Hydrogenophilus bacterium transformant capable of producing cis, cis-muconic acid or catechol

Abstract

Provided is a hydrogenophilus bacterium transformant in which a catechol 2,3-dioxygenase gene on the genome is disrupted and which has a protocatechuate decarboxylase gene and a protocatechuate decarboxylase gene, or a protocatechuate decarboxylase gene, a protocatechuate decarboxylase gene, and a catechol 1,2-dioxygenase gene, which are exogenous. The transformant can efficiently produce muconic acid or catechol by utilizing carbon dioxide as the sole carbon source.

Description

Transformant of Hydrogenophilus bacteria capable of producing cis,cis-muconic acid or catechol

[0001] The present invention relates to a transformant of a Hydrogenophilus bacterium capable of producing cis,cis-muconic acid or catechol, a method for producing cis,cis-muconic acid or catechol using the transformant, and a gene-disrupted strain of a Hydrogenophilus bacterium suitable as a host for producing the transformant.

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

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

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

[0005] Here, cis,cis-muconic acid (hereinafter sometimes abbreviated as "muconic acid") has the following structure: Due to its highly reactive double bond and two carboxyl groups, muconic acid is a highly useful dicarboxylic acid used as a starting material for the production of various polymers and pharmaceuticals. Muconic acid derivatives derived from muconic acid include adipic acid, terephthalic acid, and polyurethane. Adipic acid is a raw material for nylon-6,6, and terephthalic acid is a raw material for polyethylene terephthalate and polyethylene. These compounds are industrially important and are used in large quantities in the production of everyday products. Muconic acid can be produced by chemical processes that oxidize catechol using a copper catalyst and by biological conversion using benzoic acid. The raw materials, catechol and benzoic acid, are derived from petroleum. Currently, most muconic acid derivatives are produced from petroleum feedstocks. Specifically, adipic acid is produced by oxidizing cyclohexane derived from benzene, and terephthalic acid is produced by oxidizing xylene contained in petroleum at 250°C in the presence of a cobalt, manganese, and bromine-containing catalyst. These petroleum-dependent chemical synthesis processes place a significant burden on the environment, so bio-based muconic acid production is needed as an alternative to conventional chemical processes.

[0006] Catechol is a compound having the following structure: In addition to its use in the production of muconic acid, it is also used as an intermediate raw material for fragrances, polymerization inhibitors, antioxidants, pharmaceuticals, and pesticides. It is also used as a raw material for semiconductor photoresist strippers used in printed circuit board manufacturing, activated carbon adsorbents for deoxidation, plating agents, and rubber vulcanizing agents. Catechol is primarily produced by oxidizing phenol with hydrogen peroxide. Phenol, the raw material, is industrially produced using the cumene process, which uses petroleum-derived benzene and propylene as raw materials. This process is complex and energy-intensive due to the high-temperature, high-pressure chemical industrial process. For this reason, there is a need to move away from petroleum-based chemical synthesis processes.

[0007] In order to transition from a society dependent on petroleum, which is feared to be depleted in the future, to a sustainable society, the production of chemicals through microbial fermentation using renewable non-edible biomass resources has attracted attention. However, as mentioned above, the use of biomass has various problems, such as the need for complex processes, the disruption of stable food and feed supplies, and the large environmental impact. Therefore, there is a need for a practical method that uses microorganisms to produce muconic acid and catechol through a simpler process without disrupting the stable supply of food and feed and without imposing a burden on the environment. In particular, there is a need for a practical method that can produce muconic acid and catechol by fixing carbon dioxide.

[0008] Protocatechuate, catechol, and muconate are present in the metabolic pathways of many microorganisms. In many microorganisms, protocatechuate is produced from 3-dehydroshikimate by 3-dehydroshikimate dehydratase and from 4-hydroxybenzoate by 4-hydroxybenzoate hydroxylase. Catechol is also produced from protocatechuate by protocatechuate decarboxylase and from salicylate by salicylate 1-monooxygenase. Catechol is then degraded by ortho- or meta-cleavage and incorporated into the TCA cycle. Ortho-cleavage is catalyzed by catechol 1,2-dioxygenase to produce muconate. Meta-cleavage is catalyzed by catechol 2,3-dioxygenase to produce 2-hydroxymuconic acid semialdehyde.

[0009] Patent Document 1 discloses a technique for producing catechol from glucose using a transformed bacterium in which a transketose gene, a DAHP synthase gene, a 3-dehydroquinate synthase gene, a 3-dehydroshikimate dehydratase gene (qsuB) derived from Klebsiella pneumoniae, and a protocatechuate decarboxylase gene (aroY) derived from Klebsiella pneumoniae have been introduced into a host bacterium of the genus Escherichia or Klebsiella. Patent Document 2 discloses a technique for producing catechol from glucose using a transformed bacterium in which aroY from Lacticaseibacillus rhamnosus has been introduced, and a Corynebacterium genus host in which the functions of a protocatechuate dehydrogenase gene and a catechol 1,2-dioxygenase gene (catA) responsible for ortho-cleavage of catechol have been reduced or deleted. Patent Document 3 discloses a technology for producing muconic acid by cleaving catechol synthesized via salicylic acid using Escherichia coli in which the expression of genes encoding phosphotransferase enzymes, genes encoding pyruvate kinase, and genes encoding enzymes that synthesize L-phenylalanine is suppressed, with catA derived from Pseudomonas putida DOT-T1E. Patent Document 4 discloses a technology for producing muconic acid by using Escherichia coli, whose sugar metabolic pathway has been modified, as a host, and integrating qsuB derived from Bacillus thuringiensis, aroY derived from Klebsiella pneumoniae, and a catechol 1,2-dioxygenase gene (catA) derived from Pseudomonas putida into the chromosome. Patent Document 5 discloses a technology for producing muconic acid from lignin-derived aromatic compounds or biomass containing lignin by using Pseudomonas putida as a host in which the protocatechuate 3,4-dioxygenase gene and the muconate cycloisomerase gene that metabolizes muconic acid have been disrupted, and introducing aroY derived from Klebsiella pneumoniae.Patent Document 6 discloses a technique for producing muconic acid using a transformant in which qsuB and aroY derived from Klebsiella pneumoniae and catA derived from Acinetobacter baylyi ADP1 have been introduced into Escherichia coli in which sugar metabolism and the shikimate pathway have been modified.

[0010] Non-Patent Document 1 teaches a technique for producing catechol from glucose using a transformant in which aroY from Klebsiella pneumoniae has been introduced into Escherichia coli. Non-Patent Document 2 teaches a technique for producing muconic acid from glucose using a transformant in which qsuB from Podospora anserina, aroY from Klebsiella pneumoniae, and catA from Candida albicans have been introduced into Saccharomyces cerevisiae. Non-Patent Document 3 teaches a technique for producing muconic acid from glucose using a transformant in which qsuB from Podospora anserina, aroY from Klebsiella pneumoniae, and catA from Acinetobacter calcoaceticus have been introduced into Saccharomyces cerevisiae. Non-Patent Document 4 teaches a technique for producing muconic acid from glucose using a transformant in which Pseudomonas putida KT2440, in which the protocatechuate 3,4-dioxygenase gene and muconate cycloisomerase gene have been disrupted, has been introduced with qsuB from Bacillus cereus, aroY from Enterobacter cloacae, and catA from Pseudomonas putida. Non-Patent Document 5 teaches a technique for producing muconic acid from glucose by expressing an exogenous protocatechuate decarboxylase gene different from the endogenous qsuB and aroY in Corynebacterium glutamicum 13032, in which the protocatechuate 3,4-dioxygenase gene and muconate cycloisomerase gene have been disrupted. Non-Patent Document 6 teaches a technique for producing muconic acid from glucose by expressing endogenous qsuB and catA genes and aroY gene from Klebsiella pneumoniae in Corynebacterium glutamicum 13032, in which the protocatechuate 3,4-dioxygenase gene and the muconate cycloisomerase gene have been disrupted.Non-Patent Document 7 teaches a technique for producing muconic acid from glucose using a transformant of Escherichia coli in which qsuB and aroY from Klebsiella pneumoniae and catA with improved enzymatic properties from Acinetobacter ADP1 have been introduced. Non-Patent Document 8 teaches a technique for producing muconic acid from glucose by expressing endogenous catA in Klebsiella pneumoniae in which the sugar metabolic pathway has been modified and the protocatechuate 3,4-dioxygenase gene and muconate cycloisomerase gene have been disrupted.

[0011] However, all of these methods produce muconic acid or catechol by growing microorganisms that use sugars, which are carbon raw materials with high production costs, and are not methods for producing muconic acid or catechol using carbon dioxide as a carbon raw material.

[0012] Clostridium bacteria, which are anaerobic microorganisms, grow using chlorine-containing gaseous substrates as a carbon source (essentially carbon monoxide as a carbon source) and are used in the production of chemical products. However, their use as hosts for the production of muconic acid and catechol has not been reported.

[0013] Among bacteria that can grow using carbon dioxide as a carbon source, Cupriavidus necator strain H16 and Hydrogenobacter thermophilus strain TK-6 have been used in the production of chemical products, but their use as hosts for the production of muconic acid and catechol has not been reported.

[0014] U.S. Patent No. 5,272,073, International Publication No. 2019 / 211937, International Publication No. 2017 / 033965, Japanese Patent Application Publication No. 2020-184993, International Publication No. 2020 / 080467, Japanese Patent Application Publication No. 2019-195330

[0015] J. Am. Chem. Soc., 117(9):2395-2400 (1995)Biotechnol Bioeng., 119(2):376-387 (2022)FEMS Yeast Res., 18(2) (2018)Nat Commun., 13(1):4925 (2022)Biochem Biophys Res Commun., 499(2):279-284. (2018)Sci Rep., 8(1):18041 (2018)Sci Rep., 5:13435 (2015)Appl Microbiol Biotechnol., 99(12):5217-5225 (2015)

[0016] The present invention addresses the challenges of providing a transformant of a bacterium belonging to the genus Hydrogenophilus that can efficiently produce muconic acid or catechol using carbon dioxide as a sole carbon source, a method for efficiently producing muconic acid or catechol using the transformant, and a gene-disrupted strain of a bacterium belonging to the genus Hydrogenophilus that is suitable as a host for producing the transformant.

[0017] The present inventors focused on bacteria of the genus Hydrogenophilus as a microorganism capable of fixing carbon dioxide on an industrial scale, with the aim of avoiding the use of high-cost raw materials such as sugars and contributing to global warming countermeasures. Hydrogenophilus bacteria grow by using hydrogen as an energy source to produce organic substances from carbon dioxide. While such bacteria generally grow very slowly, Hydrogenophilus bacteria grow rapidly and have a significantly higher carbon dioxide fixation capacity than plants or photosynthetic bacteria. Hydrogenophilus bacteria do not have enzyme genes for producing muconic acid and catechol. Therefore, in order to impart the ability to produce muconic acid and catechol to Hydrogenophilus bacteria, it is necessary to introduce genes for enzymes that catalyze the reactions that produce muconic acid and catechol.

[0018] However, even when aroY, catA, and qsuB were introduced and expressed in Hydrogenophilus bacteria, muconic acid or catechol was not produced in an amount sufficient for industrial use.

[0019] The present inventors have found that, unlike Pseudomonas putida described in Patent Document 5, Hydrogenophilus bacteria do not possess genes putatively encoding protocatechuate 3,4-dioxygenase or muconate cycloisomerase. However, they do possess a putative catechol 2,3-dioxygenase gene (equivalent to HPTL_0060 in Hydrogenophilus thermorteolus) that meta-cleaves catechol to form 2-hydroxymuconic acid semialdehyde. The present inventors hypothesized that when the catechol 2,3-dioxygenase gene functions, catechol produced intracellularly in Hydrogenophilus bacteria is metabolized, resulting in reduced amounts of muconic acid or catechol secreted into the culture medium supernatant. Furthermore, the present inventors have found that introducing qsuB and aroY into a strain of Hydrogenophilus bacteria in which the putative catechol 2,3-dioxygenase gene on its genome has been disrupted significantly increases the amount of catechol secreted into the culture medium. Furthermore, we found that introducing qsuB, aroY, and catA into a strain in which the putative catechol 2,3-dioxygenase gene on the genome of a Hydrogenophilus bacterium was disrupted significantly increased the amounts of catechol and muconic acid secreted into the medium. Based on these findings, we concluded that the disrupted gene was the catechol 2,3-dioxygenase gene (xylE) and that disruption of xylE is essential for the efficient production of muconic acid or catechol in Hydrogenophilus bacteria.

[0020] The present inventors also found that a xylE-disrupted strain in which qsuB and aroY were introduced produced catechol extremely efficiently using carbon dioxide as the sole carbon source, and that a xylE-disrupted strain in which qsuB, aroY, and catA were introduced produced catechol and muconic acid extremely efficiently using carbon dioxide as the sole carbon source.

[0021] The present invention was completed based on the above findings and provides the following [1] to [8]: [1] A gene-disrupted strain of a bacterium belonging to the genus Hydrogenophilus, in which the catechol 2,3-dioxygenase gene has been disrupted. [2] The gene-disrupted strain of a bacterium belonging to the genus Hydrogenophilus according to [1], in which the catechol 2,3-dioxygenase gene is any one of the DNAs (a1) to (a5) below. (a1) DNA containing the nucleotide sequence of SEQ ID NO: 1. (a2) DNA containing a nucleotide sequence with 90% or more identity to SEQ ID NO: 1 and encoding a polypeptide with catechol 2,3-dioxygenase activity. (a3) ​​DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2. (a4) DNA encoding a polypeptide with 90% or more identity to SEQ ID NO: 2 and having catechol 2,3-dioxygenase activity. (a5) DNA encoding a polypeptide with catechol 2,3-dioxygenase activity, containing an amino acid sequence with 1 to 30 amino acids deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2. [3] A Hydrogenophilus bacterium transformant having a disrupted catechol 2,3-dioxygenase gene on its genome and having exogenous (i) 3-dehydroshikimate dehydratase gene and protocatechuate decarboxylase gene, or (ii) 3-dehydroshikimate dehydratase gene, protocatechuate decarboxylase gene, and catechol 1,2-dioxygenase gene. [4] The Hydrogenophilus bacterium transformant according to [3], wherein the protocatechuate decarboxylase gene is any one of the DNAs (b1) to (b5) below.(b1) DNA containing the base sequence of SEQ ID NO: 3; (b2) DNA containing a base sequence having 90% or more identity to SEQ ID NO: 3 and encoding a polypeptide having protocatechuate decarboxylase activity; (b3) DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 4; (b4) DNA encoding a polypeptide having 90% or more identity to SEQ ID NO: 4 and having protocatechuate decarboxylase activity; (b5) DNA encoding a polypeptide having protocatechuate decarboxylase activity and containing an amino acid sequence in which 1 to 40 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4. [5] A Hydrogenophilus bacterium transformant according to [3] or [4], wherein the catechol 1,2-dioxygenase gene is any of the DNAs (c1) to (c5) below. (c1) DNA containing the base sequence of SEQ ID NO: 5. (c2) DNA containing a base sequence having 90% or more identity to SEQ ID NO: 5 or encoding a polypeptide having catechol 1,2-dioxygenase activity. (c3) DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 6. (c4) DNA encoding a polypeptide having 90% or more identity to SEQ ID NO: 6 and having catechol 1,2-dioxygenase activity. (c5) DNA encoding a polypeptide having catechol 1,2-dioxygenase activity and containing an amino acid sequence in which 1 to 30 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6. [6] A genetically modified Hydrogenophilus bacterium transformant according to any of [3] to [5], wherein the 3-dehydroshikimate dehydratase gene is any of the DNAs (d1) to (d5) below.(d1) DNA comprising the nucleotide sequence of SEQ ID NO: 7 (d2) DNA comprising a nucleotide sequence having 90% or more identity to SEQ ID NO: 7 and encoding a polypeptide having 3-dehydroshikimate dehydratase activity (d3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 (d4) DNA encoding a polypeptide having 90% or more identity to SEQ ID NO: 8 and having 3-dehydroshikimate dehydratase activity (d5) DNA encoding a polypeptide having 3-dehydroshikimate dehydratase activity, comprising an amino acid sequence in which 1 to 60 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 8 [7] A transformant of Hydrogenophilus bacteria according to any of [3] to [6], wherein the Hydrogenophilus bacterium is Hydrogenophilus thermorteolus. [8] A method for producing muconic acid or catechol, comprising a step of culturing the transformant of Hydrogenophilus bacteria according to any of [3] to [7]. [9] A positive selection marker for Hydrogenophilus bacteria, comprising the following DNA (e1) or (e2): (e1) DNA comprising the nucleotide sequence of SEQ ID NO: 11; (e2) DNA comprising a nucleotide sequence having 90% or more identity to SEQ ID NO: 11 and encoding a polypeptide having the activity of conferring bleomycin resistance to Hydrogenophilus bacteria.

[0022] Measures to curb the increase in carbon dioxide include reducing carbon dioxide emissions and fixing emitted carbon dioxide. To reduce carbon dioxide emissions, solar, wind, geothermal, and other energy sources are being used in place of fossil fuels. However, in reality, even these energy sources have not 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 chemical products in demand while curbing global warming caused by increased carbon dioxide.

[0023] Muconic acid and catechol are industrially important chemicals. However, chemical synthesis processes from petroleum feedstocks are undesirable from an environmental perspective. In contrast, the production of muconic acid and catechol using carbon dioxide-fixing microorganisms can simultaneously address global warming caused by increased carbon dioxide emissions and ensure the availability of industrially needed muconic acid and catechol.

[0024] Bacteria that can grow using carbon dioxide as their sole carbon source, utilizing the chemical energy generated by the reaction of hydrogen and oxygen, can 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 such bacteria generally grow slowly, hydrogen-producing bacteria, the Hydrogenophilus genus, have an exceptionally fast 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."

[0025] The gene-disrupted strain of a Hydrogenophilus bacterium of the present invention, in which xylE on the genome has been disrupted, can be suitably used as a host for producing a transformant with improved muconic acid or catechol production ability by introducing a gene into the gene-disrupted strain of the present invention. Transformants obtained by introducing qsuB and aroY into the gene-disrupted strain of the present invention have significantly improved catechol production ability, and transformants obtained by introducing qsuB, aroY, and catA into the gene-disrupted strain of the present invention have significantly improved muconic acid and catechol production ability. As described above, among organisms capable of fixing carbon dioxide, Hydrogenophilus bacteria have particularly excellent carbon dioxide fixation ability. Therefore, the present invention has opened the way to the industrial-scale production of muconic acid and catechol using carbon dioxide.

[0026] Figure 1 is a diagram illustrating the steps of constructing a marker cassette and disrupting the catechol 2,3-dioxygenase gene of a host in an example. Figure 2 is a diagram illustrating the steps of constructing a marker cassette and disrupting the catechol 2,3-dioxygenase gene of a host in an example. Figure 3 is a diagram illustrating the steps of constructing a marker cassette and disrupting the catechol 2,3-dioxygenase gene of a host in an example.

[0027] The present invention will be described in detail below.

[0028] (1) Catechol 2,3-dioxygenase Gene-Disrupted Strain of Hydrogenophilus Bacteria Wild-Type Catechol 2,3-Dioxygenase Gene (Wild-Type xylE) Wild-type Hydrogenophilus bacteria have xylE on their genome. Wild-type Hydrogenophilus bacteria have the ability to metabolize catechol to 2-hydroxymuconic acid semialdehyde by producing active catechol 2,3-dioxygenase. The gene-disrupted strain of the present invention is a Hydrogenophilus bacterium in which xylE on its genome has been disrupted. In the present invention, xylE does not need to have been identified as xylE; it may be DNA encoding a polypeptide having catechol 2,3-dioxygenase activity that meta-cleaves catechol to produce 2-hydroxymuconic acid semialdehyde.

[0029] Examples of wild-type xylE from bacteria of the genus Hydrogenophilus include any of the DNAs (a1) to (a5) below. (a1) DNA comprising the nucleotide sequence of SEQ ID NO: 1 (particularly consisting of the nucleotide sequence of SEQ ID NO: 1); (a2) DNA comprising a nucleotide sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 1 (particularly consisting of a nucleotide sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 1) and encoding a polypeptide having catechol 2,3-dioxygenase activity; (a3) ​​DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (particularly consisting of the amino acid sequence of SEQ ID NO: 2); (a4) DNA encoding a polypeptide comprising an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% identity with SEQ ID NO: 2 (particularly consisting of an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 2) and having catechol 2,3-dioxygenase activity; (a5) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 in which 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: 2 in which 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 catechol 2,3-dioxygenase activity. SEQ ID NO: 1 is the nucleotide sequence of the HPTL_0060 gene of a Hydrogenophilus thermorteolus wild-type strain, and SEQ ID NO: 2 is the amino acid sequence of the polypeptide encoded by the HPTL_0060 gene of a Hydrogenophilus thermorteolus wild-type strain.

[0030] In the present invention, whether a test polypeptide has catechol 2,3-dioxygenase activity is confirmed by reacting the test polypeptide with catechol and detecting an increase in absorbance at 375 nm resulting from the product, 2-hydroxymuconic acid semialdehyde.

[0031] Disruption of the Catechol 2,3-Dioxygenase Gene (xylE) In the present invention, "disruption of the catechol 2,3-dioxygenase gene" refers to a condition in which the activity of the gene product, catechol 2,3-dioxygenase, is reduced compared to that of the parent strain before gene disruption. Preferably, the activity of the gene product is reduced to 50% or less, more preferably 10% or less, and even more preferably 1% or less, of the activity of the parent strain. A decrease in gene expression level reduces the activity of the gene product. Gene expression level can be measured by quantitative PCR (qPCR) or next-generation sequencing (NGS). Although 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 gene product's activity is reduced compared to that of the parent strain. Gene disruption can be achieved by mutating (by base deletion, substitution, insertion, addition, or a combination thereof) the coding region or gene expression regulatory region, such as the promoter region, of the gene on the chromosome of a Hydrogenophilus bacterium. Methods for gene disruption are well known, and include, for example, gene knockout methods using homologous recombination, and methods that involve the introduction of random gene mutations using mutagens and screening based on phenotypes.

[0032] For example, a disrupted xylE gene can be prepared by PCR or other methods, and the disrupted gene can be introduced into a parent strain to induce homologous recombination between the disrupted gene and a gene on the genome, thereby replacing the xylE 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 (SEQ ID NO: 10) as a gene responsible for streptomycin sensitivity that functions in bacteria of the genus Hydrogenophilus (particularly in streptomycin-resistant strains) and can be used as a counterselection marker. By using this gene, xylE in bacteria of the genus Hydrogenophilus can be disrupted by homologous recombination.

[0033] The mutant of the present invention preferably has a mutation in the coding region of xylE. 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.

[0034] The number of nucleotides to be deleted, substituted, or inserted is preferably 3 or more. It is also preferable to delete, replace, or insert nucleotides that account for 1% or more of the entire length of the gene. This ensures that each gene is disrupted. Deletion or replacement may be performed across the entire length of the gene (i.e., 100% of the nucleotides constituting the gene). The number of nucleotides to be inserted may be 100,000 or less.

[0035] Except for the case of deleting the entire region of xylE, it is desirable to avoid introducing the above mutations into the region encoding the C-terminus of catechol 2,3-dioxygenase, which is likely to eliminate or reduce the function of xylE. For example, it is desirable to introduce the above mutations into a region encoding within 95% of the entire region from the N-terminus of catechol 2,3-dioxygenase.

[0036] Hereinafter, a method for disrupting xylE will be described using a method for deleting xylE as an example.

[0037] The DNA fragment consisting of xylE from a bacterium of the genus Hydrogenophilus can be amplified by PCR using the genomic DNA of the bacterium as a template, and then ligated to form a DNA fragment consisting of the 5' upstream region of the region to be deleted and the 3' downstream region of the region to be deleted. The ligated DNA fragment from the 5' upstream region and the 3' downstream region of the region to be deleted is a DNA fragment from which xylE is completely deleted. To improve the efficiency of homologous recombination, the DNA fragments of the 5' upstream region and the 3' downstream region are preferably each 10 nucleotides or longer. Alternatively, the DNA fragments of the 5' upstream region and the 3' downstream region of the region to be deleted do not need to be ligated to DNA fragments having completely identical nucleotide sequences. The longer the region used for homologous recombination, the lower the identity between the 5' upstream region and the 3' downstream region of the region to be deleted, allowing homologous recombination to occur.

[0038] Next, a marker cassette is prepared to be inserted between the 5' upstream and 3' downstream regions of xylE. The marker cassette here comprises a streptomycin sensitivity gene that functions in Hydrogenophilus bacteria and a bleomycin resistance gene (SEQ ID NO: 11) that functions in Hydrogenophilus bacteria, linked adjacently or with a DNA fragment of approximately 10,000 base pairs or less sandwiched between them.

[0039] Next, a marker cassette is inserted between the DNA fragments in the 5' upstream and 3' downstream regions of xylE.

[0040] For example, by linking the genes so that the primers amplifying the 5' upstream region and the 3' downstream region have sequences homologous to the marker cassette gene, a labeling DNA fragment can be obtained by PCR, into which a marker cassette is inserted, in which a DNA fragment containing a gene responsible for streptomycin sensitivity and a DNA fragment containing a bleomycin resistance gene are linked.

[0041] This labeling DNA fragment is introduced into a streptomycin-resistant strain of a Hydrogenophilus bacterium. If the labeling DNA fragment is introduced into the chromosome, the strain will exhibit streptomycin sensitivity and bleomycin resistance. Therefore, using streptomycin sensitivity and bleomycin resistance as an indicator, genetic recombinants in which xylE of the streptomycin-resistant strain has been replaced with the labeling DNA fragment can be selected.

[0042] Next, to eliminate the marker cassette substituted in place of xylE, homologous recombination is carried out using the previously prepared xylE-deleted DNA fragment. Linear DNA fragments of the 5' upstream and 3' downstream regions of the xylE-deleted region are introduced into the streptomycin-sensitive strain (a genetic recombinant in which xylE has been replaced with a labeling DNA fragment), and a strain that has become streptomycin-resistant is selected. It is preferable to select a strain that is streptomycin-resistant and has lost bleomycin resistance. This results in a genetic recombinant in which the marker cassette inserted into xylE has been eliminated, i.e., a genetic recombinant in which xylE has been deleted.

[0043] The deletion of the gene can be confirmed by determining the nucleotide sequence of xylE in the resulting recombinant gene or by checking the amplified size by PCR.

[0044] The following DNAs (e1) or (e2) function in Hydrogenophilus bacteria and can be used as positive selection markers: (e1) DNA containing the nucleotide sequence of SEQ ID NO: 11 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 11); (e2) DNA containing a nucleotide sequence that is 90% or more, 95% or more, 98% or more, or 99% or more identical to SEQ ID NO: 11 and encoding a polypeptide that has the activity of conferring bleomycin resistance to Hydrogenophilus bacteria. In the present invention, the ability of a test DNA to confer bleomycin resistance to Hydrogenophilus bacteria is confirmed by showing improved growth in the presence of 50 μg / mL of bleomycin in Hydrogenophilus bacteria into which the test DNA has been introduced, compared to before the introduction; for example, by showing that the Hydrogenophilus bacteria can grow in the presence of 50 μg / mL of bleomycin, unlike the Hydrogenophilus bacteria before the introduction of the test DNA.

[0045] (2) Transformant of a Catechol 2,3-Dioxygenase Gene-Disrupted Strain The transformant (transformed cell) of the present invention is a transformant of a Hydrogenophilus bacterium in which xylE on the genome has been disrupted and which has exogenous qsuB and aroY or exogenous qsuB, aroY and catA. The transformant of the present invention may be one in which xylE on the genome of a Hydrogenophilus bacterium has been disrupted and then qsuB and aroY or qsuB, aroY and catA have been introduced, or one in which xylE on the genome has been disrupted and then qsuB and aroY or qsuB, aroY and catA have been introduced into a Hydrogenophilus bacterium.

[0046] The aroY, catA, and qsuB used in the present invention do not necessarily have to be known to have been identified as aroY, catA, and qsuB, respectively. They may be DNAs encoding a polypeptide having protocatechuate decarboxylase activity, DNAs encoding a polypeptide having catechol 1,2-dioxygenase activity, and DNAs encoding a polypeptide having 3-dehydroshikimate dehydratase activity, respectively. Protocatechuate decarboxylase activity refers to the enzyme activity that produces catechol from protocatechuate. Catechol 1,2-dioxygenase activity refers to the enzyme activity that produces muconic acid from catechol. 3-Dehydroshikimate dehydratase activity refers to the enzyme activity that produces protocatechuate from 3-dehydroshikimate. Furthermore, in the present invention, aroY, catA, and qsuB may be DNAs of aroY, catA, and qsuB isolated from naturally occurring bacteria, or may be DNAs artificially synthesized using techniques known to those skilled in the art.

[0047] Protocatechuate decarboxylase gene (aroY) In the present invention, it is preferable to use any one of the following DNAs (b1) to (b5) as aroY: (b1) DNA comprising the nucleotide sequence of SEQ ID NO: 3 (particularly consisting of the nucleotide sequence of SEQ ID NO: 3). (b2) DNA comprising a nucleotide sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 3 (particularly consisting of a nucleotide sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 3) and encoding a polypeptide having protocatechuate decarboxylase activity. (b3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (particularly consisting of the amino acid sequence of SEQ ID NO: 4). (b4) DNA encoding a polypeptide comprising an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% identity with SEQ ID NO: 4 (particularly consisting of an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 4) and having protocatechuate decarboxylase activity. (b5) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, in which 1 to 40, particularly 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: 4, in which 1 to 40, particularly 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 protocatechuate decarboxylase activity. SEQ ID NO: 3 is the nucleotide sequence of aroY of Enterobacter asburiae, and SEQ ID NO: 4 is the amino acid sequence of the polypeptide encoded by aroY of Enterobacter asburiae.

[0048] In the present invention, whether a test polypeptide has protocatechuate decarboxylase activity is confirmed by reacting the test polypeptide with protocatechuate and detecting the produced catechol by HPLC or the like.

[0049] Catechol 1,2-dioxygenase Gene (catA) In the present invention, it is preferable to use any one of the following DNAs (c1) to (c5) as catA. (c1) DNA comprising the nucleotide sequence of SEQ ID NO: 5 (particularly consisting of the nucleotide sequence of SEQ ID NO: 5). (c2) DNA comprising a nucleotide sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 5 (particularly consisting of a nucleotide sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 5) and encoding a polypeptide having catechol 1,2-dioxygenase activity. (c3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (particularly consisting of the amino acid sequence of SEQ ID NO: 6). (c4) DNA encoding a polypeptide comprising an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% identity with SEQ ID NO: 6 (particularly consisting of an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with SEQ ID NO: 6) and having catechol 1,2-dioxygenase activity. (c5) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 in which 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 30, 1 to 10, 1 to 5, 1 to 3, or 1 amino acid has been deleted, substituted, inserted, or added), and having catechol 1,2-dioxygenase activity. SEQ ID NO: 5 is the base sequence of catA of Pseudomonas thermotolerans, and SEQ ID NO: 6 is the amino acid sequence of the polypeptide encoded by catA of Pseudomonas thermotolerans.

[0050] In the present invention, whether a test polypeptide has catechol 1,2-dioxygenase activity is confirmed by reacting the test polypeptide with catechol and detecting the produced muconic acid by HPLC or the like.

[0051] 3-Dehydroshikimate dehydratase gene (qsuB) In the present invention, it is preferable to use any one of the following DNAs (d1) to (d5) as qsuB. (d1) DNA comprising the nucleotide sequence of SEQ ID NO: 7 (particularly consisting of the nucleotide sequence of SEQ ID NO: 7); (d2) DNA comprising a nucleotide sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity to SEQ ID NO: 7 (particularly consisting of a nucleotide sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity to SEQ ID NO: 7), and encoding a polypeptide having 3-dehydroshikimate dehydratase activity; (d3) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 (particularly consisting of the amino acid sequence of SEQ ID NO: 8); (d4) DNA comprising an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity to SEQ ID NO: 8 (particularly consisting of an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity to SEQ ID NO: 8), and encoding a polypeptide having 3-dehydroshikimate dehydratase activity; (d5) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, in which 1 to 60, 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: 8, in which 1 to 60, 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 3-dehydroshikimate dehydratase activity. SEQ ID NO: 7 is the base sequence of qsuB of Pseudomonas thermotolerans, and SEQ ID NO: 8 is the amino acid sequence of the polypeptide encoded by qsuB of Pseudomonas thermotolerans.

[0052] In the present invention, whether a test polypeptide has 3-dehydroshikimate dehydratase activity is confirmed by reacting the test polypeptide with 3-dehydroshikimic acid and detecting the produced protocatechuic acid by HPLC or the like.

[0053] In the present invention, the identity of the base sequence and amino acid sequence is a value calculated using GENETYX ver. 17 (GENETYX).

[0054] In the present invention, a DNA that contains a nucleotide sequence that is 90% to 100% identical to the nucleotide sequence of a certain DNA and encodes a polypeptide that has the same type of activity as the polypeptide encoded by that DNA is referred to as a "homolog" of that DNA. Furthermore, a DNA that contains an amino acid sequence that is 90% to 100% identical to the amino acid sequence of a certain polypeptide and encodes a polypeptide that has the same type of activity as that polypeptide is referred to as a homolog of the DNA encoding that polypeptide. Furthermore, a DNA that contains an amino acid sequence in which 1 to 60 amino acids have been deleted, substituted, inserted, or added in the amino acid sequence of a certain polypeptide and encodes a polypeptide that has the same type of activity as that polypeptide is referred to as a homolog of the DNA encoding that polypeptide.

[0055] Examples of Hydrogenophilus bacteria include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidans, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038. Among these, Hydrogenophilus thermoluteolus is preferred because it has the highest growth rate and carbon dioxide fixation ability among carbon dioxide fixation microorganisms. Hydrogenophilus bacteria can be easily isolated from all over the world. A preferred strain of Hydrogenophilus thermorteolus is the TH-1 (NBRC 14978) strain. 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.

[0056] The host Hydrogenophilus bacterium may be a bacterium isolated from nature, or may be a bacterium that has been genetically modified from a bacterium isolated from nature. Modifications can be performed for purposes such as enabling high gene expression. Such modifications can be achieved by, for example, removing (curing) endogenous plasmids in the Hydrogenophilus bacterium. Methods for removing endogenous plasmids are well known, including methods that involve the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide; methods that involve destabilizing the endogenous plasmid by introducing a plasmid with the same replication origin as the endogenous plasmid; and methods that utilize plasmid incompatibility, such as destabilizing the endogenous plasmid by disrupting factors involved in the plasmid partition system.

[0057] Method for Preparing Transformants A method for obtaining transformants by introducing qsuB and aroY or qsuB, aroY and catA into a host Hydrogenophilus bacterium or a xylE-disrupted strain of a Hydrogenophilus bacterium is described below.

[0058] These genes can be introduced into a host using common methods for introducing foreign genes into bacteria. These genes can be introduced directly into the host, or a vector for transformation (e.g., a plasmid vector, a viral vector, a cosmid, a fosmid, a BAC, etc.) incorporating these genes can be introduced into the host. When introducing these genes into a host, they can be cloned into the same plasmid vector or different plasmid vectors.

[0059] These genes may also be integrated into the genome of the host by homologous recombination or the like.

[0060] The vector used 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: 9) is preferred. pCAMO-6 can be prepared by those skilled in the art according to the Examples. 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 Oxford Genetics' OXB1, OXB11 to OXB20 promoters, and 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. Genes can be introduced into hosts (transformed) using standard techniques, such as the calcium chloride method, calcium phosphate method, rubidium chloride method, and electric pulse method (electroporation).

[0061] (3) Method for Producing Muconic Acid or Catechol The present invention provides a method for producing muconic acid or catechol 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 carbon dioxide-containing gas, preferably a mixed gas containing hydrogen, oxygen, and carbon dioxide. The gas supplied is preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide, but other gases may be mixed in as long as muconic acid or catechol can be efficiently produced.

[0062] 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 muconic acid or catechol 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.

[0063] The pH of the culture medium used for culture is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, bacterial growth and the solubility of the mixed gas in the culture medium are high, enabling highly efficient production of muconic acid or catechol. 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 culture medium. When performing continuous culture, the mixed gas can be continuously supplied to a sealed culture vessel while the culture is shaken, or the transformant can be cultured in a sealed culture vessel while the mixed gas is introduced into the culture 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 growth of the bacteria is favorable, and muconic acid or catechol can be efficiently produced. The supply rate of the mixed gas or raw material gas is 10 to 60 L / hour, preferably 10 to 40 L / hour, and more preferably 10 to 20 L / hour per L of medium. Within this range, the growth of the transformant is favorable, muconic acid or catechol can be efficiently produced, 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 muconic acid or catechol can be efficiently produced.

[0064] By culturing as described above, muconic acid or catechol is produced in the culture medium. Muconic acid or catechol can be recovered by recovering the reaction mixture. This recovery can be carried out by any method commonly used in the art, and is not particularly limited. For example, methods such as filtration, centrifugation, salting out, recrystallization, organic solvent extraction, esterification distillation separation, chromatographic separation, and electrodialysis can be used alone or in combination, as needed.

[0065] 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.

[0066] (1) Obtaining a streptomycin-resistant strain Hydrogenophilus thermorteolus TH-1 strain (NBRC 14978) (hereinafter referred to as "TH-1 strain") was cultured in liquid medium A [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, and CoCl2 6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0). The bacteria were inoculated into a test tube containing 5 mL of liquid medium A using a platinum loop. The tube was then gassed with a 7.5:1:1.5 H2:O2:CO2 mixture and incubated at 52°C with shaking. After 24 hours, the culture was spread onto LB solid medium containing 100 μg / mL streptomycin and incubated 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 gassed with a 7.5:1:1.5 H2:O2:CO2 mixture and incubated at 52°C. Since bacterial growth was observed after 24 hours, these strains were streptomycin-resistant variants of the TH-1 strain, and one of them was designated strain SR88.

[0067] (2) PCR, electrophoresis, DNA recovery, and seamless cloning in the construction of marker cassettes and expression plasmids. The plasmid vector pCAMO-6 for PCR, electrophoresis, DNA recovery, and seamless cloning in the construction of marker cassettes and expression plasmids was prepared as follows.

[0068] (2-1) PCR, electrophoresis, and DNA recovery PCR was performed using a Life Technologies DNA Thermal Cycler and KOD One PCR Master Mix (Toyobo Co., Ltd.) as the reaction reagent, according to standard methods. The resulting reaction mixture was then subjected to electrophoresis using a 1% agarose gel, and appropriate DNA fragments were recovered from the gel using a GEL / PCR Purification Mini Kit (FAVORGEN) as needed.

[0069] (2-2) Preparation of Plasmid Vector pCAMO-6 for Seamless Cloning To perform seamless cloning, the plasmid vector pCAMO-6 was amplified by PCR using the DNA of SEQ ID NO: 9 as a template. The following primers were used for PCR. Primers used for amplification of plasmid vector pCAMO-6: (a-1) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 12) (b-1) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 13) As a result of electrophoresis, a DNA fragment of approximately 5.2 kbp corresponding to the vector gene was detected, and the DNA fragment was recovered from the gel.

[0070] (2-3) Ligation of the DNA fragment of the vector pCAMO-6 with other DNA fragments Using recombinase extracted from Escherichia coli JM109, the DNA fragment of the vector pCAMO-6 synthesized above was ligated to the DNA fragment to be inserted. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the transformed cells were plated on LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours.

[0071] (2-4) Plasmid extraction and gene sequence confirmation Each strain growing on LB medium was inoculated using a platinum loop into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL of kanamycin, and cultured with shaking at 37°C, and plasmid DNA was extracted from the culture medium. The sequence of the gene inserted into each plasmid was analyzed by the Sanger method at Eurofins Genomics, and it was confirmed to match the sequence in the database.

[0072] The steps for constructing the marker cassette and disrupting the host's catechol 2,3-dioxygenase gene are shown in Figures 1 to 3. (3) Construction of the Marker Cassette (3-1) Preparation of Counterselection Marker Genomic DNA was extracted from the wild-type TH-1 strain (a streptomycin-sensitive strain) according to standard methods. Using the extracted genomic DNA as a template, a DNA fragment containing the rpsL gene, which encodes the S12 ribosomal protein and is responsible for streptomycin sensitivity, was amplified by PCR. The following primers were used for PCR: Primers for amplifying the wild-type rpsL gene of the TH-1 strain: (a-2) 5'-CTGGAGGAGAAACGCATATGCCAACCATCAACCAGTTGGTG-3' (SEQ ID NO: 14) (b-2) 5'-CGACGGAGCTCGAATTCTTATTTCTTGCCCGCAGCGGC-3' (SEQ ID NO: 15) Primers (a-2) and (b-2) contained sequences homologous to the vector pCAMO-6. Electrophoresis detected a DNA fragment of approximately 0.4 kbp corresponding to the rpsL gene of the wild-type TH-1 strain, and the DNA fragment was recovered from the gel.

[0073] The DNA fragment of vector pCAMO-6 and the DNA fragment of rpsL were ligated together and used to transform Escherichia coli JM109 strain, after which the plasmid was extracted and the sequence of the rpsL gene was confirmed.

[0074] The rpsL gene containing the tac promoter region was amplified using pCAMO-6 containing the rpsL gene as a template. Primers for amplifying the rpsL gene containing the tac promoter region were: (a-3) 5'-CCAAAGTAAAGGAGAGTAGCACATAACGGTTCTGGCAAATATTC-3' (SEQ ID NO: 16) and (b-3) 5'-GCCATATGCGATACTCCTCCTCATTTCTTGCCCGCAGCGGCGCC-3' (SEQ ID NO: 17). Primer (a-3) contained a sequence homologous to the 5' region of the HPTL_0060 gene DNA, while primer (b-3) contained a sequence required for binding to the bleomycin resistance gene. Electrophoresis detected a DNA fragment of approximately 0.4 kbp corresponding to the rpsL gene containing the tac promoter region, and the DNA fragment was recovered from the gel.

[0075] (3-2) Preparation of Positive Selection Marker. A DNA fragment of the bleomycin resistance gene sequence was amplified by PCR using artificially synthesized (partially codon-replaced) DNA from Streptoalloteichus hindustanus as a template using techniques known to those skilled in the art. The following primers were used for PCR: (a-4) 5'-GAAATGAGGAGGAGTATCGCATATGGCTAAACTTACTTCTGCTG-3' (SEQ ID NO: 18) (b-4) 5'-CGGTGAAGCTCGAAACTCAATCTTGTTCCTCTGCAACAAAATG-3' (SEQ ID NO: 19). Primer (a-4) contained a sequence required for binding to the rpsL gene, and primer (b-4) contained a sequence homologous to the DNA in the 3'-flanking region of the HPTL_0060 gene. As a result of electrophoresis, a DNA fragment of approximately 0.4 kbp corresponding to the bleomycin resistance gene was detected, and the DNA fragment was recovered from the gel.

[0076] (3-3) Preparation of Marker Cassette The rpsL gene fragment containing the tac promoter region and the bleomycin resistance gene fragment were mixed, ligated, and amplified by PCR. The following primers were used for PCR: (a-3) 5'-CCAAAGTAAAGGAGAGTAGCACATAACGGTTCTGGCAAATATTC-3' (SEQ ID NO: 20) (b-4) 5'-CGGTGAAGCTCGAAACTCAATCTTGTTCCTCTGCAACAAAATG-3' (SEQ ID NO: 21) Electrophoresis detected a DNA fragment of approximately 1.0 kbp corresponding to the ligated marker cassette gene, and the DNA fragment was recovered from the gel.

[0077] (4) Disruption of the Host Catechol 2,3-Dioxygenase Gene (4-1) Construction of DNA for Disrupting the HPTL_0060 Gene Using the genomic DNA of the wild-type TH-1 strain as a template, DNA fragments corresponding to the 5' and 3' regions of the HPTL_0060 gene were amplified by PCR. The following primers were used for PCR: Primers for Amplifying the 5' Region of the HPTL_0060 Gene: (a-5) 5'-CTACAACACGGATTCCTCTTTCCACTGGCAAG-3' (SEQ ID NO: 22) (b-5) 5'-CTTCGGTGAAGCTCGAAACTGCTACTCTCCTTTACTTTGGGTGG-3' (SEQ ID NO: 23). Primer (b-5) contained a sequence necessary for binding to the 3' region of the gene. Primers for amplifying the 3' region of the HPTL_0060 gene: (a-6) 5'-GTAAAGGAGAGTAGCAGTTTCGAGCTTCACCGAAGTCTATAC-3' (SEQ ID NO: 24) (b-6) 5'-GTTTTACAATTTAATGCAGATGTTCTTGAGTTCCGTGTAGAAC-3' (SEQ ID NO: 25) Primer (a-6) contains a sequence necessary for binding to the 5' region gene. The resulting reaction solution was subjected to electrophoresis using a 1% agarose gel, and DNA fragments of approximately 1.5 kbp and approximately 2.0 kbp corresponding to the 5' and 3' regions of the HPTL_0060 gene, respectively, were detected.

[0078] The DNA fragments from the 5' and 3' regions of the HPTL_0060 gene prepared in this manner were mixed and ligated and amplified by PCR. The following primers were used for PCR: (a-5) 5'-CTACAACACGGATTCCTCTTTCCACTGGCAAG-3' (SEQ ID NO: 22) (b-6) 5'-GTTTTACAATTTAATGCAGATGTTCTTGAGTTCCGTGTAGAAC-3' (SEQ ID NO: 25) Electrophoresis detected a DNA fragment of approximately 3.5 kbp corresponding to the ligated DNA fragment containing the 5' and 3' regions of the HPTL_0060 gene, and the DNA fragment was recovered from the gel.

[0079] (4-2) Construction of DNA for HPTL_0060 gene labeling Using the genomic DNA of the TH-1 strain as a template, DNA fragments corresponding to the 5' and 3' regions of the HPTL_0060 gene were amplified by PCR. The following primers were used for PCR: Primers for amplifying the 5' region of the HPTL_0060 gene: (a-5) 5'-CTACAACACGGATTCCTCTTTCCACTGGCAAG-3' (SEQ ID NO: 22) (b-7) 5'-CAGAACCGTTATGTGCTACTCTCCTTTACTTTGGGTGGATAAAC-3' (SEQ ID NO: 26). Primer (b-7) contained a sequence necessary for binding to the marker cassette. Primers for amplifying the 3' region of the HPTL_0060 gene: (a-7) 5'-GCAGAGGAACAAGATTGAGTTTCGAGCTTCACCGAAGTCTATAC-3' (SEQ ID NO: 27) (b-6) 5'-GTTTTACAATTTAATGCAGATGTTCTTGAGTTCCGTGTAGAAC-3' (SEQ ID NO: 25). Primer (a-7) contained a sequence necessary for binding to the marker cassette. Electrophoresis detected approximately 1.5 kbp and approximately 2.0 kbp DNA fragments corresponding to the 5' and 3' regions of the HPTL_0060 gene, respectively, and the DNA fragments were recovered from the gel.

[0080] The DNA fragments from the 5' and 3' regions of the HPTL_0060 gene prepared in this manner and the marker cassette prepared in (3-3) were mixed and ligated by PCR for amplification. The following primers were used for PCR: (a-5) 5'-CTACAACACGGATTCCTCTTTCCACTGGCAAG-3' (SEQ ID NO: 22) (b-6) 5'-GTTTTACAATTTAATGCAGATGTTCTTGAGTTCCGTGTAGAAC-3' (SEQ ID NO: 25). Electrophoresis detected a DNA fragment of approximately 4.3 kbp corresponding to the DNA fragment containing the DNA fragments from the 5' and 3' regions and the marker cassette DNA fragment, and the DNA fragment was recovered from the gel. This DNA fragment was used for labeling the HPTL_0060 gene.

[0081] (4-3) Labeling of the HPTL_0060 Gene in Streptomycin-Resistant Strains (Positive Selection) The streptomycin-resistant SR88 strain of the TH-1 strain was transformed with the HPTL_0060 gene-labeling DNA prepared in (4-2) by electroporation. The transformants were plated onto LB medium containing 50 μg / mL of the bleomycin antibiotic Zeocin and cultured at 52°C for 48 hours. Each strain grown on LB medium was restreaked onto LB medium containing 50 μg / mL of the bleomycin antibiotic Zeocin (trade name) and cultured at 52°C for 24 hours. Using the resulting genomic DNA of the Zeocin-resistant strain as a template, the DNA region containing the HPTL_0060 gene was amplified by PCR. The following primers were used for PCR: When the HPTL_0060 gene is replaced with the marker cassette, PCR using the primer combination (a-3) and (b-8) amplifies a DNA region of approximately 1.2 kbp. (a-3) 5'-CCAAAGTAAAGGAGAGTAGCACATAACGGTTCTGGCAAATATTC-3' (SEQ ID NO: 20) (b-8) 5'-CGGCGACGTTGATCCGTATCCCCAAATTCCGTG-3' (SEQ ID NO: 28) Electrophoresis detected a DNA fragment of approximately 1.2 kbp corresponding to the sequence in which the HPTL_0060 gene was replaced with the marker cassette. In this strain, the HPTL_0060 gene is labeled with a marker.

[0082] (4-4) Disruption of the HPTL_0060 Gene (Counterselection) The HPTL_0060 gene-labeled strain obtained in (4-3) was transformed with the DNA fragment for HPTL_0060 gene disruption obtained in (4-1) by electroporation. The transformation was then plated onto LB medium containing 400 μg / mL streptomycin and cultured at 52°C for 48 hours. Each strain grown on LB medium was restreaked onto LB medium containing 400 μg / mL streptomycin and cultured at 52°C for 24 hours. The resulting streptomycin-resistant strain was used as a template to amplify the DNA region containing the HPTL_0060 gene by PCR. The following primers were used for PCR: When PCR was performed using the combination of primers (a-8) and (b-8), a DNA fragment of approximately 0.3 kbp was amplified if homologous recombination had occurred with the HPTL_0060 gene disruption DNA fragment, which contained only the 5' and 3' regions and lacked the HPTL_0060 gene. PCR was performed using standard methods using EmeraldAmp PCR Master Mix (Takara Bio Inc.) as the reaction reagent. (a-8) 5'-GTGGTTGGAAAAATGAGCAAACCGTTTCTTGCCTTG-3' (SEQ ID NO: 29) (b-8) 5'-CGGCGACGTTGATCCGTATCCCCAAATTCCGTG-3' (SEQ ID NO: 28). Electrophoresis detected a DNA fragment of approximately 0.3 kbp that would be amplified if the HPTL_0060 gene had been deleted and the marker cassette had not been present. In other words, a strain in which the HPTL_0060 gene had been deleted and disrupted was obtained. One of them was designated as strain Δ0060.

[0083] (5) Construction of an Expression Plasmid for 3-Dehydroshikimate Dehydratase A DNA fragment of qsuB (SEQ ID NO: 7) derived from Pseudomonas thermotolerans was amplified by PCR. Primers for amplifying Pseudomonas thermotolerans qsuB: (a-9) 5'-CTGGAGGAGAAACGCATATGCAGCGTTCGATCGCCACCGTCTC-3' (SEQ ID NO: 30) (b-9) 5'-CGACGGAGCTCGAATTCTCAGAGCCTGGGCTGACGAGCGGCGC-3' (SEQ ID NO: 31). Each primer (a-9) and (b-9) contained a sequence homologous to the vector pCAMO-6. Electrophoresis detected a DNA fragment of approximately 1.9 kbp corresponding to qsuB, and the DNA was recovered from the gel.

[0084] The DNA fragment of vector pCAMO-6 and the DNA fragment of qsuB were ligated together and used to transform Escherichia coli JM109 strain, after which the plasmid was extracted and the sequence of the qsuB gene was confirmed.

[0085] (6) Construction of a Protocatechuate Decarboxylase Expression Plasmid. A DNA fragment of aroY (SEQ ID NO: 3) derived from Enterobacter azubriae was amplified by PCR. Enterobacter azubriae aroY amplification primers: (a-10) 5'-CAGATCTGGAGGAGAAACGCATATGGAAAACCCGATCAACGATC-3' (SEQ ID NO: 32); (b-10) 5'-CTTGTCGACGGAGCTCGAATTCCTATTTCTTATCAGCGAACAGC-3' (SEQ ID NO: 33). Each primer (a-10) and (b-10) contained a sequence homologous to the pCAMO-6 vector. Electrophoresis detected a DNA fragment of approximately 1.5 kbp corresponding to aroY, and the DNA was recovered from the gel.

[0086] The DNA fragment of vector pCAMO-6 and the DNA fragment of aroY were ligated together and used to transform Escherichia coli JM109 strain. Then, the plasmid was extracted and the sequence of the aroY gene was confirmed.

[0087] (7) Construction of an Expression Plasmid for Catechol 1,2-Dioxygenase A DNA fragment of catA (SEQ ID NO: 5) derived from Pseudomonas thermotolerans was amplified by PCR. Primers for amplifying Pseudomonas thermotolerans catA: (a-11) 5'-CTGGAGGAGAAACGCATATGACCGTGAAAATCACTCACACTG-3' (SEQ ID NO: 34) (b-11) 5'-CGACGGAGCTCGAATTCTCAGGCGCCCACGACCTGCAGTGCACG-3' (SEQ ID NO: 35). Each primer (a-11) and (b-11) contained a sequence homologous to the vector pCAMO-6. Electrophoresis detected a DNA fragment of approximately 0.9 kbp corresponding to catA, and the DNA was recovered from the gel.

[0088] The DNA fragment of vector pCAMO-6 and the DNA fragment of catA were ligated to each other, and the Escherichia coli JM109 strain was transformed with the ligated DNA. Then, the plasmid was extracted and the sequence of the catA gene was confirmed.

[0089] (8) Construction of a plasmid for producing catechol A plasmid for producing catechol was constructed by seamless cloning, linking qsuB and aroY via a ribosome binding sequence using PCR. qsuB derived from Pseudomonas thermotolerans was amplified by PCR using the expression vector obtained in (5) as a template and the following primers: (a-12) 5'-GGCTCGTATAATGTGTGGAATTGTGAGCGGATAAC -3' (SEQ ID NO: 36) (b-12) 5'-CATATGCGATACTCCTCCTCAGAGCCTGGGCTGACGAGCG-3' (SEQ ID NO: 37) Primer (b-12) contains a ribosome binding sequence and a sequence homologous to aroY. Electrophoresis detected a DNA fragment of approximately 1.9 kbp corresponding to qsuB, and the DNA was recovered from the gel.

[0090] Enterobacter asbriae aroY was amplified by PCR using the expression vector obtained in (6) as a template and the following primers: (a-13) 5'-CTGAGGAGGAGTATCGCATATGGAAAACCCGATCAACGATCTC-3' (SEQ ID NO: 38) (b-13) 5'-CCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACC-3' (SEQ ID NO: 39) Primer (a-13) contains a ribosome binding sequence and a sequence homologous to qsuB. Electrophoresis detected a DNA fragment of approximately 1.5 kbp corresponding to aroY, and the DNA was recovered from the gel.

[0091] The qsuB and aroY DNA fragments thus generated were mixed, ligated, and amplified using the following primers. The following primers were used for PCR: (a-12) 5'-GGCTCGTATAATGTGTGGAATTGTGAGCGGATAAC -3' (SEQ ID NO: 36) (b-13) 5'-CCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACC-3' (SEQ ID NO: 39) A DNA fragment of approximately 3.5 kbp corresponding to the ligated qsuB and aroY gene was detected by electrophoresis, and the DNA was recovered from the gel.

[0092] The DNA fragment of vector pCAMO-6 and the qsuB and aroY ligated genes were ligated together and used to transform Escherichia coli JM109. The plasmid was then extracted and the sequences of the qsuB and aroY genes were confirmed. The resulting plasmid was used for catechol production.

[0093] (9) Construction of a Muconic Acid Production Plasmid The muconic acid production plasmid was constructed by seamless cloning of qsuB, aroY, and catA genes linked via a ribosome binding sequence using PCR. The catechol production plasmid prepared in (8) was used as a template to amplify the qsuB-aroY-linked DNA fragment. The following primers were used for PCR: (a-12) 5'-GGCTCGTATAATGTGTGGAATTGTGAGCGGATAAC -3' (SEQ ID NO: 36) (b-14) 5'-ATGCGAGTCTCCTCCCTATTTCTTATCAGCGAACAGCTCCGGCG-3' (SEQ ID NO: 40). Primer (b-24) contained a sequence homologous to the ribosome binding sequence. Electrophoresis detected a DNA fragment of approximately 3.5 kbp corresponding to the qsuB-aroY-linked DNA, and the DNA was recovered from the gel.

[0094] Pseudomonas thermotolerans catA was amplified by PCR using the expression vector obtained in (7) as a template and the following primers: (a-14) 5'-GAAATAGGGAGGAGACTCGCATATGACCGTGAAAATCACTCAC-3' (SEQ ID NO: 41) (b-13) 5'-CCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACC-3' (SEQ ID NO: 39). Primer (a-14) contained a ribosome binding sequence and a sequence homologous to aroY. A DNA fragment of approximately 1.5 kbp corresponding to catA was detected by electrophoresis, and the DNA was recovered from the gel.

[0095] The resulting qsuB and aroY linked DNA fragment and the catA-containing DNA fragment were mixed and ligated by PCR using the following primers: (a-12) 5'-GGCTCGTATAATGTGTGGAATTGTGAGCGGATAAC -3' (SEQ ID NO: 36) (b-13) 5'-CCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACC-3' (SEQ ID NO: 39) Electrophoresis detected a DNA fragment of approximately 4.5 kbp corresponding to the gene formed by linking qsuB, aroY, and catA.

[0096] The DNA fragment of vector pCAMO-6 and the ligated DNA fragment of qsuB, aroY, and catA were ligated together and used to transform Escherichia coli JM109. Plasmids were then extracted and the sequences of the qsuB, aroY, and catA genes were confirmed.

[0097] (10) Gene Introduction into Wild-Type Strains of Hydrogenophilus thermorteolus Transformants (10-1). The Hydrogenophilus thermorteolus TH-1 strain was transformed by electroporation with the qsuB-containing plasmid obtained in (5), the qsuB and aroY-containing plasmid obtained in (8), and the qsuB, aroY, and catA-containing plasmid obtained in (9). The transformations were then spread onto LB solid medium containing 50 μg / mL kanamycin and cultured at 52°C for 24 hours. Similarly, the TH-1 strain was transformed with pCAMO-6. Each strain grown 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. Amplification of the insert fragments of each plasmid was confirmed for each strain grown on LB solid medium by PCR. The amplification of DNA fragments corresponding to the respective genes was confirmed. Each plasmid and the TH-1 strain transformant transformed with that plasmid were named as shown in Table 1.

[0098] The plasmid-transfected strain prepared as described above was inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop, and cultured with shaking at 52°C for 72 hours while supplying a gas mixture of H2:O2:CO2 = 7.5:1:1.5. After cultivation, the culture supernatant was obtained by centrifugation (4°C, 5,000 g, 10 minutes).

[0099] (10-2) Gene Introduction into the HPTL_0060 Gene Disruption Strain (Δ0060 Strain) The Δ0060 strain was transformed by electroporation with the qsuB-containing plasmid obtained in (5), the qsuB and aroY-containing plasmid obtained in (8), and the qsuB, aroY, and catA-containing plasmid obtained in (9). The transformed strain was then plated onto LB solid medium containing 100 μg / mL kanamycin and 400 μg / mL streptomycin and cultured at 52°C for 24 hours. Each strain grown on LB solid medium was inoculated onto LB solid medium containing 100 μg / mL kanamycin and 400 μg / mL streptomycin using a platinum loop and cultured at 52°C for 24 hours. Similarly, the Δ0060 strain was transformed with pCAMO-6. Amplification of the insert fragments of each plasmid was confirmed for each strain grown on LB solid medium by PCR. The amplification of DNA fragments corresponding to the respective genes was confirmed. Each plasmid and the Δ0060 strain transformant transformed with that plasmid were named as shown in Table 1.

[0100] The plasmid-transfected strain prepared as described above was inoculated into liquid medium A containing 100 μg / mL kanamycin and 400 μg / mL streptomycin using a platinum loop, and cultured with shaking at 52°C for 72 hours while supplying a gas mixture of H2:O2:CO2 = 7.5:1:1.5. After cultivation, the culture supernatant was obtained by centrifugation (4°C, 5,000 g, 10 minutes).

[0101] (10-3) Production of Catechol and Protocatechuic Acid The concentrations of catechol and protocatechuic acid in the culture supernatant were measured by high-performance liquid chromatography (Shimadzu Corporation) under the following conditions: Mobile phase A: 0.1% phosphoric acid in water Mobile phase B: 50% acetonitrile Flow rate: 1 mL / min Column: CAOCELLPAK MGII, 5.0 μm, 4.6 mm ID x 150 mm Column temperature: 40°C PDA temperature: 40°C PDA: 200-300 nm Reference wavelength: 210 nm (10-4) Production of Muconic Acid The concentrations of muconic acid in the culture supernatant were measured by high-performance liquid chromatography (Shimadzu Corporation) under the following conditions. Mobile phase A: 5 mmol / L p-toluenesulfonic acid:ethanol 90:10 (v / v) Mobile phase B: 5 mmol / L p-toluenesulfonic acid, 20 mmol / L Bis-Tris, 0.1 mmol / L EDTA:ethanol 90:10 (v / v) Flow rate: 0.7 mL / min for both mobile phases A and B Column: Shim-pack SCR-102H Column temperature: 45°C Detector: CDD-10A VP Sample injection volume: 20 μL The results are shown in Table 1. Protocatechuic acid was detected in the culture supernatant of the TH-1 strain in which qsuB was introduced and the Δ0060 strain in which qsuB was introduced. On the other hand, when qsuB and aroY or qsuB, aroY, and catA were introduced, catechol and muconic acid were not detected in the culture supernatant when the host was the TH-1 strain, but catechol or catechol and muconic acid were detected in the culture supernatant only when the host was the Δ0060 strain. From these results, we concluded that HPTL_0060 is a catechol 2,3-dioxygenase gene and that disruption of HPTL_0060 is essential for efficient production of muconic acid or catechol in Hydrogenophilus bacteria.

[0102]

[0103] 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.

[0104] SEQ ID NOs: 1 to 11 are shown below.

[0105] The transformant of the present invention can produce muconic acid or catechol with high efficiency using carbon dioxide as the sole carbon source, thereby contributing to the industrial production of chemical products with high efficiency while resolving global warming caused by increased carbon dioxide.

Claims

1. A Hydrogenophilus bacterial gene disruption strain in which the catechol 2,3-dioxygenase gene is disrupted.

2. The Hydrogenophilus bacterial gene disruption strain according to claim 1, wherein the catechol 2,3-dioxygenase gene is any one of the following DNAs (a1) to (a5): (a1) A DNA containing the nucleotide sequence of SEQ ID NO: 1; (a2) A DNA containing a nucleotide sequence having 90% or more identity with SEQ ID NO: 1 and encoding a polypeptide having catechol 2,3-dioxygenase activity; (a3) A DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2; (a4) A DNA encoding a polypeptide containing an amino acid sequence having 90% or more identity with SEQ ID NO: 2 and having catechol 2,3-dioxygenase activity; (a5) A DNA encoding a polypeptide containing an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 2 and having catechol 2,3-dioxygenase activity.

3. A Hydrogenophilus bacterial transformant in which the catechol 2,3-dioxygenase gene on the genome is disrupted and has an exogenous (i) 3-dehydroshikimate dehydratase gene and protocatechuate decarboxylase gene, or (ii) 3-dehydroshikimate dehydratase gene, protocatechuate decarboxylase gene, and catechol 1,2-dioxygenase gene.

4. The Hydrogenophilus bacterial transformant according to claim 3, wherein the protocatechuate decarboxylase gene is any one of the following DNAs (b1) to (b5): (b1) A DNA containing the nucleotide sequence of SEQ ID NO: 3; (b2) A DNA containing a nucleotide sequence having 90% or more identity with SEQ ID NO: 3 and encoding a polypeptide having protocatechuate decarboxylase activity; (b3) A DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 4; (b4) A DNA encoding a polypeptide containing an amino acid sequence having 90% or more identity with SEQ ID NO: 4 and having protocatechuate decarboxylase activity; (b5) A DNA encoding a polypeptide containing an amino acid sequence in which 1 to 40 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 4 and having protocatechuate decarboxylase activity.

5. The Hydrogenophilus bacterium transformant according to claim 3 or 4, wherein the catechol 1,2-dioxygenase gene is any one of the following DNAs (c1) to (c5): (c1) a DNA containing the nucleotide sequence of SEQ ID NO: 5; (c2) a DNA containing a nucleotide sequence having 90% or more identity with SEQ ID NO: 5 and encoding a polypeptide having catechol 1,2-dioxygenase activity; (c3) a DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 6; (c4) a DNA encoding a polypeptide containing an amino acid sequence having 90% or more identity with SEQ ID NO: 6 and having catechol 1,2-dioxygenase activity; (c5) a DNA encoding a polypeptide containing an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6 and having catechol 1,2-dioxygenase activity 6. The genetically recombinant Hydrogenophilus bacterium transformant according to any one of claims 3 to 5, wherein the 3-dehydroshikimate dehydratase gene is any one of the following DNAs (d1) to (d5): (d1) a DNA containing the nucleotide sequence of SEQ ID NO: 7; (d2) a DNA containing a nucleotide sequence having 90% or more identity with SEQ ID NO: 7 and encoding a polypeptide having 3-dehydroshikimate dehydratase activity; (d3) a DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 8; (d4) a DNA encoding a polypeptide containing an amino acid sequence having 90% or more identity with SEQ ID NO: 8 and having 3-dehydroshikimate dehydratase activity; (d5) a DNA encoding a polypeptide containing an amino acid sequence in which 1 to 60 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 8 and having 3-dehydroshikimate dehydratase activity 7. The Hydrogenophilus bacterium transformant according to any one of claims 3 to 6, wherein the Hydrogenophilus bacterium is Hydrogenophilus thermoluteolus 8. A method for producing muconic acid or catechol, comprising the step of culturing the Hydrogenophilus bacterium transformant according to any one of claims 3 to 7 using carbon dioxide as substantially the sole carbon source 9. A positive selection marker for Hydrogenophilus bacteria containing the DNA of the following (e1) or (e2). (e1) DNA containing the nucleotide sequence of SEQ ID NO: 11 (e2) DNA containing a nucleotide sequence having 90% or more identity with SEQ ID NO: 11 and encoding a polypeptide having an activity of conferring bleomycin resistance to Hydrogenophilus bacteria

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