Hydrogenophilus bacterium transformant capable of decarboxylating 4-hydroxybenzoic acid and method for producing phenol

Abstract

The present invention enables efficient production of phenol from 4-hydroxybenzoic acid by using a transformant obtained by introducing the DNA(s) described in (a) or (c) below or homologous DNA(s) thereof into a bacterium of the genus Hydrogenophilus. (a) A set of three DNAs including the nucleotide sequences of SEQ ID NOs: 2 to 4, a set of three DNAs including the nucleotide sequences of SEQ ID NOs: 9 to 11, a set of three DNAs including the nucleotide sequences of SEQ ID NOs: 16 to 18, a set of three DNAs including the nucleotide sequences of SEQ ID NOs: 23 to 25, a set of three DNAs including the nucleotide sequences of SEQ ID NOs: 30 to 32, or a DNA including the nucleotide sequence of SEQ ID NO: 5, 12, 19, 26, or 33. (c) A set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 6 to 8, a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 13 to 15, a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 20 to 22, a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 27 to 29, or a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 34 to 36.

Description

Transformant of Hydrogenophilus bacterium capable of decarboxylating 4-hydroxybenzoic acid and method for producing phenol

[0001] The present invention relates to a transformant of a bacterium belonging to the genus Hydrogenophilus that has been imparted with the ability to decarboxylate 4-hydroxybenzoic acid, and to an efficient method for producing phenol using the transformant.

[0002] The Paris Agreement, adopted in 2015, calls for rapid reductions in global greenhouse gas emissions. The Sixth Assessment Report of the Intergovernmental Panel on Climate Change (IPCC), published in 2023, pointed out that "if greenhouse gas emissions continue to rise, there is a very high possibility that global average temperatures will rise by more than 1.5°C above pre-industrial levels between 2021 and 2040, and that this could exceed 1.5°C even if emissions are kept low." The report also estimated that a 2°C rise in temperature would result in distinctly different climate changes in different regions of the world compared to a 1.5°C rise. It called for achieving carbon neutrality, meaning that net anthropogenic CO2 emissions worldwide must be reduced by approximately 45% by 2030 compared to 2010 levels and reach zero by around 2050. In accordance with this report, drastically reducing greenhouse gas emissions, such as carbon dioxide and methane, has become a global challenge, and technological development is becoming increasingly necessary to achieve this.

[0003] The materials currently supplied by the chemical industry are essential for maintaining the world's current science, culture, and standard of living, and a continuous, stable supply is required. However, the majority of chemical production worldwide is done using petroleum-based raw materials, which increases greenhouse gas emissions. Therefore, there is a need to shift chemical production methods from the current methods that consume large amounts of petroleum resources to carbon recycling methods that make effective use of carbon resources. For this reason, research and development of biorefineries that use microbial fermentation to produce green chemicals from biomass is being actively conducted in various countries.

[0004] Phenol is widely used as a raw material for the production of resins such as phenolic resins and epoxy resins, dyes, synthetic fragrances, pesticides, surfactants, and compounds such as picric acid, salicylic acid, phenacetin, bisphenol A, aniline, p-phenolsulfonic acid, and 2-phenoxyethanol. Phenol itself is also used as a disinfectant, dental anesthetic, and analytical reagent.

[0005] Among these, phenolic resins are an important compound with high demand. Phenolic resins are the oldest synthetic resins and are characterized by excellent heat resistance and durability. Currently, phenolic resins are used as raw materials for automotive parts, electronic products such as semiconductor components and electronic circuit components, and are also used in a wide range of fields, including medicine, agriculture, and construction. Furthermore, new functional materials are being created by blending phenolic resins with other materials such as glass fiber and organic or inorganic fillers, and the demand for phenolic resins is increasing.

[0006] Currently, phenol is industrially produced using the cumene process, which uses petroleum-derived (coal tar) benzene and propylene as raw materials. This process is complex and requires high temperature and pressure, resulting in a large amount of energy consumption. 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, phenol production by microbial fermentation using renewable non-edible biomass resources is attracting attention.

[0008] For example, Patent Document 1 discloses a method for producing phenol from glucose, a carbon source, by introducing a 4-hydroxybenzoic acid (hereinafter sometimes referred to as "4-HBA") decarboxylase gene derived from a bacterium of the genus Bacillus, Citrobacter, Enterobacter, Escherichia, Paenibacillus, or Pantoea into Corynebacterium glutamicum. Non-Patent Document 1 teaches a method for producing phenol from glucose by introducing a 4-HBA decarboxylase gene derived from Escherichia coli into Escherichia coli. Non-Patent Document 2 teaches a method for producing phenol from glucose by co-culturing Escherichia coli into which the 4-HBA decarboxylase gene derived from Escherichia coli has been introduced and Escherichia coli into which the tyrosine phenol-lyase gene derived from Pasteurella multocida has been introduced. Non-Patent Document 3 teaches a method for producing phenol from glucose by introducing the 4-HBA decarboxylase gene derived from Klebsialla pneumoniae and the salicylic acid decarboxylase gene derived from Trichosporon moniliiforme into Escherichia coli. Non-Patent Document 4 teaches a method for producing phenol from glucose by introducing into Escherichia coli the tyrosine phenol-lyase gene derived from Citrobacter braakii, the 4-HBA decarboxylase gene derived from Klebsiella pneumoniae, and the salicylic acid decarboxylase gene derived from Trichosporon moniliforme.

[0009] However, the product, phenol, is highly cytotoxic due to its protein-denaturing properties. For this reason, industrial production of phenol by bacterial growth has been difficult because the phenol produced inhibits bacterial growth. While 4-HBA inhibits cell viability, this inhibitory effect is weaker than that of phenol. Therefore, if a transformant carrying a 4-HBA decarboxylase gene is grown in advance and used as a reaction catalyst to produce phenol from 4-HBA, phenol can be produced without being affected by phenol's growth inhibition.

[0010] As an attempt to achieve this, Patent Document 2 discloses a method for the prevention and treatment of bacterial infections caused by bacteria such as Bacillus subtilis, Bacillus atrophaeus, Bacillus subtilis subsp. spizizenii, Citrobacter koseri, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter hormaechei, Enterobacter sakazakii, Escherichia coli, Escherichia fergusonii, Paenibacillus polymyxa, or Pantoea ananatis. The paper discloses that by first growing Corynebacterium glutamicum into which the 4-HBA decarboxylase gene of Corynebacterium ananatis has been introduced, and then using the grown bacteria as a reaction catalyst to produce phenol from the raw material 4-HBA, phenol can be produced efficiently without being affected by growth inhibition by phenol.

[0011] However, Corynebacterium glutamicum grows using sugars as a carbon source, and converting biomass into sugars requires a complex process, resulting in high costs. Furthermore, using biomass that can be used as food or feed for chemical production hinders the stable supply of food and feed. Furthermore, consuming large amounts of biomass for chemical production can actually lead to environmental destruction. Therefore, the method of Patent Document 2 is insufficient as a measure to protect the global environment, as it utilizes sugars produced from biomass.

[0012] In research into carbon recycling, which reduces fossil fuel consumption and greenhouse gas emissions, gases such as carbon dioxide, methane, and carbon monoxide are attracting attention as highly sustainable carbon raw materials. If these gases present in the atmosphere could be fixed by microorganisms and used industrially, it would contribute greatly to carbon recycling and lead to the achievement of the goal of carbon neutrality. Therefore, there is growing interest in technologies that use microorganisms that grow on these gases to produce valuable chemicals and biofuels. In particular, there are high expectations for fixing and effectively utilizing carbon dioxide, which contributes significantly to global warming.

[0013] Among the bacteria that can grow using carbon dioxide as a carbon source, Cupriavidus necator strain H16 and Hydrogenobacter thermophilus strain TK-6 are used in the production of chemical products.

[0014] However, no method has been reported for efficiently producing phenol from 4-HBA by imparting 4-HBA decarboxylation activity to bacteria that can grow using carbon dioxide as a carbon source.

[0015] Re-table 2012 / 063862 Re-table 2012 / 063860

[0016] Appl Microbiol Biotechnol., 99(12):5163-5173(2015)Biotechnol Bioeng., 116(12):3349-3359(2019)Biotechnol Bioeng., 113(8):1745-1754(2016)Fermentation., 7(4):216(2021)

[0017] An objective of the present invention is to provide a transformant of a bacterium belonging to the genus Hydrogenophilus that can efficiently produce phenol from 4-HBA, and a method for efficiently producing phenol using the transformant.

[0018] 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.

[0019] Hydrogenophilus bacteria do not have the 4-HBA decarboxylase gene required for producing phenol from 4-HBA, so to produce phenol from 4-HBA, it is necessary to introduce the 4-HBA decarboxylase gene into Hydrogenophilus bacteria.

[0020] The present inventors have found that heterologous genes that are expressed in bacteria other than those of the genus Hydrogenophilus are often not expressed or expressed insufficiently in bacteria of the genus Hydrogenophilus. Therefore, it is generally not useful to use genes that can be introduced into other bacteria to induce enzymatic reactions in bacteria of the genus Hydrogenophilus for the purpose of enzymatic reactions.

[0021] Under these circumstances, in order to obtain a 4-HBA decarboxylase gene that is expressed in bacteria of the genus Hydrogenophilus, the present inventors examined genes that are predicted to be 4-HBA decarboxylase genes present in the genomes of other microorganisms and found that the 4-HBA decarboxylase genes of Alicyclobacillus acidiphilus, Alicyclobacillus suci, Bacillus licheniformis, Saccharopolyspora rectivirgula, and Cronobacter sakazakii express functional 4-HBA decarboxylase in bacteria of the genus Hydrogenophilus.

[0022] Furthermore, they found that transformants obtained by introducing these 4-HBA decarboxylase genes into Hydrogenophilus bacteria could efficiently produce phenol by reacting it with 4-HBA in the reaction solution.

[0023] The present invention was completed based on the above findings and provides the following transformant and method for producing phenol: [1] A transformant obtained by introducing the following DNA (a), (b1), (b2), (c), (d), or (e) into a bacterium of the genus Hydrogenophilus: (a) A set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 2 to 4, a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 9 to 11, a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 16 to 18, a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 23 to 25, a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 30 to 32, or a DNA containing the nucleotide sequence of SEQ ID NO: 5, 12, 19, 26, or 33 (b1) A set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOs: 2 to 4 have been replaced with a nucleotide sequence having 90% or more identity to the nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; a set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOs: 9 to 11 have been replaced with a nucleotide sequence having 90% or more identity to the nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; a set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOs: 16 to 18 have been replaced with a nucleotide sequence having 90% or more identity to the nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; A set of three DNAs in which NA contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; a set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOS: 23 to 25 have been replaced with a nucleotide sequence having 90% or more identity to that nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; or a set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOS: 30 to 32 have been replaced with a nucleotide sequence having 90% or more identity to that nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity.(b2) DNAs encoding three subunits of a polypeptide having 4-HBA decarboxylase activity, comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 5, 12, 19, 26, or 33. (c) A set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 6 to 8, a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 13 to 15, a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 20 to 22, a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 27 to 29, or a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 34 to 36.(d) A set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 6 to 8 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 13 to 15 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 20 to 22 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences that are the amino acid sequences of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which any one or more of the amino acid sequences of SEQ ID NOS: 27 to 29 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase; or a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which any one or more of the amino acid sequences of SEQ ID NOS: 34 to 36 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase.(e) A set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 6 to 8 have one to five amino acids deleted, substituted, inserted, or added, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 13 to 15 have one to five amino acids deleted, substituted, inserted, or added, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase; and a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 20 to 22 have one to five amino acids deleted, substituted, inserted, or added, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase. a set of three DNAs encoding three polypeptides each comprising one or more of the amino acid sequences of SEQ ID NOS: 27 to 29, in which one to five amino acids have been deleted, substituted, inserted, or added relative to the amino acid sequence, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase; or a set of three DNAs encoding three polypeptides each comprising one or more of the amino acid sequences of SEQ ID NOS: 34 to 36, in which one to five amino acids have been deleted, substituted, inserted, or added relative to the amino acid sequence, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase. [2] The transformant according to [1], wherein the Hydrogenophilus bacterium is Hydrogenophilus thermorteolus. [3] A method for producing phenol, comprising a step of reacting the transformant according to [1] or [2] with 4-hydroxybenzoic acid in a reaction solution. [4] The method according to [3], comprising a step of growing the transformant according to [1] or [2] before the reaction step.

[0024] 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, the use of these energy sources has not sufficiently curbed the increase in carbon dioxide. Therefore, it is necessary to promote the fixation or resource recovery of emitted carbon dioxide. Carbon dioxide can be fixed physically or chemically, but if it is fixed using living organisms, it can be used to produce organic matter that can be used as food, feed, fuel, and other products. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve both the two problems of global warming caused by increased carbon dioxide and the difficulty in securing food, feed, and fuel. Furthermore, it can produce chemical products in demand while curbing global warming caused by increased carbon dioxide.

[0025] 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."

[0026] Microbial growth is typically strongly inhibited by the cytotoxicity of phenol, making the production of phenol by growing microorganisms endowed with phenol-producing ability impractical. In contrast, if a pre-grown transformant of the present invention is used as a reaction catalyst to produce phenol using 4-HBA present in the reaction solution as a substrate, phenol can be produced efficiently without the influence of phenol growth inhibition. Hydrogenophilus bacteria have excellent carbon dioxide fixation or intracellular metabolic capabilities and a fast growth rate, making it easy to prepare a reaction catalyst. Furthermore, if 4-HBA is produced from carbon dioxide using Hydrogenophilus bacteria (for example, by the method described in WO 2024 / 100776), and phenol is produced using this 4-HBA as a raw material and the transformant of the present invention as a reaction catalyst, phenol can essentially be produced from carbon dioxide. The present invention opens a way to secure industrially necessary phenol while suppressing global warming due to increased carbon dioxide.

[0027] The present invention is described in detail below. (1) Transformant Having 4-HBA Decarboxylase Activity The transformant of the present invention is a transformant obtainable by introducing genes encoding the three subunits of 4-hydroxybenzoate decarboxylase from Alicyclobacillus acidophilus, Alicyclobacillus suchii, Bacillus licheniformis, Saccharopolyspora rectivirgula, or Cronobacter sakazakii, or homologs thereof, into a host Hydrogenophilus bacterium. That is, the transformant of the present invention is a Hydrogenophilus bacterium transformant carrying exogenous genes encoding the three subunits of 4-HBA decarboxylase, such as genes encoding the three subunits of 4-HBA decarboxylase from Alicyclobacillus acidophilus, Alicyclobacillus suchii, Bacillus licheniformis, Saccharopolyspora rectivirgula, or Cronobacter sakazakii, or homologs thereof. The 4-HBA decarboxylase subunit gene used in the present invention does not need to have been identified as a 4-HBA decarboxylase subunit gene, but may be DNA encoding a polypeptide having 4-HBA decarboxylase subunit activity. The 4-HBA decarboxylase subunit activity refers to the activity of producing phenol by decarboxylating 4-HBA together with two other subunits. Furthermore, in the present invention, the 4-HBA decarboxylase subunit gene may be DNA of a 4-HBA decarboxylase subunit gene isolated from a naturally occurring bacterium, or may be DNA artificially synthesized using techniques known to those skilled in the art.

[0028] The transformant of the present invention may be any bacterium belonging to the genus Hydrogenophilus into which the genes encoding the three subunits of 4-HBA decarboxylase from Alicyclobacillus acidophilus, the genes encoding the three subunits of 4-HBA decarboxylase from Alicyclobacillus suchii, the genes encoding the three subunits of 4-HBA decarboxylase from Bacillus licheniformis, the genes encoding the three subunits of 4-HBA decarboxylase from Saccharopolyspora rectivirgra, or the genes encoding the three subunits of 4-HBA decarboxylase from Cronobacter sakazakii, or homologs thereof have been introduced. The transformant also includes a bacterium belonging to the genus Hydrogenophilus into which two or more of these three gene sets have been introduced, and a bacterium belonging to the genus Hydrogenophilus into which other genes have been introduced in addition to these genes.

[0029] 4-HBA decarboxylase gene The 4-HBA decarboxylase gene consists of three genes encoding each of the three subunits of 4-HBA decarboxylase. Genes encoding enzymes with 4-HBA acid decarboxylase activity are referred to by various abbreviations depending on their origin. In the present invention, the 4-HBA decarboxylase gene may be abbreviated as "hbdc" regardless of its origin.

[0030] The genes to be introduced into the host can be any of three subunit genes: DNA containing the nucleotide sequence of SEQ ID NO: 2 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 2), DNA containing the nucleotide sequence of SEQ ID NO: 3 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 3), and DNA containing the nucleotide sequence of SEQ ID NO: 4 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 4). DNA containing the nucleotide sequence of SEQ ID NO: 5 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 5), which is a preferred example of hbdc containing the subunit genes of SEQ ID NOs: 2, 3, and 4, can also be introduced. SEQ ID NOs: 2 to 5 are the nucleotide sequences of the subunit genes or hbdc of Alicyclobacillus acidophilus.

[0031] The genes that can be introduced into the host include three subunit genes: DNA containing the nucleotide sequence of SEQ ID NO: 9 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 9), DNA containing the nucleotide sequence of SEQ ID NO: 10 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 10), and DNA containing the nucleotide sequence of SEQ ID NO: 11 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 11). DNA containing the nucleotide sequence of SEQ ID NO: 12 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 12), which is a preferred example of hbdc containing the subunit genes of SEQ ID NOs: 9, 10, and 11, can also be introduced. SEQ ID NOs: 9 to 12 are the nucleotide sequences of the subunit genes or hbdc of Alicyclobacillus subtilis.

[0032] The genes to be introduced into the host can be any of the three subunit genes: DNA containing the nucleotide sequence of SEQ ID NO: 16 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 16), DNA containing the nucleotide sequence of SEQ ID NO: 17 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 17), and DNA containing the nucleotide sequence of SEQ ID NO: 18 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 18). DNA containing the nucleotide sequence of SEQ ID NO: 19 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 19), which is a preferred example of hbdc containing the subunit genes of SEQ ID NOs: 16, 17, and 18, can also be introduced. SEQ ID NOs: 16 to 19 are the nucleotide sequences of the subunit genes or hbdc of Bacillus licheniformis.

[0033] The genes to be introduced into the host can be any of the three subunit genes: DNA containing the nucleotide sequence of SEQ ID NO: 23 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 23), DNA containing the nucleotide sequence of SEQ ID NO: 24 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 24), and DNA containing the nucleotide sequence of SEQ ID NO: 25 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 25). DNA containing the nucleotide sequence of SEQ ID NO: 26 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 26), which is a preferred example of hbdc containing the subunit genes of SEQ ID NOs: 23, 24, and 25, can also be introduced. SEQ ID NOs: 23 to 26 are the nucleotide sequences of the subunit genes or hbdc of Saccharopolyspora rectivirgula.

[0034] The genes to be introduced into the host can be any of the three subunit genes: DNA containing the nucleotide sequence of SEQ ID NO: 30 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 30), DNA containing the nucleotide sequence of SEQ ID NO: 31 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 31), and DNA containing the nucleotide sequence of SEQ ID NO: 32 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 32). DNA containing the nucleotide sequence of SEQ ID NO: 33 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 33), which is a preferred example of hbdc containing the subunit genes of SEQ ID NOs: 30, 31, and 32, can also be introduced. SEQ ID NOs: 30 to 33 are the nucleotide sequences of the subunit genes or hbdc of Cronobacter sakazakii.

[0035] The three subunit genes can be introduced into a host as adjacently linked DNA. For example, a suitable promoter can be incorporated into the 5'-end upstream of each gene, and a terminator can also be incorporated into the 3'-end downstream. Examples of promoters include the tac promoter, lac promoter, trc promoter, or Oxford Genetics' OXB1, OXB11-OXB20 promoters. Examples of terminators include the rrnB T1T2 terminator of the E. coli rRNA operon, the bacteriophage λt0 transcription terminator, and the T7 terminator. Alternatively, the three subunit genes can be introduced into a host as DNA linked via a ribosome binding sequence found upstream of the start codon. This "ribosome binding site" refers to a ribosome binding site that allows mRNA transcribed from DNA to bind to ribosomes in the host cell when protein biosynthesis begins. Those skilled in the art can appropriately select the ribosome binding site from known sequences. In either case, the arrangement of the three subunit genes in the introduced DNA is not limited, and the three subunit genes can also be inserted separately into two or three vectors and then introduced into the host.

[0036] SEQ ID NO: 5 is a sequence in which SEQ ID NOs: 2 to 4 are linked adjacently in the stated order, SEQ ID NO: 12 is a sequence in which SEQ ID NOs: 9 to 11 are linked adjacently in the stated order, SEQ ID NO: 19 is a sequence in which SEQ ID NOs: 16 to 18 are linked adjacently in the stated order, SEQ ID NO: 26 is a sequence in which SEQ ID NOs: 23 to 25 are linked adjacently in the stated order, and SEQ ID NO: 33 is a sequence in which SEQ ID NOs: 30 to 32 are linked adjacently in the stated order.

[0037] In the present invention, DNAs that contain a nucleotide sequence that has 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the nucleotide sequence of SEQ ID NO: 2, 3, 4, 9, 10, 11, 16, 17, 18, 23, 24, 25, 30, 31, or 32 (particularly, a nucleotide sequence that has 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the nucleotide sequence of SEQ ID NO: 2, 3, 4, 9, 10, 11, 16, 17, 18, 23, 24, 25, 30, 31, or 32) and encode a subunit of a polypeptide having 4-HBA decarboxylase activity can also be used.

[0038] In this case, one, two, or three of the three subunit genes introduced into the host can be replaced with DNA containing a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 2, 3, 4, 9, 10, 11, 16, 17, 18, 23, 24, 25, 30, 31, or 32 (particularly, DNA consisting of a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 2, 3, 4, 9, 10, 11, 16, 17, 18, 23, 24, 25, 30, 31, or 32). Taking 4-HBA decarboxylase from Alicyclobacillus acidophilus as an example, for example, three DNAs can be introduced into the host: DNA of SEQ ID NO: 2, DNA having a nucleotide sequence in which five nucleotides have been substituted in SEQ ID NO: 3, and DNA having a nucleotide sequence in which two nucleotides have been substituted in SEQ ID NO: 4. In the present invention, a different base sequence that has 90% or more identity with a certain base sequence and encodes a polypeptide having the same type of activity as the polypeptide encoded by the original base sequence is referred to as a "homologous base sequence."

[0039] Alternatively, DNA containing a nucleotide sequence having 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the nucleotide sequence of SEQ ID NO: 5, 12, 19, 26, or 33 (particularly, consisting of a nucleotide sequence having 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to the nucleotide sequence of SEQ ID NO: 5, 12, 19, 26, or 33) and encoding three subunits of a polypeptide having 4-HBA decarboxylase activity can also be used.

[0040] Furthermore, three subunit genes can be used as genes to be introduced into a host: DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6), DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7), and DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8). SEQ ID NOs: 6 to 8 are the amino acid sequences of the subunits of 4-HBA decarboxylase from Alicyclobacillus acidophilus.

[0041] Three subunit genes can be used as genes to be introduced into a host: DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 13 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 13), DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 14), and DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 15 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 15). SEQ ID NOs: 13 to 15 are the amino acid sequences of the subunits of 4-HBA decarboxylase from Alicyclobacillus subtilis.

[0042] Three subunit genes can be used as genes to be introduced into a host: DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 20 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 20), DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 21 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 21), and DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 22). SEQ ID NOs: 20 to 22 are the amino acid sequences of the subunits of 4-HBA decarboxylase from Bacillus licheniformis.

[0043] Three subunit genes can be used as genes to be introduced into a host: DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 27), DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 28 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 28), and DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 29 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 29). SEQ ID NOs: 27 to 29 are the amino acid sequences of the subunits of 4-HBA decarboxylase from Saccharopolyspora rectivirgula.

[0044] The genes that can be introduced into the host include three subunit genes: DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 34 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 34), DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 35 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 35), and DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 36 (particularly, DNA encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 36). SEQ ID NOs: 34 to 36 are the amino acid sequences of the subunits of 4-HBA decarboxylase from Cronobacter sakazakii.

[0045] DNAs that contain an amino acid sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 6, 7, 8, 13, 14, 15, 20, 21, 22, 27, 28, 29, 34, 35, or 36 (particularly, consisting of an amino acid sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identical to the amino acid sequence of SEQ ID NO: 6, 7, 8, 13, 14, 15, 20, 21, 22, 27, 28, 29, 34, 35, or 36) and that encode a subunit of a polypeptide that has 4-HBA decarboxylase activity can also be used. Furthermore, DNAs that contain an amino acid sequence in which 1 to 5, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6, 7, 8, 13, 14, 15, 20, 21, 22, 27, 28, 29, 34, 35, or 36 (particularly, consisting of an amino acid sequence in which 1 to 5, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 6, 7, 8, 13, 14, 15, 20, 21, 22, 27, 28, 29, 34, 35, or 36) and encode a subunit of a polypeptide that has 4-HBA decarboxylase activity can also be used.

[0046] In these cases, one, two, or three of the three subunit genes introduced into the host can be replaced with DNA encoding a polypeptide comprising an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 6, 7, 8, 13, 14, 15, 20, 21, 22, 27, 28, 29, 34, 35, or 36 (particularly, DNA encoding a polypeptide consisting of an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 6, 7, 8, 13, 14, 15, 20, 21, 22, 27, 28, 29, 34, 35, or 36). Taking the 4-HBA decarboxylase of Alicyclobacillus acidophilus as an example, for example, three DNAs can be introduced into the host: DNA encoding the polypeptide of SEQ ID NO: 6, DNA encoding a polypeptide having an amino acid sequence in which five amino acids have been substituted in SEQ ID NO: 7, and DNA encoding a polypeptide having an amino acid sequence in which two amino acids have been substituted in SEQ ID NO: 8. In the present invention, the amino acid sequence of a polypeptide that has the same type of activity as a polypeptide consisting of a given amino acid sequence, but is a different amino acid sequence that has 90% or more identity with that amino acid sequence, is referred to as a "homologous amino acid sequence." Also, in the present invention, the amino acid sequence of a polypeptide that has 1 to 5 amino acids deleted, substituted, inserted, or added from a given amino acid sequence, but is a polypeptide that has the same type of activity as a polypeptide consisting of the given amino acid sequence, is referred to as a "homologous amino acid sequence."

[0047] In the present invention, the DNA encoding the 4-HBA decarboxylase subunits and the hbdc containing the three subunit genes described above can be introduced into a host in a state where they are bound to different DNAs, to the extent that the 4-HBA decarboxylase subunit activity and the 4-HBA decarboxylase activity are not impaired. Examples of such DNAs include DNA encoding protein tags such as GFP (Green Fluorescent Protein). The expressed protein can be a fusion protein.

[0048] A DNA sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 6, 7, 8, 13, 14, 15, 20, 21, 22, 27, 28, 29, 34, 35, or 36 may have various base substitutions in the coding region, taking into account codon degeneracy or codon preference in Hydrogenophilus bacteria, as long as the amino acid sequence of the protein expressed from the coding region is not changed.

[0049] In the present invention, whether a test polypeptide is a subunit of a polypeptide having 4-HBA decarboxylase activity is confirmed by expressing the test polypeptide as a complex with two other 4-HBA decarboxylase subunits, reacting the resulting complex with 4-HBA, and detecting the produced phenol by high-performance liquid chromatography (HPLC).

[0050] Examples of Hydrogenophilus bacteria include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidans, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038. Among these, Hydrogenophilus thermoluteolus is preferred because it has the highest growth rate and carbon dioxide fixation ability of any carbon dioxide-fixing microorganism. Hydrogenophilus bacteria can be easily isolated from all over the world. A preferred strain of Hydrogenophilus thermorteolus is the TH-1 (NBRC 14978) strain. Hydrogenophilus thermorteolus TH-1 (NBRC 14978) exhibits the highest growth rate of any carbon-fixing microorganism (Agricultural and Biological Chemistry, 41, 685-690 (1977)) (doubling in one hour). The Hydrogenophilus thermorteolus NBRC 14978 strain has been internationally deposited under the Budapest Treaty and is publicly available.

[0051] In addition, the host Hydrogenophilus bacterium of the present invention may be a bacterium isolated from nature, or a bacterium obtained by genetically modifying a bacterium isolated from nature. Modifications can be performed for purposes such as enabling high expression of the introduced hbdc or increasing phenol production. Such modifications can be performed by removing (curing) endogenous plasmids in the Hydrogenophilus bacterium, introducing a gene that increases 4-HBA production, or disrupting a gene on the genome of the Hydrogenophilus bacterium.

[0052] Method for Preparing Transformants A method for obtaining transformants by introducing hbdc into Hydrogenophilus bacteria is described below. Introduction of hbdc into Hydrogenophilus bacteria can be carried out using a general method for introducing foreign genes into bacteria. hbdc may be directly introduced into Hydrogenophilus bacteria, or a vector incorporating the hbdc gene into a transformation vector (e.g., a plasmid vector, a viral vector, a cosmid, a fosmid, a BAC, etc.) may be introduced into Hydrogenophilus bacteria. The vector for transformation may contain DNA capable of autonomous replication in Hydrogenophilus bacteria. Examples include broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI Reference Sequence: NC_004444.1), pC194 (NCBI Reference Sequence: NC_002013.1), pK18mobsacB (GenBank: FJ437239.1), and pUB110 (NCBI Reference Sequence: NC_001384.1), as well as genetically modified versions of these vectors (e.g., pCAMO-6 (SEQ ID NO: 1)). Among these, pCAMO-6 is preferred. pCAMO-6 can be prepared from its DNA sequence by those skilled in the art using gene synthesis services, etc. Examples of promoters contained in the vector include the tac promoter, lac promoter, trc promoter, and 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 λt0 transcription terminator, the T7 terminator, etc. hbdc can be introduced (transformed) into Hydrogenophilus bacteria using conventional methods, such as the calcium chloride method, calcium phosphate method, rubidium chloride method, and electric pulse method (electroporation).

[0053] (2) Method for Producing Phenol

[0033] Phenol can be produced from 4-HBA using the transformant of the present invention. Phenol is preferably produced by reacting the transformant of the present invention in a reaction solution containing 4-HBA. This method is described in detail below.

[0054] Pre-Growth: It is desirable to grow the transformant of the present invention in advance before subjecting it to the reaction with 4-HBA. This suppresses the effect of phenol, which inhibits bacterial growth, and ensures the number of bacteria necessary for the reaction. Pre-growth can be carried out by culturing the transformant of the present invention in an inorganic or organic medium while supplying a gas containing carbon dioxide, 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 the number of bacteria necessary for the reaction can be ensured and phenol can be efficiently produced from 4-HBA.

[0055] Hydrogenophilus bacteria can grow using hydrogen as an energy source and carbon dioxide as the sole carbon source, and therefore, by culturing the transformant using substantially only carbon dioxide as a carbon source (especially using only carbon dioxide), carbon dioxide can be efficiently fixed. Therefore, when culturing the transformant 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, it is preferable to culture the transformant 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.

[0056] 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, the growth of the transformant and the solubility of the mixed gas in the medium are high. When performing batch culture, the mixed gas can be sealed in a sealed culture vessel and cultured by static or shaking. Shaking culture is preferred because it improves the solubility of the mixed gas in the medium. When performing continuous culture, the mixed gas can be continuously supplied to a sealed culture vessel while the culture is shaken, or the transformant can be cultured in a sealed culture vessel while introducing the mixed gas into the medium by bubbling. The volume ratio of hydrogen, oxygen, and carbon dioxide (hydrogen:oxygen:carbon dioxide) in the supply gas is preferably 1.75 to 7.5:1:0.25 to 3, more preferably 5 to 7.5:1:1 to 2, and even more preferably 6.25 to 7.5:1:1.5. Within this range, the growth of the transformant is good. 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 liter of medium. Within this range, the growth of the transformant is favorable 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.

[0057] When the grown bacterial cells are subjected to the reaction, the bacterial cells may be collected by centrifugation, filtration, or the like and then added to a separately prepared reaction solution, or 4-HBA may be added to the culture solution in which the bacterial cells have been grown.

[0058] The reaction solution can be water, a buffer solution, an inorganic salt medium, or the like containing 4-HBA. When 4-HBA is added to pre-grown cells, the reaction solution is a culture supernatant containing 4-HBA. The concentration of 4-HBA in the reaction solution at the start of the reaction can be 0.5 w / v% or more, 1 w / v% or more, or 2 w / v% or more, and can be 20 w / v% or less, 10 w / v% or less, or 5 w / v% or less. Within this range, phenol is efficiently produced.

[0059] In addition to the free acid form, 4-HBA can take the form of salts such as sodium salts and potassium salts, and esters with alcohols having 1 to 4 carbon atoms. In the present invention, "4-HBA" encompasses the free acid form, salts, and esters with alcohols having 1 to 4 carbon atoms, and one or more types of 4-HBA can be used. In addition, the "4-HBA concentration" in the case of a salt or ester is a value calculated based on the weight converted into the weight of 4-HBA as a free acid.

[0060] Examples of buffer solutions that can be used include phosphate buffer, Tris buffer, and carbonate buffer. Examples of inorganic salt media include inorganic media containing one or more inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, cobalt chloride, calcium carbonate, calcium chloride, ammonium sulfate, iron sulfate, molybdenum oxide, and copper sulfate. The pH of the reaction solution is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within these ranges, phenol can be produced efficiently.

[0061] The reaction temperature between the transformant and 4-HBA can be 20°C or higher, 30°C or higher, 40°C or higher, or 50°C or higher, and 65°C or lower, 60°C or lower, 55°C or lower, or 52°C or lower. This temperature range allows for efficient production of phenol. The reaction time can be approximately 0.5 to 7 days. The density of the transformant at the start of the reaction, converted into dry cell weight, can be 0.2 g / L or higher, 1 g / L or higher, 5 g / L or higher, 25 g / L or higher, or 50 g / L or higher. The upper limit can be set within a technically feasible range, for example, 200 g / L. The reaction can be performed batchwise, fed-batch, or continuous. When the transformant is grown prior to the reaction, it is not necessary to grow the transformant during the reaction. Therefore, a carbon dioxide-containing gas may or may not be introduced into the reaction solution. The carbon dioxide-containing gas is preferably a mixed gas containing hydrogen, oxygen, and carbon dioxide, and more preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide.

[0062] By reacting 4-HBA with the transformant of the present invention as described above, phenol is produced in the reaction solution. Phenol can be recovered by recovering the reaction solution, but it can also be separated from the reaction solution by known methods such as distillation, membrane permeation, and organic solvent extraction.

[0063] 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.

[0064] (1) Methods for PCR, electrophoresis, DNA recovery, and seamless cloning in constructing expression plasmids. PCR, electrophoresis, DNA recovery, and preparation of the plasmid vector pCAMO-6 for seamless cloning in constructing the marker cassette and expression plasmid were performed using the following methods.

[0065] (1-1) 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 excised from the gel and recovered as needed using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0066] (1-2) Preparation of Plasmid Vector pCAMO-6 for Seamless Cloning To perform seamless cloning, the plasmid vector pCAMO-6 (SEQ ID NO: 1) was amplified by PCR using the DNA of SEQ ID NO: 1 as a template. The following primers were used for PCR: Primers for amplifying the plasmid vector pCAMO-6: (a-1) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 37) (b-1) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 38) As a result of electrophoresis, a DNA fragment of approximately 5.2 kbp corresponding to the vector gene was detected, and this DNA fragment was recovered from the gel.

[0067] (1-3) Ligation of the vector pCAMO-6 with the DNA fragment to be inserted: The DNA fragment of the vector pCAMO-6 synthesized above was ligated to the DNA fragment to be inserted using recombinase extracted from Escherichia coli JM109 strain. The resulting reaction mixture was transformed into Escherichia coli JM109 strain 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.

[0068] (1-4) 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. 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.

[0069] (2) Construction of hbdc expression plasmid DNA fragments consisting of the three subunits that make up hbdc from the following bacteria were amplified by PCR: Alicyclobacillus acidophilus (SEQ ID NO: 5), Alicyclobacillus sutchi (SEQ ID NO: 12), Bacillus licheniformis (SEQ ID NO: 19), Saccharopolyspora rectivirgra (SEQ ID NO: 26), and Cronobacter sakazakii (SEQ ID NO: 33).

[0070] The following primers were used for PCR: Primers for amplifying Alicyclobacillus acidophilus hbdc (a-2) 5'-GATCTGGAGGAGAAACGCATATGAGAATCGTCGTCGGTATTAC-3' (SEQ ID NO: 39) (b-2) 5'-GTCGACGGAGCTCGAATTCTTACTTGCGCTGTGCAATCTCGGGC-3' (SEQ ID NO: 40) Primers (a-2) and (b-2) contained sequences homologous to the vector pCAMO-6. Primers for amplifying Alicyclobacillus stiphi hbdc: (a-3) 5'-CAGATCTGGAGGAGAAACGCATATGAAGGAAGGTGGGAGATCCG-3' (SEQ ID NO: 41) (b-3) 5'-GTCGACGGAGCTCGAATTCTCAGTGGTTTGTCCGCGATGGCAC -3' (SEQ ID NO: 42) Primers (a-3) and (b-3) contain sequences homologous to the vector pCAMO-6. Primers for amplifying Bacillus licheniformis hbdc: (a-4) 5'-CAGATCTGGAGGAGAAACGCATATGAACATCATCGTCGGAATC-3' (SEQ ID NO: 43) (b-4) 5'-CTTGTCGACGGAGCTCGAATTCTTAGATTTTCCGGTCTGGAATC-3' (SEQ ID NO: 44) Primers (a-4) and (b-4) contain sequences homologous to the vector pCAMO-6. Primers for amplifying Saccharopolyspora rectivirgra hbdc (a-5) 5'-GATCTGGAGGAGAAACGCATATGCGGCTGGTCCTCGGGATCACC-3' (SEQ ID NO: 45) (b-5) 5'-GTCGACGGAGCTCGAATTCTCACGCGCTCCCGGTGCGCAGCGG-3' (SEQ ID NO: 46) Primers (a-5) and (b-5) contain sequences homologous to the vector pCAMO-6.Primers for amplifying Cronobacter sakazakii hbdc: (a-6) 5'-CAGATCTGGAGGAGAAACGCATATGAGGCTAATTGTCGGAATG-3' (SEQ ID NO: 47) (b-6) 5'-GTCGACGGAGCTCGAATTCTTAACGCTTACCATCCGCCAGCAGC-3' (SEQ ID NO: 48). Primers (a-6) and (b-6) contain sequences homologous to the pCAMO-6 vector. Electrophoresis detected a DNA fragment of approximately 2.3 kbp for the hbdc from each strain, and the DNA fragment was recovered from the gel.

[0071] The DNA fragment of vector pCAMO-6 and each hbdc DNA fragment were ligated to each other, and the Escherichia coli JM109 strain was transformed with the resulting DNA. Plasmids were then extracted and the sequences of each hbdc were confirmed.

[0072] (3) Hydrogenophilus thermorteolus Transformants (3-1) Gene Introduction: Hydrogenophilus thermorteolus TH-1 was transformed with each of the plasmids obtained in section (2) "Construction of hbdc Expression Plasmids" by electroporation. The transformants were then plated onto LB solid medium containing 50 μg / mL kanamycin and cultured at 52°C for 24 hours. Each strain grown on LB solid medium was inoculated 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. PCR was performed using a Life Technologies DNA Thermal Cycler with KOD One PCR Master Mix (Toyobo Co., Ltd.) as the reaction reagent. As a result, amplification of a DNA fragment of approximately 2.3 kbp in length corresponding to the hbdc gene consisting of three subunits was confirmed. Plasmids containing hbdc (DNA consisting of three subunit genes) derived from each bacterium, and the Hydrogenophilus thermorteolus TH-1 strain transformants transformed with these plasmids, were named as shown in Table 1.

[0073] (3-2) Confirmation of 4-HBA decarboxylase activity The hbdc-transfected strain prepared as described above was cultured in liquid medium A containing 50 μg / mL of kanamycin [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, and CoCl2 6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0). The cells were inoculated into a 1000-well plate (7.0) using a platinum loop, and a gas mixture of H2:O2:CO2 (7.5:1:1.5) was supplied during the culture, followed by shaking at 52°C for 72 hours. Hydrogenophilus thermorteolus strain TH-1, carrying the empty vector pCAMO-6 (which lacks the hbdc gene), was also cultured in the same manner. After cultivation, the cells were harvested by centrifugation (4°C, 5,000 g, 10 minutes). The cells were suspended in 100 mM Tris-HCl (pH 7.5) (cell density calculated as dry weight: 5 g / L) and then sonicated. 4-HBA dissolved in 100 mM Tris-HCl (pH 7.5) was added to the resulting cell lysate and incubated at 52°C for 8 hours. Free 4-HBA was used. The final concentration of 4-HBA in the reaction mixture at the start of the reaction was 4 mM. The phenol produced by the reaction was quantified using high-performance liquid chromatography (Shimadzu Corporation) under the following conditions: Mobile phase A: 0.1% aqueous phosphoric acid; Mobile phase B: 50% acetonitrile; Flow rate: 1 mL / min; Column: CAPCELLPAK 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. As shown in Table 1, in the case of the hbdc005 strain, which had been transformed with the Cronobacter sakazakii hbdc gene, all of the 4-HBA used in the reaction was converted to phenol.Conversion of 4-HBA to phenol was also observed in the hbdcl01 strain, into which the hbdc from Alicyclobacillus acidophilus had been introduced, the hbdcl02 strain, into which the hbdc from Alicyclobacillus schii had been introduced, the hbdcl03 strain, into which the hbdc from Bacillus licheniformis had been introduced, and the hbdcl04 strain, into which the hbdc from Saccharopolyspora rectivirgra had been introduced.

[0074]

[0075] (4) Production of phenol from 4-HBA using a transformant. The Cronobacter sakazakii strain hbdc-introduced hbdc005 cells (undisrupted cells) prepared as described above were suspended in a reaction solution containing 4-HBA dissolved in Tris-HCl (pH 7.5) (cell density converted to dry weight: 13 g / L), and the reaction was carried out at 52°C for 8 hours without introducing a mixed gas. The final concentration of 4-HBA in the reaction solution at the start of the reaction was 30 mM. 4-HBA was used in its free form. The phenol produced was quantified by high-performance liquid chromatography under the conditions described above.

[0076] As a result, approximately 27 mM of 4-HBA was converted to phenol.

[0077] 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.

[0078] The sequences of SEQ ID NOs: 1 to 36 are shown below.

[0079] The transformant of the present invention can produce 4-HBA decarboxylase and thus phenol with high efficiency without using sugar as a carbon source, thereby contributing to the efficient industrial production of phenol and chemical products using phenol as a raw material while resolving global warming caused by increased carbon dioxide.

Claims

1. A transformant obtained by introducing the following DNA (a), (b1), (b2), (c), (d), or (e) into a bacterium belonging to the genus Hydrogenophilus: (a) a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 2 to 4, a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 9 to 11, a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 16 to 18, a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 23 to 25, a set of three DNAs containing the nucleotide sequences of SEQ ID NOs: 30 to 32, or a DNA containing the nucleotide sequence of SEQ ID NO: 5, 12, 19, 26, or 33 (b1) A set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOs: 2 to 4 have been replaced with a nucleotide sequence having 90% or more identity to the nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; a set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOs: 9 to 11 have been replaced with a nucleotide sequence having 90% or more identity to the nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; a set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOs: 16 to 18 have been replaced with a nucleotide sequence having 90% or more identity to the nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; A set of three DNAs in which NA contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; a set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOS: 23 to 25 have been replaced with a nucleotide sequence having 90% or more identity to that nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity; or a set of three DNAs in which one or more of the nucleotide sequences of SEQ ID NOS: 30 to 32 have been replaced with a nucleotide sequence having 90% or more identity to that nucleotide sequence, and the DNA after the replacement contains a nucleotide sequence encoding a subunit of a polypeptide having 4-HBA decarboxylase activity.(b2) DNAs encoding three subunits of a polypeptide having 4-HBA decarboxylase activity, comprising a nucleotide sequence having 90% or more identity to the nucleotide sequence of SEQ ID NO: 5, 12, 19, 26, or 33; (c) a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 6 to 8; a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 13 to 15; a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 20 to 22; a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 27 to 29; or a set of three DNAs encoding three polypeptides comprising the amino acid sequences of SEQ ID NOs: 34 to 36.(d) A set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 6 to 8 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 13 to 15 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 20 to 22 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences that are the amino acid sequences of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which any one or more of the amino acid sequences of SEQ ID NOS: 27 to 29 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase; or a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which any one or more of the amino acid sequences of SEQ ID NOS: 34 to 36 have been replaced with an amino acid sequence having 90% or more identity to the amino acid sequence, and the amino acid sequence after the replacement is the amino acid sequence of a subunit of 4-HBA decarboxylase.(e) A set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 6 to 8 have one to five amino acids deleted, substituted, inserted, or added, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase; a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 13 to 15 have one to five amino acids deleted, substituted, inserted, or added, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase; and a set of three DNAs encoding three polypeptides each containing three amino acid sequences in which one or more of the amino acid sequences of SEQ ID NOS: 20 to 22 have one to five amino acids deleted, substituted, inserted, or added, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase. a set of three DNAs encoding three polypeptides each comprising one of three amino acid sequences of SEQ ID NOS: 27 to 29, in which one or more of the amino acid sequences have been deleted, substituted, inserted or added with 1 to 5 amino acids, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase; or a set of three DNAs encoding three polypeptides each comprising one of three amino acid sequences of SEQ ID NOS: 34 to 36, in which one or more of the amino acid sequences have been deleted, substituted, inserted or added with 1 to 5 amino acids, and the amino acid sequence after the substitution is the amino acid sequence of a subunit of 4-HBA decarboxylase.

2. The transformant according to claim 1, wherein the Hydrogenophilus bacterium is Hydrogenophilus thermorteolus.

3. A method for producing phenol, comprising the step of reacting the transformant according to claim 1 or 2 with 4-hydroxybenzoic acid in a reaction solution.

4. The method according to claim 3, which comprises a step of growing the transformant according to claim 1 or 2 before the reaction step.

Images