoding an enzyme that catalyzes the fatty alcohol synthesis reaction, but they can produce fatty alcohols by introducing a gene encoding an enzyme that catalyzes the fatty alcohol synthesis reaction.
  • the present invention has been completed based on the above findings, and provides the following [1]-[7].
  • [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.
  • DNA containing the nucleotide sequence of SEQ ID NO: 1 (a2) A DNA comprising a nucleotide sequence having 90% or more identity to SEQ ID NO: 1 and encoding a polypeptide having thioesterase activity that synthesizes a fatty acid from an acyl-ACP. (a3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 (a4) A DNA encoding a polypeptide comprising an amino acid sequence having 90% or more identity to SEQ ID NO: 2 and having thioesterase activity for synthesizing fatty acids from acyl-ACP.
  • (a5) A 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: 2, and having a thioesterase activity for synthesizing a fatty acid from an acyl-ACP.
  • the transformant according to [1] or [2], wherein the fatty acyl-CoA reductase gene that produces a fatty alcohol from fatty acyl-CoA is DNA of the following (b1), (b2), (b3), (b4), or (b5).
  • (b1) DNA containing the nucleotide sequence of SEQ ID NO: 3
  • (b2) A DNA encoding a polypeptide having a nucleotide sequence having 90% or more identity to SEQ ID NO: 3 and having fatty acyl-CoA reductase activity that produces a fatty alcohol from fatty acyl-CoA.
  • (b3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:4
  • acyl-CoA dehydrogenase gene is DNA of the following (c1), (c2), (c3), (c4), or (c5): (c1) DNA containing the base sequence of SEQ ID NO:5 (c2) A DNA containing a nucleotide sequence having 90% 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) A DNA encoding a polypeptide comprising an amino acid sequence having 90% or more identity to SEQ ID NO:6 and having acyl-CoA dehydrogenase activity.
  • (c5) A DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 80 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6 and having acyl-CoA dehydrogenase activity.
  • a method for producing an aliphatic alcohol comprising a step of culturing the transformant described in any one of [1] to [6].
  • Measures to curb the increase in carbon dioxide include reducing carbon dioxide emissions and fixing the emitted carbon dioxide.
  • energy sources such as solar, wind, and geothermal energy are being used in place of fossil energy.
  • the increase in carbon dioxide cannot be sufficiently curbed even by using these types of energy. 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 produce organic matter that can be used as food, feed, fuel, etc. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve two problems at the same time: global warming caused by an increase in carbon dioxide, and the difficulty in securing food, feed, and fuel. It can also produce in-demand raw materials for chemical products while suppressing global warming caused by an increase in carbon dioxide.
  • Wild-type Hydrogenophilus bacteria have the gene for acyl-CoA dehydrogenase, an enzyme that breaks down fatty acids, on their genome, and have the ability to decompose fatty acids by producing active acyl-CoA dehydrogenase.
  • acyl-CoA dehydrogenase an enzyme that breaks down fatty acids, is disrupted, the decomposition of fatty acids, which are precursors of fatty alcohols, is suppressed, and the productivity of fatty alcohols is improved.
  • Fatty alcohols are widely used as raw materials for detergents, additives for cosmetics, medicines, foods, etc., and as diesel fuel.
  • the transformant of the present invention it is possible to produce fatty alcohols on an industrial scale, which can solve the various problems associated with the mass production of palm oil, the raw material for fatty alcohols.
  • Gas chromatography chromatograms showing that cell lysate from 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.
  • the transformant (transformed cell) of the present invention is a transformant in which a gene encoding a thioesterase that produces fatty acid from acyl-ACP and a gene encoding fatty acyl-CoA reductase that produces fatty alcohol from fatty acyl-CoA are introduced into a host Hydrogenophilus bacterium.
  • the transformant of the present invention is a Hydrogenophilus bacterium having an exogenous thioesterase gene that produces fatty acid from acyl-ACP and an exogenous fatty acyl-CoA reductase gene that produces fatty alcohol from fatty acyl-CoA.
  • the range of acyl groups in the acyl-ACP that can be used as a substrate may vary depending on the amino acid sequence of the thioesterase, but for example, a linear or branched, saturated or unsaturated acyl group having 8 to 18 carbon atoms (particularly, any number of carbon atoms from 8 to 18) can be used as a substrate.
  • the range of fatty acyl groups in the fatty acyl-CoA that can be used as a substrate may vary depending on the amino acid sequence of the fatty acyl-CoA reductase, but for example, a fatty acyl-CoA having a linear or branched, saturated or unsaturated fatty acyl group having 12 to 18 carbon atoms (particularly, any number of carbon atoms from 8 to 18) can be used as a substrate.
  • the above-mentioned 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 a method known to those skilled in the art.
  • 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, and it is sufficient if it is a DNA that encodes a polypeptide having this thioesterase activity.
  • a polypeptide has thioesterase activity using acyl-ACP as a substrate is confirmed by reacting the test polypeptide with RCO-ACP (R represents a linear saturated alkyl group with 11 carbon atoms) and confirming by SDS-PAGE that RCO-ACP is decomposed and consumed.
  • RCO-ACP represents a linear saturated alkyl group with 11 carbon atoms
  • SDS-PAGE SDS-PAGE
  • a preferred example of the thioesterase gene is the thioesterase gene of Tepidiphilus thermophilus, and in particular, the thioesterase gene of Tepidiphilus lacking a DNA region encoding an N-terminal peptide presumed to be a periplasmic transport signal sequence.
  • the thioesterase gene of Tepidiphilus lacking a presumed signal sequence is a DNA consisting of the base sequence of SEQ ID NO: 1.
  • a DNA that contains a base sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO:1 (particularly, consisting of a base sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO:1) and encodes a polypeptide having thioesterase activity that synthesizes fatty acids from acyl-ACP.
  • a preferred example of the thioesterase gene is a DNA encoding a thioesterase from Tepidophilus thermophilus, particularly a DNA encoding a thioesterase from Tepidophilus thermophilus lacking an N-terminal peptide presumed to be a periplasmic transport signal sequence.
  • the thioesterase from Tepidophilus thermophilus lacking a presumed signal sequence is a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
  • a DNA that contains an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO:2 (particularly, consisting of an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO:2) and encodes a polypeptide having thioesterase activity that produces fatty acids from acyl-ACP.
  • DNA can also be used that encodes 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 acid from acyl-ACP.
  • the fatty acyl-CoA reductase gene does not need to be known to have been identified as a fatty acyl-CoA reductase gene, and may be any DNA that encodes a polypeptide having fatty acyl-CoA reductase activity.
  • test polypeptide has fatty acyl-CoA reductase activity using fatty acyl-CoA as a substrate is confirmed by reacting the test polypeptide with lauroyl-CoA in the presence of NADPH and detecting the generated 1-dodecanol by gas chromatography or the like.
  • a preferred example of the fatty acyl-CoA reductase gene is the fatty acyl-CoA reductase gene of Marinobacter lutaoensis.
  • the fatty acyl-CoA reductase gene of Marinobacter lutaoensis is a DNA having the base sequence of SEQ ID NO:3.
  • a DNA that contains a base 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, it consists of a base sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO:3) and encodes a polypeptide having fatty acyl-CoA reductase activity that produces fatty alcohols from fatty acyl-CoA.
  • a preferred example of the fatty acyl-CoA reductase gene is a 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 that encodes a polypeptide comprising an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO:4 (particularly, consisting of an amino acid sequence having 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 fatty alcohols from fatty acyl-CoA.
  • DNA can also be used that encodes 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, 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 has fatty acyl-CoA reductase activity that produces fatty alcohols from fatty acyl-CoA.
  • a DNA sequence encoding a 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 substitutions do not change the amino acid sequence of the protein expressed from the coding region.
  • the identity of base sequences and amino acid sequences is a value calculated using GENETYX ver.17 (GENETYX).
  • Hydrogenophilus bacteria examples include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidans, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038.
  • Hydrogenophilus thermoluteolus is preferred because it has a top-level growth rate and carbon dioxide fixation ability as a carbon dioxide fixation microorganism. Hydrogenophilus bacteria can be easily isolated from all over the world.
  • a preferred strain of Hydrogenophilus thermorteolus is the TH-1 (NBRC 14978) strain.
  • Hydrogenophilus thermorteolus TH-1 (NBRC 14978) strain shows the highest growth rate among carbon fixation microorganisms [Agricultural and Biological Chemistry, 41, 685-690 (1977)] (doubling in one hour). Hydrogenophilus thermorteolus NBRC 14978 strain has been internationally deposited under the Budapest Treaty and is publicly available.
  • the host Hydrogenophilus bacterium may be a bacterium isolated from nature, or may be a bacterium obtained by genetically modifying a bacterium isolated from nature, for example, to enable high expression of the introduced thioesterase gene and fatty acyl-CoA reductase gene. Such modification can be achieved by removing (curing) endogenous plasmids in Hydrogenophilus bacteria, etc.
  • Methods for removing endogenous plasmids are well known, and include methods that utilize plasmid incompatibility, such as a method of applying chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide, a method of destabilizing the endogenous plasmid by introducing a plasmid having the same replication origin as the endogenous plasmid, and a method of destabilizing the endogenous plasmid by destroying factors involved in the plasmid partition system.
  • plasmid incompatibility such as a method of applying chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide, a method of destabilizing the endogenous plasmid by introducing a plasmid having the same replication origin as the endogenous plasmid, and a method of destabilizing the endogenous plasmid by destroying factors involved in the plasmid partition system.
  • a method for obtaining a transformant by introducing a thioesterase gene and a fatty acyl-CoA reductase gene into a Hydrogenophilus bacterium is described below. These genes can be introduced into Hydrogenophilus bacteria by using a general method for introducing foreign genes into bacteria. These genes may be directly introduced into Hydrogenophilus bacteria, or a vector for transformation (e.g., a plasmid vector, a virus vector, a cosmid, a fosmid, a BAC, etc.) incorporating the thioesterase gene and the fatty acyl-CoA reductase gene may be introduced into Hydrogenophilus bacteria.
  • a vector for transformation e.g., a plasmid vector, a virus vector, a cosmid, a fosmid, a BAC, etc.
  • the vector for transformation may contain DNA capable of autonomously replicating in Hydrogenophilus bacteria, and examples of such vectors include broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI Reference Sequence: NC_004444.1), pC194 (NCBI Reference Sequence: NC_002013.1), pK18mobsacB (GenBank: FJ437239.1), and pUB110 (NCBI Reference Sequence: NC_001384.1), as well as genetically modified versions of these vectors (e.g., pCAMO-6).
  • broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI
  • pCAMO-6 (SEQ ID NO: 7) is preferable.
  • pCAMO-6 can be prepared from the DNA sequence by those skilled in the art using gene synthesis services or the like.
  • promoters contained in the vector include tac promoter, lac promoter, trc promoter, and each of the OXB1, OXB11 to OXB20 promoters from Oxford Genetics, and examples of terminators contained in the vector include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the bacteriophage ⁇ t0 transcription terminator, and the T7 terminator.
  • the thioesterase gene and the fatty acyl-CoA reductase gene can be introduced (transformed) into a Hydrogenophilus bacterium by a general method, such as the calcium chloride method or the electric pulse method (electroporation method).
  • the transformant of the present invention preferably has an exogenous thioesterase gene and an exogenous fatty acyl-CoA reductase gene, and also has the acyl-CoA dehydrogenase gene on the chromosome of a Hydrogenophilus bacterium disrupted.
  • the acyl-CoA dehydrogenase gene in the genome of a Hydrogenophilus bacterium may be disrupted and then a thioesterase gene and a fatty acyl-CoA reductase gene may be introduced thereinto, or the thioesterase gene and a fatty acyl-CoA reductase gene may be introduced into the Hydrogenophilus bacterium and then the acyl-CoA dehydrogenase gene in the genome may be disrupted.
  • disruption of the acyl-CoA dehydrogenase gene means that the activity of the acyl-CoA dehydrogenase product of this gene is lower than that of a parent strain in which the acyl-CoA dehydrogenase gene is not disrupted.
  • the activity of the product of this gene is 50% or less, particularly 10% or less, particularly 1% or less of the activity of the parent strain.
  • the expression level of a gene can be measured by quantitative PCR (qPCR) or next-generation sequencing (NGS).
  • Gene disruption can be achieved by mutating (base deletion, substitution, insertion, addition, or a combination thereof) the coding region or gene expression regulatory region of the gene on the chromosome of the Hydrogenophilus bacterium. Methods for gene disruption are well known, including, for example, gene knockout methods using homologous recombination, and methods of randomly introducing gene mutations using a mutagen and screening based on phenotypes.
  • a disrupted acyl-CoA dehydrogenase gene can be created by PCR or other techniques, and this disrupted gene can be introduced into a parent strain to cause homologous recombination between the disrupted gene and a gene on the genome, thereby replacing the acyl-CoA dehydrogenase gene on the chromosome with this disrupted gene.
  • Gene disruption methods by homologous recombination are well known.
  • the present inventors have found that the rpsL gene is a gene responsible for streptomycin sensitivity that functions in Hydrogenophilus bacteria (especially 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.
  • the mutant of the present invention is preferably one in which the coding region of the acyl-CoA dehydrogenase gene has been mutated.
  • mutate the coding region typically, all or a portion of each gene may be deleted. Alternatively, a gene may be destroyed by inserting a certain nucleotide, oligonucleotide, or polynucleotide into the gene. Alternatively, all or a portion of a gene may be replaced with another nucleotide, oligonucleotide, or polynucleotide.
  • multiple nucleotide deletions, substitutions, and/or insertions may be introduced at one location in the gene, or may be distributed over multiple locations.
  • 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 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 more preferably 50% or more of the nucleotides of the entire length of the gene. This ensures that each gene is destroyed. It is also possible to delete or replace the entire length of the gene (i.e., 100% of the number of nucleotides constituting the gene).
  • the number of nucleotides to be inserted can be 100,000 or less, 1,000 or less, or 100 or less.
  • a DNA fragment containing the DNA of the region encoding the acyl-CoA dehydrogenase gene of a bacterium of the genus Hydrogenophilus, the DNA of the 5' upstream region of the region encoding the acyl-CoA dehydrogenase gene, and the DNA of the 3' downstream region thereof is amplified by PCR using the genomic DNA of a bacterium of the genus Hydrogenophilus as a template, and the amplified DNA fragment is then ligated to a marker cassette containing a streptomycin sensitivity gene (counter selection marker) that functions in bacteria of the genus Hydrogenophilus and a kanamycin resistance gene (positive selection marker) that functions in bacteria of the genus Hydrogenophilus to prepare a plasmid.
  • a streptomycin sensitivity gene counter selection marker
  • kanamycin resistance gene positive selection marker
  • the DNA of the 5' upstream region and the DNA of the 3' downstream region are each 10 nucleotides or more, preferably 50 nucleotides or more, more preferably 100 nucleotides or more.
  • a gene for disruption is created in which the acyl-CoA dehydrogenase gene is deleted.
  • a DNA fragment in which the acyl-CoA dehydrogenase gene is deleted is amplified by PCR, and then circularized using T4 polynucleotide kinase and T4 DNA ligase.
  • the disruption gene is introduced into a streptomycin-resistant strain of Hydrogenophilus bacteria, and a strain that is resistant to streptomycin and 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.
  • Examples of the wild-type acyl-CoA dehydrogenase gene include the following DNAs (c1) to (c5).
  • (c1) DNA containing the base sequence of SEQ ID NO:5 (c2) A DNA containing a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity with 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 with SEQ ID NO:5), and encoding a polypeptide having acyl-CoA dehydrogenase activity.
  • DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:6 (c4) DNA encoding a polypeptide comprising an amino acid sequence having 90% or more, particularly 95% or more, particularly 98% or more, particularly 99% or more identity with 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 with SEQ ID NO:6) and having acyl-CoA dehydrogenase activity.
  • (c5) a DNA encoding a polypeptide comprising an amino acid sequence in which 1 to 80, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6 (particularly, consisting of an amino acid sequence in which 1 to 80, 1 to 50, 1 to 30, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6), 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 of a Hydrogenophilus thermorteolus wild-type strain
  • SEQ ID NO: 6 is the amino acid sequence of a polypeptide encoded by the HPTL_0940 gene of a Hydrogenophilus thermorteolus wild-type strain.
  • acyl-CoA dehydrogenase activity in a test polypeptide is confirmed by spectrophotometric analysis of the redox reaction using ferrocenium hexafluorophosphate.
  • the 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 cell lysate of Escherichia coli JM109 strain in which the test polypeptide is overexpressed.
  • 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.”
  • the present invention provides a method for producing an 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 in 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 the aliphatic alcohol 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 aliphatic alcohols using substantially only carbon dioxide as the carbon source (particularly, 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, that is, to culture using substantially only carbon dioxide as the carbon source (particularly, using only carbon dioxide as the carbon source).
  • "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 aliphatic alcohols can be produced with high efficiency.
  • the mixed gas can be sealed in a sealed culture vessel and cultured by stationary culture or shaking, with shaking culture being preferred since it improves the dissolution of the mixed gas into the medium.
  • the mixed gas can be continuously supplied to a sealed culture vessel while cultured by shaking, or the transformant can be cultured by introducing the mixed gas into the medium by bubbling using a sealed culture vessel.
  • the volume ratio of hydrogen, oxygen, and carbon dioxide in the feed gas 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.
  • the supply rate of the mixed gas or raw 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.
  • the culture temperature is preferably 35 to 55° C., more preferably 37 to 52° C., and even more preferably 50 to 52° C. Within this range, the transformant grows well and aliphatic alcohols can be produced efficiently.
  • 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.
  • FAVORGEN GEL/PCR Purification Mini Kit
  • the primers (a-1) and (b-1) contain sequences homologous to the vector pCAMO-6.
  • Primer (a-2) for amplifying the fatty acyl-CoA reductase gene of Marinobacter lutaoensis (SEQ ID NO: 3): 5'-GATCTGGAGGAGAAACGCATATGGCAACACCGCATGTATCCACC-3' (SEQ ID NO: 10)
  • the primers (a-2) and (b-2) contain sequences homologous to the vector pCAMO-6.
  • DNA fragments of about 0.6 kbp were detected for the mutant thioesterase gene and about 1.5 kbp for the fatty acyl-CoA reductase gene, and were recovered from agarose gel.
  • 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.
  • a DNA fragment of about 5.2 kbp corresponding to the vector gene was detected and recovered from the agarose gel.
  • the above DNA fragment of vector pCAMO-6 and the above DNA fragment of the thioesterase gene or the above DNA fragment of the fatty acyl-CoA reductase gene were ligated to each other.
  • the resulting reaction solution was used to transform Escherichia coli JM109 strain by the heat shock method, which was then spread onto LB medium containing 50 ⁇ g/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 ⁇ g/mL kanamycin using a platinum loop, cultured with shaking at 37°C, and plasmid DNA was extracted from the culture medium.
  • the base sequence of the gene inserted into each plasmid was analyzed by Eurofins Genomics Co., Ltd. using the Sanger method, and it was confirmed that it matched the sequence in the database.
  • TH-1 strain Gene introduction Hydrogenophilus thermorteolus TH-1 strain (NBRC 14978) (hereinafter sometimes referred to as "TH-1 strain") was transformed with each of the plasmids obtained in the section "(1) Construction of gene expression plasmid” by the electric pulse method (electroporation method), and the transformed strain was inoculated onto LB solid medium containing 50 ⁇ g/mL kanamycin and cultured at 52°C for 24 hours.
  • Each of the strains grown on the LB solid medium was inoculated onto LB solid medium containing 50 ⁇ g/mL kanamycin using a platinum loop and cultured at 52°C for 24 hours.
  • amplification of the insert fragment of each plasmid was confirmed by PCR.
  • amplification of DNA fragments of the length corresponding to each gene was confirmed.
  • the TH-1 transformant transformed with a plasmid containing the mutant thioesterase gene of Tepidophilus thermophilus was named TE strain, and the TH-1 transformant transformed with a plasmid containing the fatty acyl-CoA reductase gene of Marinobacter lutaoensis was named FAR strain.
  • C12:0-acyl-ACP from Escherichia coli, which contains a structure similar to that of C12:0-acyl-ACP from Hydrogenophilus thermorteolus (C12:0-acyl refers to a linear saturated acyl group having 12 carbon atoms), was prepared as a substrate.
  • a crude solution of C12:0-acyl-ACP was prepared by binding lauroyl-CoA to ACP from Escherichia coli using holo-ACP synthase from Escherichia coli as a catalyst. Specific preparation conditions can be appropriately set 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 crude purified C12:0-acyl-ACP solution in the same volume, and the reaction was carried out for 15 minutes at 52° C.
  • the reaction solution was analyzed by SDS-PAGE, and the thioesterase activity was evaluated by confirming the disappearance of the C12:0-acyl-ACP band due to hydrolysis.
  • a cultured cell lysate was obtained from a negative control (NC) strain obtained by introducing the empty vector pCAMO-6 into the Hydrogenophilus thermorteolus TH-1 strain, and the thioesterase activity was evaluated.
  • NC negative control
  • NC is the negative control strain transformed with an empty vector
  • Std is the crude purified solution of C12:0-acyl-ACP used in the reaction.
  • a band of approximately 10 kDa of C12:0-acyl-ACP was present in the NC strain, but disappeared in the TE strain. It can be seen that when DNA of sequence number 1 was introduced into the TH-1 strain, an enzyme that functions as a thioesterase was produced, and C12:0-acyl-ACP was decomposed.
  • the cell lysate was prepared by the method described in "(2-2) Measurement of thioesterase activity" for the fatty acyl-CoA reductase gene-introduced strain (FAR strain) of Marinobacter lutaoensis prepared as described above.
  • the cell lysate supernatant was used as a crude enzyme solution to measure fatty acyl-CoA reductase activity.
  • the crude enzyme solution was added with reaction buffer, mixed with 5 mM NADPH as a coenzyme and 1 mM lauroyl-CoA or myristoyl-CoA as a substrate, and reacted at 52°C for 60 minutes.
  • reaction solution was analyzed by gas chromatography (Shimadzu Corporation: GC-2014, Polar-WAX column), and the generated 1-dodecanol or 1-tetradodecanol was identified by detecting a peak showing the same column retention time as the standard.
  • Figure 2 shows the chromatogram obtained for the transformant with the fatty acyl-CoA reductase gene of Marinobacter lutaoensis.
  • NC is the negative control strain transformed with an empty vector
  • Std is 1-dodecanol and 1-tetradecanol standard (100 ⁇ M).
  • the FAR strain in which DNA of sequence number 3 was introduced into the TH-1 strain, produced 1-dodecanol and 1-tetradecanol, indicating that an enzyme that functions as fatty acyl-CoA reductase was produced.
  • Genomic DNA was extracted from the wild type TH-1 strain (a streptomycin-sensitive strain) according to a standard method. Using the extracted genomic DNA as a template, a DNA fragment containing the rpsL gene, a streptomycin-sensitive gene that codes for the S12 ribosomal protein, 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-4) 5'-CTGGAGGAGAAACGCATATGCCAACCATCAACCAGTTGGTG-3' (SEQ ID NO: 14) (b-4) 5'-CGACGGAGCTCGAATTCTTATTTCTTGCCCGCAGCGGC-3' (SEQ ID NO: 15)
  • the primers (a-4) and (b-4) contain sequences homologous to the vector pCAMO-6.
  • a DNA fragment of about 0.4 kbp was detected for the rpsL gene derived from the TH-1 strain, and was recovered from the agarose gel.
  • 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.
  • the DNA fragment of the vector pCAMO-6 synthesized above and the DNA fragment of the rpsL gene were ligated to each other by Gibson Assembly.
  • the resulting reaction solution was used to transform Escherichia coli JM109 strain by the heat shock method, which was then spread onto LB medium containing 50 ⁇ g/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 ⁇ g/mL kanamycin using a platinum loop, cultured with shaking at 37°C, and plasmid DNA was extracted from the culture medium.
  • pCAMO-7 has a streptomycin sensitivity gene (rpsL gene) and a kanamycin resistance gene derived from pCAMO-6.
  • the primers (a-5) and (b-5) contain sequences homologous to the vector pCAMO-7.
  • the plasmid vector pCAMO-7 was amplified by PCR using the following primers: Primer (a-6) for amplifying the plasmid vector pCAMO-7: 5'-GGACTCCTGTTGATAGATCCAGTAATGACC-3' (SEQ ID NO: 18) (b-6) 5'-GGCCTTATTTCGCGATTCCCAAGAAGACAG-3' (SEQ ID NO: 19) As a result of electrophoresis, a DNA fragment of approximately 3.4 kbp was detected and recovered from the agarose gel.
  • the DNA fragment of the vector pCAMO-7 synthesized above and the DNA fragment of the gene to be disrupted containing the HPTL_0940 gene and both flanking regions thereof were ligated to each other by Gibson Assembly.
  • the resulting reaction solution was used to transform Escherichia coli JM109 strain by the heat shock method, which was then spread onto LB medium containing 50 ⁇ g/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 ⁇ g/mL kanamycin using a platinum loop, cultured with shaking at 37°C, and plasmid DNA was extracted from the culture medium.
  • the base sequence of the gene inserted into the plasmid was analyzed by the Sanger method and confirmed to match the sequence in the database.
  • the DNA fragment of the gene lacking HPTL_0940 was amplified by PCR using the plasmid prepared in this manner as a template.
  • 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)
  • a DNA fragment of about 6.3 kbp was detected.
  • This DNA fragment was ligated by the ligation method using T4 DNA ligase and T4 DNA kinase (Takara Bio Inc.) to prepare a circular plasmid lacking the HPTL_0940 gene.
  • the resulting reaction solution was transformed into Escherichia coli JM109 strain by the heat shock method, and the transformed plasmid was applied to LB medium containing 50 ⁇ g/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 ⁇ g/mL kanamycin using a platinum loop, cultured with shaking at 37°C, and plasmid DNA was extracted from the culture medium.
  • the base sequence of the gene inserted into the plasmid was analyzed by the Sanger method and confirmed to match the sequence in the database.
  • the resulting plasmid was named pCAMO-8, which 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).
  • the resulting reaction mixture was transformed into the streptomycin-resistant SR88 strain of Hydrogenophilus thermorteolus by the electric pulse method (electroporation method), and the transformed strain was applied to LB solid medium containing 50 ⁇ g/mL kanamycin and cultured at 52°C for 48 hours.
  • the strain that grew in the kanamycin-containing medium was not a strain that grew due to the kanamycin-resistant gene of the circular plasmid, but a strain that grew due to the incorporation of the kanamycin-resistant gene into the genome of Hydrogenophilus thermorteolus by the first homologous recombination.
  • the entire introduced circular plasmid is integrated into the genome of Hydrogenophilus thermorteolus by a single crossover.
  • the resulting culture solution was applied to LB solid medium containing 100 ⁇ g/mL streptomycin and cultured at 52° C. for 24 hours.
  • the strains grown on the streptomycin-containing medium are either strains in which the marker cassette consisting of the kanamycin resistance gene and the streptomycin sensitivity gene has been lost due to a second homologous recombination between the 5' upstream region or the 3' downstream region in the genome of Hydrogenophilus thermorteolus, and the 5' upstream region and the 3' downstream region of the HPTL_0940 gene region are adjacent to each other, i.e., strains in which the HPTL_0940 gene that was present between them has been lost, or are wild type.
  • Strains growing on LB solid medium were screened for strains in which the HPTL_0940 gene had been disrupted by colony PCR.
  • Colony PCR was performed by standard methods using primers (a-5) and (b-5).
  • a DNA fragment of approximately 5.9 kbp was amplified in wild-type strains, and a DNA fragment of approximately 3.3 kbp was amplified in strains in which the HPTL_0940 gene had been deleted.
  • the strain in which a DNA fragment of approximately 3.3 kbp had been amplified in which the HPTL_0940 gene had been deleted was designated the ⁇ 0940 strain.
  • Each strain growing on the LB solid medium is inoculated into LB solid medium containing 50 ⁇ g/mL kanamycin using a platinum loop, and cultured at 52°C for 24 hours.
  • amplification of the inserted fragment of the plasmid is confirmed by PCR, and amplification of DNA fragments of the length corresponding to each gene is confirmed.
  • the obtained transformed strain is named FA-2 strain.
  • the culture supernatant of the FA-2 strain contains more 1-dodecanol and 1-tetradecanol than the culture supernatant of the FA-1 strain, which is a wild-type Hydrogenophilus thermorteolus TH-1 strain into which a mutant thioesterase gene of Tepidophilus thermophilus and a fatty acyl-CoA reductase gene of Marinobacter lutaoensis have been introduced.

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Abstract

Les bactéries appartenant au genre Hydrogenophilus ne présentent pas de gène codant pour une enzyme catalysant une réaction de production d'alcool aliphatique et ne produisent pas intrinsèquement d'alcool aliphatique. Cependant, en introduisant un gène codant pour une thioestérase produisant un acide gras à partir d'un acyl-ACP et un gène codant pour une acyl-CoA réductase aliphatique produisant un alcool aliphatique à partir d'un acyl-CoA aliphatique dans une bactérie appartenant au genre Hydrogenophilus, ladite bactérie appartenant au genre Hydrogenophilus produit un alcool aliphatique. En outre, l'efficacité de la production d'un alcool d'acide gras est améliorée en perturbant le gène de l'acyl-CoA déshydrogénase sur le chromosome de la bactérie appartenant au genre Hydrogenophilus.
PCT/JP2024/043666 2023-12-21 2024-12-10 Transformant producteur d'alcools aliphatiques d'une bactérie appartenant au genre hydrogenophilus Pending WO2025134868A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160138061A1 (en) * 2014-11-17 2016-05-19 Evonik Degussa Gmbh Fatty acid and derivatives production
WO2019207812A1 (fr) * 2018-04-27 2019-10-31 株式会社Ｃｏ２資源化研究所 Transformant de bactérie du genre hydrogenophilus
WO2020110300A1 (fr) * 2018-11-30 2020-06-04 株式会社Ｃｏ２資源化研究所 Transformant produisant de l'acide lactique d'une bactérie du genre hydrogenophilus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160138061A1 (en) * 2014-11-17 2016-05-19 Evonik Degussa Gmbh Fatty acid and derivatives production
WO2019207812A1 (fr) * 2018-04-27 2019-10-31 株式会社Ｃｏ２資源化研究所 Transformant de bactérie du genre hydrogenophilus
WO2020110300A1 (fr) * 2018-11-30 2020-06-04 株式会社Ｃｏ２資源化研究所 Transformant produisant de l'acide lactique d'une bactérie du genre hydrogenophilus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Uniprot 10 October 2018 (2018-10-10), ANONYMOUS: "RecName: Full=Acyl-coenzyme A dehydrogenase {ECO:0000256|ARBA:ARBA00020144}; EC=1.3.8.7 {ECO:0000256|ARBA:ARBA00012033};EC=1.3.8.8 {ECO:0000256|ARBA:ARBA00012040};", XP093324545, retrieved from UniprotKB Database accession no. A0A2Z6DY07 *
DATABASE UniProt 11 November 2015 (2015-11-11), ANONYMOUS: "SubName: Full=Lysophospholipase L1 or related esterase {ECO:0000313|EMBL:CUB05634.1};", XP093324540, retrieved from UniProtKB Database accession no. A0A0K6IRC5 *
DATABASE UniProt 7 June 2017 (2017-06-07), ANONYMOUS: "SubName: Full=Dehydrogenase {ECO:0000313|EMBL:ONF42868.1};", XP093324543, retrieved from UniProtKB Database accession no. A0A1V2DQE8 *

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