Phenol-generating hydrogenophilus bacterium transformant

Abstract

This transformant is obtained by introducing the gene for tyrosine phenol-lyase of (a), (b), (c), (d), or (e) to a bacterium of the genus Hydrogenophilus and can efficiently produce phenol by utilizing carbon dioxide as the sole carbon source. (a) DNA that includes the base sequence of SEQ ID NO: 2 or 4; (b) DNA that includes a base sequence that has at least 90% identity with SEQ ID NO: 2 or 4 and codes for a polypeptide that has tyrosine phenol-lyase activity; (c) DNA that codes for a polypeptide that includes the amino acid sequence of SEQ ID NO: 3 or 5; (d) DNA that codes for a polypeptide that includes an amino acid sequence that has at least 90% identity with SEQ ID NO: 3 or 5 and that codes for a polypeptide that has tyrosine phenol-lyase activity; and (e) DNA that codes for a polypeptide that includes the amino acid sequence of SEQ ID NO: 3 or 5 with 1 to 40 amino acids deleted, substituted, inserted, or added and that codes for a polypeptide that has tyrosine phenol-lyase activity.

Description

Phenol-producing transformants of Hydrogenophilus sp.

[0001] The present invention relates to a transformant of a bacterium belonging to the genus Hydrogenophilus having phenol-producing ability, and a 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 would result in distinctly different climate changes in different regions of the world compared to a 1.5°C rise. It called for a reduction of global anthropogenic CO2 emissions by approximately 45% compared to 2010 levels by 2030, reaching zero by around 2050—a carbon-neutral goal. 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 goal.

[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 stable and continuous supply is required. However, the majority of chemical production worldwide is based on petroleum-based raw materials, which increases greenhouse gas emissions. Therefore, chemical production methods must shift from traditional methods that consume large amounts of petroleum resources to carbon recycling methods that effectively utilize carbon resources. To this end, research and development into biorefineries that use microbial fermentation to produce green chemicals from biomass is being actively pursued around the world. However, converting biomass into sugars, which are the feedstock for microbial fermentation, requires complex processes and is expensive. Furthermore, using biomass that can be used as food or feed for chemical production hinders the stable supply of food and feed. Furthermore, the large-scale consumption of biomass for chemical production can actually lead to environmental damage.

[0004] 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.

[0005] 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, solvent, and analytical reagent. Among these, phenolic resins are in high demand and are an important compound. Phenolic resins are the oldest synthetic resins and have 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 in a wide range of fields, including medicine, agriculture, and construction. Furthermore, the blending of phenolic resins with other materials, such as glass fiber and organic or inorganic fillers, has led to the creation of new functional materials, increasing the demand for phenolic resins.

[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 expected to run out in the future, to a sustainable society, phenol production by 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 complex process required, the disruption of stable food and feed supplies, and the large environmental impact. Therefore, a practical method for producing phenol using microorganisms through a simpler process that does not disrupt the stable supply of food and feed and does not burden the environment is needed. In particular, a practical method for producing phenol by fixing carbon dioxide is needed.

[0008] Many microorganisms have a metabolic pathway that produces phenol from the aromatic amino acid tyrosine through the catalytic action of tyrosine phenol-lyase, and another metabolic pathway that produces decarboxylase from 4-hydroxybenzoic acid or salicylic acid on the shikimate pathway. However, no microorganisms have been reported to actually produce phenol.

[0009] As a technique for producing phenol by fermentation using genetically modified microorganisms, Patent Document 1 discloses that a tyrosine phenol-lyase gene derived from Pantoea agglomerans, Citrobacter braakii, Desulfitobacterium hafniense, Chloroflexus aurantiacus, Nostoc punctiforme, or Treponema denticola introduced into Corynebacterium glutamicum expresses an active polypeptide, and discloses a method for producing phenol from glucose, a carbon source, using a transformant into which the tyrosine phenol-lyase gene derived from Pantoea agglomerans has been introduced. Non-Patent Documents 1 to 4 teach a method for producing phenol from glucose using a transformant of Escherichia coli introduced with a tyrosine phenol-lyase gene derived from Pasteurella multocida, and a method for producing phenol from glucose using a transformant of Pseudomonas putida or Pseudomonas taiwanensis introduced with a tyrosine phenol-lyase gene derived from Pantoea agglomerans. Non-Patent Document 5 teaches a method for producing phenol from glucose by microbial co-cultivating Escherichia coli introduced with a 4-hydroxybenzoate decarboxylase gene derived from Escherichia coli and Escherichia coli introduced with a tyrosine phenol-lyase gene derived from Pasteurella multocida.Non-Patent Document 6 teaches a method for producing phenol from glucose using a transformant of Escherichia coli into which the tyrosine phenol-lyase gene derived from Citrobacter Braakii, the 4-hydroxybenzoate decarboxylase gene derived from Klebsiella pneumoniae, and the salicylate decarboxylase gene derived from Trichosporon moniliiforme have been introduced.

[0010] However, all of these methods involve growing microorganisms using sugars, which are carbon raw materials with high production costs, to produce phenol, and are not methods of producing phenol using carbon dioxide as a carbon raw material.

[0011] Re-table 2012 / 033112 issue

[0012] J Bacteriol., 190 (8): 2822-2830 (2008)Appl Environ Microbiol., 71(12):8221-8227 (2005)Metab Eng., 47 : 121- 133 (2018)Biotechnol J., 9(5):621-629(2014)Biotechnol Bioeng., 116(12):3349-3359(2019)Fermentation., 7(4):216(2021)

[0013] An object of the present invention is to provide a transformant of a Hydrogenophilus bacterium that can efficiently produce phenol using carbon dioxide as a sole carbon source, and a method for efficiently producing phenol using the transformant.

[0014] 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 expensive 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 possess an enzyme for producing phenol. Therefore, to impart the ability to produce phenol on an industrial scale to Hydrogenophilus bacteria, it is necessary to introduce a gene for an enzyme that catalyzes the reaction that produces phenol.

[0015] The present inventors have found that even 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 produce substances in bacteria of the genus Hydrogenophilus for the purpose of substance production.

[0016] Under these circumstances, the present inventors investigated genes predicted to be tyrosine phenol-lyase genes present in the genomes of other microorganisms in order to obtain a tyrosine phenol-lyase gene that can be expressed in Hydrogenophilus bacteria. They found that the tyrosine phenol-lyase genes of Chloroflexus aggregans and Chloroflex aurantiacus express functional tyrosine phenol-lyase in Hydrogenophilus bacteria. They also found that transformants obtained by introducing these genes into Hydrogenophilus bacteria can efficiently produce phenol using carbon dioxide as the sole carbon source.

[0017] 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 tyrosine phenol-lyase gene (a), (b), (c), (d), or (e) into a bacterium belonging to the genus Hydrogenophilus: (a) DNA comprising the nucleotide sequence of SEQ ID NO: 2 or 4; (b) DNA comprising a nucleotide sequence having 90% or more identity to SEQ ID NO: 2 or 4 and encoding a polypeptide having tyrosine phenol-lyase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 5; (d) DNA encoding a polypeptide having 90% or more identity to SEQ ID NO: 3 or 5 and having tyrosine phenol-lyase activity; (e) DNA encoding a polypeptide having tyrosine phenol-lyase activity and comprising 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: 3 or 5. [2] The transformant according to [1], wherein the bacterium of the genus Hydrogenophilus is Hydrogenophilus thermorteolus. [3] A method for producing phenol, comprising a step of culturing the transformant according to [1] or [2].

[0018] 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.

[0019] Phenol is an aromatic compound that is important industrially. However, its chemical synthesis from petroleum is undesirable from an environmental perspective. In contrast, the production of phenol using carbon dioxide-fixing microorganisms can simultaneously address global warming caused by increased carbon dioxide emissions and ensure the availability of industrially needed phenol.

[0020] 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."

[0021] According to the present invention, by introducing a specific tyrosine phenol-lyase gene into a bacterium belonging to the genus Hydrogenophilus, a tyrosine phenol-lyase that functions in the bacterium can be expressed, enabling phenol to be produced on an industrial scale. As described above, among organisms capable of fixing carbon dioxide, bacteria belonging to the genus Hydrogenophilus have an especially excellent ability to fix carbon dioxide. Therefore, the present invention has opened the way to producing phenol on an industrial scale using carbon dioxide.

[0022] The present invention is described in detail below. (1) Transformant Capable of Producing Phenol The transformant (transformed bacterium) of the present invention is a transformant obtained by introducing a tyrosine phenol-lyase gene (hereinafter sometimes abbreviated as "tpl gene") from Chloroflex agregans or Chloroflex aurantiacus, or a homolog thereof, into a host Hydrogenophilus bacterium. That is, the transformant of the present invention is a Hydrogenophilus bacterium transformant harboring an exogenous tpl gene, such as a tpl gene from Chloroflex agregans or Chloroflex aurantiacus, or a homolog thereof. The tpl gene used in the present invention does not need to be known to have been identified as a tpl gene; it may be DNA encoding a polypeptide having tyrosine phenol-lyase activity. Tyrosine phenol-lyase activity refers to the enzymatic activity of producing phenol from tyrosine. In addition, in the present invention, the tpl gene may be DNA of a tpl gene isolated from a naturally occurring bacterium, or DNA artificially synthesized using techniques known to those skilled in the art.

[0023] The transformant of the present invention may be any bacterium of the genus Hydrogenophilus in which the tpl gene of Chloroflex agregans, the tpl gene of Chloroflex aurantiacus, or a homolog thereof has been introduced, and includes transformants in which two or more of these genes have been introduced into a bacterium of the genus Hydrogenophilus, and transformants in which other genes have been introduced into a bacterium of the genus Hydrogenophilus in addition to these genes.

[0024] Tyrosine phenol-lyase gene (tpl gene) In the present invention, DNA containing the nucleotide sequence of SEQ ID NO: 2 or 4 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4) can be used as the tpl gene. SEQ ID NO: 2 is the nucleotide sequence of the tpl gene of Chloroflexus agregans, and SEQ ID NO: 4 is the nucleotide sequence of the tpl gene of Chloroflexus aurantiacus. In the present invention, DNA containing a nucleotide sequence having 90% or more, preferably 95% or more, preferably 98% or more, and particularly 99% or more identity to SEQ ID NO: 2 or 4 (particularly, consisting of a nucleotide sequence having 90% or more, preferably 95% or more, preferably 98% or more, and particularly 99% or more identity to SEQ ID NO: 2 or 4) and encoding a polypeptide having tyrosine phenol-lyase activity can also be used.

[0025] Furthermore, in the present invention, a DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 5 (particularly, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or 5) can be used as the tpl gene. SEQ ID NO: 3 is the amino acid sequence of tyrosine phenol-lyase from Chloroflexus agregans, and SEQ ID NO: 5 is the amino acid sequence of tyrosine phenol-lyase from Chloroflexus aurantiacus.

[0026] DNAs that encode a polypeptide having tyrosine phenol-lyase activity and 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 SEQ ID NO: 3 or 5 (particularly, an amino acid sequence that is 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identical to SEQ ID NO: 3 or 5) can also be used. Furthermore, DNAs that encode a polypeptide having tyrosine phenol-lyase activity and that contain an amino acid sequence in which 1 to 40, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 3 or 5 (particularly, an amino acid sequence in which 1 to 40, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 3 or 5) can also be used.

[0027] A DNA sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 3 or 5 may have various base substitutions in the coding region, taking into account codon degeneracy or preferred codons in Hydrogenophilus bacteria, as long as the amino acid sequence of the protein expressed from the coding region is not changed.

[0028] In the present invention, the identities of base sequences and amino acid sequences are values ​​calculated using GENETYX ver. 17 (GENETYX).

[0029] In the present invention, whether a test polypeptide has tyrosine phenol-lyase activity is confirmed by reacting the test polypeptide with tyrosine and detecting the produced phenol by high performance liquid chromatography (HPLC).

[0030] 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 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 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 40 amino acids have been deleted, substituted, inserted, or added from the amino acid sequence of a certain polypeptide and encodes a polypeptide that has the same activity as that polypeptide is referred to as a homolog of the DNA encoding that polypeptide.

[0031] Examples of Hydrogenophilus bacteria include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidance, 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.

[0032] In the present invention, the host Hydrogenophilus bacterium may be a bacterium isolated from nature, or a bacterium that has been genetically modified from a bacterium isolated from nature. Modification can be performed for purposes such as enabling high expression of the introduced tpl gene. Such modification can be achieved by removing (curing) the endogenous plasmid in the Hydrogenophilus bacterium or by disrupting a gene on the genome of the Hydrogenophilus bacterium. Methods for removing endogenous plasmids are well known, including methods that utilize plasmid incompatibility, such as the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide; destabilization by introducing a plasmid with the same replication origin as the endogenous plasmid; or destabilization by disrupting factors involved in the plasmid partition system.

[0033] A method for preparing a transformant by introducing the tpl gene into a bacterium belonging to the genus Hydrogenophilus to obtain a transformant is described below. The tpl gene can be introduced into a bacterium belonging to the genus Hydrogenophilus using a general method for introducing foreign genes into bacteria. The tpl gene may be directly introduced into a bacterium belonging to the genus Hydrogenophilus, or a vector containing the tpl gene for transformation (e.g., a plasmid vector, a viral vector, a cosmid, a fosmid, a BAC, etc.) may be introduced into a bacterium belonging to the genus Hydrogenophilus. The vector for transformation may contain DNA capable of autonomous replication in Hydrogenophilus bacteria. Examples include broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI Reference Sequence: NC_004444.1), pC194 (NCBI Reference Sequence: NC_002013.1), pK18mobsacB (GenBank: FJ437239.1), and pUB110 (NCBI Reference Sequence: NC_001384.1), as well as genetically modified versions of these vectors (e.g., pCAMO-6). Among these, pCAMO-6 (SEQ ID NO: 1) 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. The tpl gene can be introduced (transformed) into Hydrogenophilus bacteria using conventional techniques, such as the calcium chloride method, calcium phosphate method, rubidium chloride method, and electric pulse method (electroporation).

[0034] (2) Method for Producing Phenol The present invention provides a method for producing phenol using the transformant of the present invention described above. This method includes a step of culturing the transformant of the present invention, particularly a step of culturing the transformant of the present invention using an inorganic or organic medium while supplying a 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 phenol can be efficiently produced.

[0035] 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 phenol 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.

[0036] 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 phenol production. 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 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 introducing the mixed gas 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, bacterial growth is favorable and phenol 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, phenol can be produced efficiently, and waste of the mixed gas is reduced. The culture temperature is preferably 35 to 55°C, more preferably 37 to 52°C, and even more preferably 50 to 52°C. Within this range, the growth of the transformant is favorable, and phenol can be produced efficiently.

[0037] By culturing as described above, phenol is produced in the culture 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.

[0038] 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.

[0039] (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.

[0040] (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 recovered from the gel using a GEL / PCR Purification Mini Kit (FAVORGEN) as needed.

[0041] (1-2) 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: 1 as a template. The following primers were used for PCR: Primers for amplifying plasmid vector pCAMO-6: (a-1) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 6) (b-1) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 7) 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.

[0042] (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.

[0043] (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.

[0044] (2) Construction of expression plasmid for tpl gene DNA fragments of the tpl gene from the following bacteria were amplified by PCR: Chloroflexus agregans (SEQ ID NO: 2), Chloroflexus aurantiacus (SEQ ID NO: 4), Chryseobacterium gleum (SEQ ID NO: 8), and Serratia plymuthica (SEQ ID NO: 9).

[0045] The following primers were used for PCR: Primers for amplifying the Chloroflexus agregans tpl gene: (a-2) 5'-CTGGAGGAGAAACGCATATGGAGATGGAACCAGACTTCCCAC-3' (SEQ ID NO: 10) (b-2) 5'-CGACGGAGCTCGAATTCTCACTCCGTAACCGGTTCAAAGCGGGC-3' (SEQ ID NO: 11) Primers (a-2) and (b-2) contained sequences homologous to the vector pCAMO-6. Primers for amplifying the tpl gene of Chloroflexus aurantiacus: (a-3) 5'-CTGGAGGAGAAACGCATATGCAGGAACAAGACTACCCCCGTAC-3' (SEQ ID NO: 12) (b-3) 5'-CGACGGAGCTCGAATTCTCATTCCACCGGTTCAAACCGGGCCTG-3' (SEQ ID NO: 13). Primers (a-3) and (b-3) contain sequences homologous to the vector pCAMO-6. Primers for amplifying the tpl gene of Chryseobacterium gleum: (a-4) 5'-CTGGAGGAGAAACGCATATGAATTTACCGTACGCGGAAC-3' (SEQ ID NO: 14) (b-4) 5'-CGACGGAGCTCGAATTCTTAGGCTTTTTCAAGCTGAACAG-3' (SEQ ID NO: 15). Primers (a-4) and (b-4) contain sequences homologous to the vector pCAMO-6. Primers for amplifying the Serratia prymusica tpl gene: (a-5) 5'-CTGGAGGAGAAACGCATATGTCCTACCCAGCTGAACC-3' (SEQ ID NO: 16) (b-5) 5'-CGACGGAGCTCGAATTCTCACAAATATTCAAAGCGCGCG-3' (SEQ ID NO: 17) Primers (a-5) and (b-5) contain sequences homologous to the vector pCAMO-6. Electrophoresis detected a DNA fragment of approximately 1.4 kbp for the tpl gene derived from each strain, and the DNA fragment was recovered from an agarose gel.

[0046] The DNA fragment of vector pCAMO-6 and each tpl gene DNA fragment were ligated together and used to transform Escherichia coli JM109, after which the plasmid was extracted and the tpl gene sequence was confirmed.

[0047] (3) Hydrogenophilus thermorteolus Transformants (3-1) Gene Introduction: The Hydrogenophilus thermorteolus TH-1 strain was transformed with each of the plasmids obtained in section "(2) Construction of Expression Plasmids for the tpl Gene" 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 1.4 kbp corresponding to the tpl gene was confirmed. The plasmids containing the tpl genes derived from each bacterium and the Hydrogenophilus thermorteolus TH-1 strain transformants transformed with these plasmids were named as shown in Table 1.

[0048] (3-2) Production of phenol The tpl gene-transformed 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 cultivation. The culture was then cultured at 52°C for 72 hours with shaking. The Hydrogenophilus thermorteolus TH-1 strain, carrying the empty vector pCAMO-6 lacking the tpl gene, was also cultured in the same manner. After cultivation, the culture supernatant was obtained by centrifugation (4°C, 5,000 g, 10 minutes). The phenol concentration in the culture supernatant was measured 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: CAOCELLPAK MGII, 5.0 μm, 4.6 mm ID x 150 mm. Column temperature: 40°C. PDA temperature: 40°C. PDA wavelength: 200-300 nm. Reference wavelength: 210 nm. As shown in Table 1, 0.3 mM phenol was detected in the culture supernatant of strain Tpl01, which contained the tpl gene of Chloroflexus agregans. 0.15 mM phenol was detected in strain Tpl02, which contained the tpl gene of Chloroflexus aurantiacus. In contrast, phenol was not detected in strains containing the tpl gene of Chryseobacterium gleum or Serratia prymusica, or in strains without the tpl gene.

[0049]

[0050] 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.

[0051] SEQ ID NOs: 1, 2, 3, 4, 5, 8, and 9 are shown below.

[0052] The transformant of the present invention can produce phenol with high efficiency using carbon dioxide as the sole carbon source, and therefore contributes to the industrial production of phenol and chemical products using phenol as a raw material with high efficiency while resolving global warming caused by increased carbon dioxide.

Claims

1. A transformant obtained by introducing a tyrosine phenol-lyase gene of the following (a), (b), (c), (d), or (e) into a bacterium belonging to the genus Hydrogenophilus: (a) DNA comprising the nucleotide sequence of SEQ ID NO: 2 or 4; (b) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 2 or 4, and encoding a polypeptide having tyrosine phenol-lyase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or 5; (d) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 3 or 5, and having tyrosine phenol-lyase activity; (e) DNA encoding a polypeptide having 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: 3 or 5, and having tyrosine phenol-lyase activity.

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

3. A method for producing phenol, comprising a step of culturing the transformant according to claim 1 or 2.

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