Hydrogenophilus bacterium transformant capable of producing salicylic acid

Abstract

Provided is a transformant produced by introducing a specific salicylate synthase gene or introducing a specific isochorismate synthase gene and a specific isochorismate-pyruvate lyase gene into a bacterium belonging to the genus Hydrogenophilus. The transformant can efficiently produce salicylic acid by using carbon dioxide as a sole carbon source.

Description

Hydrogenophilus transformants producing salicylic acid

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

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

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

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

[0005] Salicylic acid is an important compound used in pharmaceuticals, cosmetics, and food. In pharmaceuticals, salicylic acid is used as a precursor for the production of aspirin and lamivudine (an anti-HIV drug). In cosmetics, it is incorporated as a peeling agent in skin care cosmetics and facial cleansers. Recently, microbial and chemical conversion of salicylic acid into adipic acid, a raw material for nylon-6,6, has been reported. The Kolbe-Schmitt process, an industrial method for producing salicylic acid, involves reacting phenol with sodium hydroxide to obtain sodium phenoxide, which is then reacted with carbon dioxide at high temperature and pressure to obtain sodium salicylate, which is then converted to salicylic acid under acidic conditions. Phenol, the raw material, is industrially produced by the cumene process, which uses petroleum-derived benzene and propylene. This process is complex and energy-intensive due to the high-temperature, high-pressure chemical industrial process. Therefore, there is a need to move away from petroleum-dependent chemical synthesis processes.

[0006] In order to transition from a society dependent on petroleum, which is expected to run out in the future, to a sustainable society, the production of chemicals through microbial fermentation using renewable non-edible biomass resources has attracted attention. However, as mentioned above, the use of biomass has various problems, such as the need for complex processes, the disruption of stable food and feed supplies, and the large environmental impact. Therefore, there is a need for a practical method for producing salicylic acid using microorganisms in a simpler process that does not disrupt the stable supply of food and feed and does not burden the environment. In particular, there is a need for a practical method for producing salicylic acid by fixing carbon dioxide.

[0007] In many microorganisms, isochorismate is produced from chorismate by the catalytic action of isochorismate synthase in the shikimate pathway, which is involved in the synthesis of aromatic amino acids, and salicylic acid is produced from isochorismate by the catalytic action of isochorismate-pyruvate lyase.

[0008] As a technique for producing salicylic acid by fermentation using genetically modified microorganisms, Patent Document 1 discloses a method for producing salicylic acid from glucose, a carbon source, by introducing isochorismate synthase derived from Escherichia coli or Pseudomonas aeruginosa and isochorismate-pyruvate lyase derived from Pseudomonas aeruginosa into Escherichia coli and suppressing the pathway that metabolizes phosphoenolpyruvate to other compounds upstream of salicylic acid production. Non-Patent Documents 1 and 2 teach methods for producing salicylic acid from glucose, a carbon source, by introducing isochorismate synthase derived from Escherichia coli and isochorismate-pyruvate lyase derived from Pseudomonas fluroescens into Escherichia coli.

[0009] However, all of these methods involve growing microorganisms using sugars, which are carbon sources with high production costs, to produce salicylic acid, and are not methods of producing salicylic acid using carbon dioxide as a carbon source.

[0010] Furthermore, Patent Document 2 discloses a method for producing salicylic acid by culturing a transformant of Clostridium bacteria, an anaerobic microorganism, into which isochorismate synthase and isochorismate-pyruvate lyase derived from Pseudomonas aeruginosa have been introduced. Clostridium bacteria grow using chlorine-containing gaseous substrates as a carbon source, but essentially require carbon monoxide as a carbon source to produce salicylic acid. Therefore, the method of Patent Document 2 does not produce salicylic acid using carbon dioxide as a carbon source, and therefore does not contribute sufficiently to combating global warming.

[0011] Among 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, but their use as hosts for salicylic acid production has not been reported.

[0012] WO2017 / 033965A1 special table 2018-522536

[0013] Microb Cell Fact., 18(1):18(2019) Metab Eng., 23:62-69 (2014)

[0014] An object of the present invention is to provide a transformant of a Hydrogenophilus bacterium that can efficiently produce salicylic acid using carbon dioxide as a sole carbon source, and a method for efficiently producing salicylic acid using the transformant.

[0015] 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 salicylic acid. Therefore, to impart the ability to produce salicylic acid on an industrial scale to Hydrogenophilus bacteria, it is necessary to introduce a gene for an enzyme that catalyzes the reaction that produces salicylic acid.

[0016] The present inventors have also 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 produce substances in bacteria of the genus Hydrogenophilus for the purpose of substance production.

[0017] Under these circumstances, the present inventors investigated genes predicted to be salicylate synthase genes, isochorismate synthase genes, and isochorismate-pyruvate lyase genes present in the genomes of other microorganisms in order to obtain salicylate synthase genes, isochorismate synthase genes, and isochorismate-pyruvate lyase genes that are expressed in Hydrogenophilus bacteria. As a result, they found that the salicylate synthase gene from Laceyella tengchongensis expresses a salicylate synthase that functions in Hydrogenophilus bacteria. They also found that the isochorismate synthase gene from Alicyclobacillus contaminans expresses an isochorismate synthase that functions in Hydrogenophilus bacteria. They also found that the isochorismate-pyruvate lyase gene from Pseudomonas aeruginosa can express isochorismate-pyruvate lyase that functions in Hydrogenophilus bacteria. They also found that transformants obtained by introducing the salicylate synthase gene from Raceella tenchogensis into Hydrogenophilus bacteria, and transformants obtained by introducing the isochorismate synthase gene from Alisicolobacillus contamins and the isochorismate-pyruvate lyase gene from Pseudomonas aeruginosa into Hydrogenophilus bacteria, can produce salicylic acid using carbon dioxide as the sole carbon source.

[0018] Salicylate synthase is a bifunctional enzyme that catalyzes the reaction of chorismate to salicylic acid, possessing both isochorismate synthase activity, which produces isochorismate from chorismate, and isochorismate-pyruvate lyase activity, which produces salicylic acid from isochorismate.

[0019] The present invention was completed based on the above findings and provides the following transformant and method for producing salicylic acid: [1] A transformant obtained by introducing the following salicylic acid synthase 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; (b) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 2, and encoding a polypeptide having salicylic acid synthase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3; (d) DNA encoding a polypeptide having 90% or more identity with SEQ ID NO: 3, and having salicylic acid synthase activity; (e) DNA encoding a polypeptide having salicylic acid synthase 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. [2] A transformant obtained by introducing the following isochorismate synthase gene (f), (g), (h), (i), or (j) and the isochorismate-pyruvate lyase gene (k), (l), (m), (n), or (o) into a bacterium of the genus Hydrogenophilus.(f) DNA comprising the nucleotide sequence of SEQ ID NO: 4; (g) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 4 and encoding a polypeptide having isochorismate synthase activity; (h) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 5; (i) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 5 and having isochorismate synthase activity; (j) 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: 5 and having isochorismate synthase activity; (k) DNA comprising the nucleotide sequence of SEQ ID NO: 6; (l) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 6 and encoding a polypeptide having isochorismate-pyruvate lyase activity; (m) DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO: 7; (n) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO: 7 and having isochorismate-pyruvate lyase activity; (o) [3] A DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 in which 1 to 10 amino acids have been deleted, substituted, inserted, or added, and having isochorismate-pyruvate lyase activity. [4] A method for producing salicylic acid, comprising a step of culturing the transformant of [1], [2], or [3].

[0020] Measures to curb the increase in carbon dioxide include reducing carbon dioxide emissions and fixing emitted carbon dioxide. To reduce carbon dioxide emissions, solar, wind, geothermal, and other energy sources are being used in place of fossil fuels. However, in reality, even these energy sources have not sufficiently curbed the increase in carbon dioxide. Therefore, it is necessary to promote the fixation or resource recovery of emitted carbon dioxide. Carbon dioxide can be fixed physically or chemically, but if it is fixed using living organisms, it can be used to produce organic matter that can be used as food, feed, fuel, and other products. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve both the two problems of global warming caused by increased carbon dioxide and the difficulty in securing food, feed, and fuel. Furthermore, it can produce chemical products in demand while curbing global warming caused by increased carbon dioxide.

[0021] Salicylic acid 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 salicylic acid using carbon dioxide-fixing microorganisms can simultaneously address global warming caused by increased carbon dioxide emissions and ensure the supply of salicylic acid needed industrially.

[0022] 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."

[0023] According to the present invention, by introducing a specific salicylate synthase gene into a bacterium belonging to the genus Hydrogenophilus, a salicylate synthase that functions in the bacterium can be expressed, enabling industrial-scale production of salicylic acid. Furthermore, by introducing a specific isochorismate synthase gene and a specific isochorismate-pyruvate lyase gene into a bacterium belonging to the genus Hydrogenophilus, isochorismate synthase and isochorismate-pyruvate lyase that function in the bacterium can be expressed, enabling industrial-scale production of salicylic acid. As described above, among organisms capable of fixing carbon dioxide, Hydrogenophilus bacteria have particularly excellent carbon dioxide fixation capabilities. Therefore, the present invention has opened the way to industrial-scale production of salicylic acid using carbon dioxide.

[0024] The present invention is described in detail below. (1) Transformant The first transformant (transformed bacterium) of the present invention is a transformant obtained by introducing the Raceella tenchonggensis salicylate synthase gene 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 Raceella tenchonggensis salicylate synthase gene or a homolog thereof. The salicylate synthase gene used in the present invention does not need to be known to have been identified as a salicylate synthase gene; it may be DNA encoding a polypeptide having salicylate synthase activity. Salicylate synthase activity refers to the enzymatic activity of producing salicylic acid from chorismate. In addition, in the present invention, the salicylate synthase gene may be DNA of a salicylate synthase gene isolated from a naturally occurring bacterium, or DNA artificially synthesized using techniques known to those skilled in the art. The first transformant of the present invention may have two or more of the salicylic acid synthase gene of Raceella tenchongensis or its homologs, and may also have other foreign genes in addition to these.

[0025] The second transformant (transformed bacterium) of the present invention is a transformant obtained by introducing the Alisicolobacillus contamins isochorismate synthase gene or a homolog thereof and the Pseudomonas aeruginosa isochorismate-pyruvate lyase gene or a homolog thereof into a host Hydrogenophilus bacterium. That is, the transformant of the present invention is a Hydrogenophilus bacterium transformant having an exogenous Alisicolobacillus contamins isochorismate synthase gene or a homolog thereof and an exogenous Pseudomonas aeruginosa isochorismate-pyruvate lyase gene or a homolog thereof.

[0026] The isochorismate synthase gene used in the present invention does not need to have been identified as an isochorismate synthase gene, but may be DNA encoding a polypeptide having isochorismate synthase activity. Isochorismate synthase refers to the enzyme activity that produces isochorismate from chorismate. Furthermore, the isochorismate-pyruvate lyase gene does not need to have been identified as an isochorismate-pyruvate lyase gene, but may be DNA encoding a polypeptide having isochorismate-pyruvate lyase activity. Isochorismate-pyruvate lyase activity refers to the enzyme activity that produces salicylic acid from isochorismate.

[0027] In the present invention, the isochorismate synthase gene and the isochorismate-pyruvate lyase gene may be DNA of an isochorismate synthase gene and DNA of an isochorismate-pyruvate lyase gene isolated from a naturally occurring bacterium, respectively, or may be DNA artificially synthesized using techniques known to those skilled in the art. The second transformant of the present invention may have two or more Alisicolobacillus contamins isochorismate synthase genes or homologs thereof and two or more Pseudomonas aeruginosa isochorismate-pyruvate lyase genes or homologs thereof, and may also have other foreign genes in addition to these.

[0028] Salicylate Synthase Gene In the present invention, DNA containing the nucleotide sequence of SEQ ID NO: 2 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 2) can be used as the salicylate synthase gene. SEQ ID NO: 2 is the nucleotide sequence of the salicylate synthase gene of Raceella tenchongensis. 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 (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) and encoding a polypeptide having salicylate synthase activity can also be used.

[0029] Furthermore, in the present invention, a DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 3 (particularly a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3) can be used as the salicylic acid synthase gene. SEQ ID NO: 3 is the amino acid sequence of salicylic acid synthase from Raceella tenchongensis.

[0030] DNA encoding a polypeptide having salicylate synthase activity and comprising an amino acid sequence having at least 90%, preferably at least 95%, preferably at least 98%, and more preferably at least 99% identity to SEQ ID NO: 3 (particularly, consisting of an amino acid sequence having at least 90%, preferably at least 95%, preferably at least 98%, and more preferably at least 99% identity to SEQ ID NO: 3) can also be used. Furthermore, DNA encoding a polypeptide having salicylate synthase activity and comprising an amino acid sequence having 1 to 40, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 3 (particularly, consisting of an amino acid sequence having 1 to 40, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 3).

[0031] In the present invention, whether a test polypeptide has salicylate synthase activity is confirmed by reacting the test polypeptide with chorismate and detecting the produced salicylic acid by liquid chromatography or the like.

[0032] Isochorismate Synthase Gene In the present invention, DNA containing the nucleotide sequence of SEQ ID NO: 4 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 4) can be used as the isochorismate synthase gene. SEQ ID NO: 4 is the nucleotide sequence of the isochorismate synthase gene of Alisicolobacillus contamins. In the present invention, DNA containing a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 4 (particularly, consisting of a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 4) and encoding a polypeptide having isochorismate synthase activity can also be used.

[0033] Furthermore, in the present invention, a DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 5 (particularly, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5) can be used as the isochorismate synthase gene. SEQ ID NO: 5 is the amino acid sequence of isochorismate synthase from Alisicolobacillus contamins.

[0034] DNA encoding a polypeptide having isochorismate synthase activity and comprising an amino acid sequence having 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to SEQ ID NO: 5 (particularly, consisting of an amino acid sequence having 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to SEQ ID NO: 5). Furthermore, DNA encoding a polypeptide having isochorismate synthase activity and comprising an amino acid sequence in which 1 to 40, 1 to 20, 1 to 10, 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: 5 (particularly, consisting of an amino acid sequence in which 1 to 40, 1 to 20, 1 to 10, 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: 5).

[0035] In the present invention, whether a test polypeptide has isochorismate synthase activity is confirmed by reacting the test polypeptide with chorismate and detecting the produced isochorismate by liquid chromatography or the like.

[0036] Isochorismate-pyruvate lyase gene In the present invention, DNA containing the nucleotide sequence of SEQ ID NO: 6 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 6) can be used as the isochorismate-pyruvate lyase gene. SEQ ID NO: 6 is the nucleotide sequence of the isochorismate-pyruvate lyase gene of Pseudomonas aeruginosa. In the present invention, DNA containing a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 6 (particularly, consisting of a nucleotide sequence having 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identity to SEQ ID NO: 6) and encoding a polypeptide having isochorismate-pyruvate lyase activity can also be used.

[0037] Furthermore, in the present invention, a DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 7 (particularly, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7) can be used as the isochorismate-pyruvate lyase gene. SEQ ID NO: 7 is the amino acid sequence of isochorismate-pyruvate lyase from Pseudomonas aeruginosa.

[0038] DNA encoding a polypeptide having isochorismate-pyruvate lyase activity and comprising an amino acid sequence having 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to SEQ ID NO: 7 (particularly, consisting of an amino acid sequence having 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more identity to SEQ ID NO: 7). Furthermore, DNA encoding a polypeptide having isochorismate-pyruvate lyase activity and comprising an amino acid sequence having 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 7 (particularly, consisting of an amino acid sequence having 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 7).

[0039] In the present invention, whether a test polypeptide has isochorismate-pyruvate lyase activity is confirmed by reacting the test polypeptide with isochorismate and detecting the produced salicylic acid by liquid chromatography or the like.

[0040] In the present invention, the identities of base sequences and amino acid sequences are values ​​calculated using GENETYX ver. 17 (GENETYX).

[0041] 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.

[0042] 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.

[0043] The host Hydrogenophilus bacterium may be a bacterium isolated from nature, or a bacterium that has been genetically modified from a bacterium isolated from nature. Modifications can be performed for purposes such as enabling high expression of the introduced salicylate synthase gene, or the isochorismate synthase gene and isochorismate-pyruvate lyase gene. Such modifications can be achieved by removing (curing) endogenous plasmids in the Hydrogenophilus bacterium or by disrupting genes on the genome of the Hydrogenophilus bacterium. Methods for removing endogenous plasmids are well known, including methods that involve the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide; methods that involve destabilizing the plasmid by introducing a plasmid with the same replication origin as the endogenous plasmid; and methods that exploit plasmid incompatibility, such as destabilizing the plasmid by disrupting factors involved in the plasmid partition system.

[0044] Method for Preparing Transformants A method for obtaining transformants by introducing a salicylate synthase gene, or an isochorismate synthase gene and an isochorismate-pyruvate lyase gene into a bacterium of the genus Hydrogenophilus is described below. These genes can be introduced into a bacterium of the genus Hydrogenophilus using a general method for introducing foreign genes into bacteria. These genes may be introduced directly into a bacterium of the genus Hydrogenophilus, or a transformation vector (e.g., a plasmid vector, a viral vector, a cosmid, a fosmid, a BAC, etc.) incorporating these genes may be introduced into a bacterium of 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 is preferred. pCAMO-6 can be prepared from its DNA sequence (SEQ ID NO: 1) by those skilled in the art using gene synthesis services, etc. Examples of promoters contained in the vector include the tac promoter, lac promoter, trc promoter, and each of the OXB1, OXB11 to OXB20 promoters from Oxford Genetics. Examples of terminators contained in the vector include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the bacteriophage λt0 transcription terminator, and the T7 terminator.The salicylate synthase gene, or the isochorismate synthase gene and the isochorismate-pyruvate lyase gene can be introduced (transformed) into a Hydrogenophilus bacterium by a conventional method, such as the calcium chloride method, the rubidium chloride method, or the electric pulse method (electroporation).

[0045] (2) Method for Producing Salicylic Acid The present invention provides a method for producing salicylic acid 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 salicylic acid can be efficiently produced.

[0046] Hydrogenophilus bacteria can grow using hydrogen as an energy source and carbon dioxide as the sole carbon source, and therefore can efficiently fix carbon dioxide by producing salicylic acid using substantially only carbon dioxide as a carbon source (especially using only carbon dioxide). Therefore, in the method of the present invention, it is preferable to use an inorganic medium that does not contain carbon sources such as organic matter or carbonates, i.e., to culture using substantially only carbon dioxide as a carbon source (especially using carbon dioxide as the only carbon source). In the present invention, "using carbon dioxide as the sole carbon source" includes cases where unavoidable amounts of other carbon sources are mixed in.

[0047] The pH of the culture medium used for culture is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, bacterial growth and the solubility of the mixed gas in the culture medium are high, enabling highly efficient production of salicylic acid. 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 the mixed gas is introduced 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 bacteria is favorable and salicylic acid can be produced efficiently. The supply rate of the mixed gas or raw material gas is 10 to 60 L / hour, preferably 10 to 40 L / hour, and more preferably 10 to 20 L / hour per L of medium. Within this range, the growth of the transformant is favorable, salicylic acid 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 salicylic acid can be produced efficiently.

[0048] By culturing as described above, salicylic acid is produced in the culture medium. The reaction mixture can be recovered to recover salicylic acid, and the salicylic acid can be separated from the reaction mixture by known methods. Such known methods include purification methods using filtration, centrifugation, various types of chromatography, etc., which can be used alone or in combination.

[0049] The present invention will now be described with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0050] (1) 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.

[0051] (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.

[0052] (1-2) Preparation of Plasmid Vector pCAMO-6 for Seamless Cloning To perform seamless cloning, the plasmid vector pCAMO-6 was amplified by PCR. PCR was performed using the DNA consisting of the base sequence of SEQ ID NO: 1 as a template and the following primers. Primers used to amplify the plasmid vector pCAMO-6: (a-11) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 8) (b-11) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 9) Electrophoresis detected a DNA fragment of approximately 5.2 kbp corresponding to the vector gene, and the DNA fragment was recovered from the gel.

[0053] (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.

[0054] (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.

[0055] (2) Construction of Expression Plasmid for Salicylic Acid Synthase Gene DNA fragments of salicylic acid synthase genes from the following bacteria were amplified by PCR: Raceella tenchongensis (SEQ ID NO: 2) Klebsiella pneumoniae (SEQ ID NO: 10)

[0056] The following primers were used for PCR: Primers for amplifying the Raceella tenchongensis salicylic acid synthase gene (a-12) 5'-CTGGAGGAGAAACGCATATGAAGAAAACCCCAATGTATGAAG-3' (SEQ ID NO: 11) (b-12) 5'-GTCGACGGAGCTCGAATTCTCACCCATGTTTCAGCACCAACGTG-3' (SEQ ID NO: 12) Primers (a-12) and (b-12) contained sequences homologous to the vector pCAMO-6. Primers for amplifying the Klebsiella pneumoniae salicylate synthase gene: (a-13) 5'-GATCTGGAGGAGAAACGCATATGAAAATCAGTGAATTTTTACAC-3' (SEQ ID NO: 13) (b-13) 5'-CGACGGAGCTCGAATTCCTACACCATTAAATAGGGCGCAATGC-3' (SEQ ID NO: 14) Primers (a-13) and (b-13) contain sequences homologous to the vector pCAMO-6. Electrophoresis detected a DNA fragment of approximately 1.7 kbp for the salicylate synthase gene derived from each strain, and the DNA fragment was recovered from an agarose gel.

[0057] The DNA fragment of vector pCAMO-6 and each DNA fragment of the salicylate synthase gene were ligated to each other, and the Escherichia coli JM109 strain was transformed with the resulting DNA. The plasmid was then extracted and the sequence of the salicylate synthase gene was confirmed.

[0058] (3) Construction of an Expression Plasmid for the Isochorismate Synthase Gene The DNA fragment (SEQ ID NO: 4) of the isochorismate synthase gene derived from Alisicolobacillus contamins was amplified by PCR. The following primers were used for PCR: (a-14) 5'-GATCTGGAGGAGAAACGCATATGGCTGGACAGCAAGTGATG-3' (SEQ ID NO: 15) (b-14) 5'-GTCGACGGAGCTCGAATTCTCACGGGCTGTACCTCCCTTCGAGC-3' (SEQ ID NO: 16). Primers (a-14) and (b-14) contained sequences homologous to the vector pCAMO-6. Electrophoresis detected a DNA fragment of approximately 1.5 kbp for the isochorismate synthase gene, which was then recovered from an agarose gel.

[0059] The DNA fragment of the vector pCAMO-6 and the DNA fragment of the isochorismate synthase gene were ligated to each other and used to transform Escherichia coli JM109 strain, after which the plasmid was extracted and the sequence of the isochorismate synthase gene was confirmed.

[0060] (4) Construction of an Expression Plasmid for the Isochorismate-Pyruvate Lyase Gene The DNA fragment (SEQ ID NO: 6) of the isochorismate-pyruvate lyase gene from Pseudomonas aeruginosa was amplified by PCR. The following primers were used for PCR: (a-15) 5'-GATCTGGAGGAGAAACGCATATGAAAACTCCCGAAGACTGCAC-3' (SEQ ID NO: 17) (b-15) 5'-GTCGACGGAGCTCGAATTCTCATGCGGCACCCCGTGTCTGGCG-3' (SEQ ID NO: 18). Primers (a-15) and (b-15) contained sequences homologous to the pCAMO-6 vector. Electrophoresis detected a DNA fragment of approximately 0.3 kbp for the isochorismate-pyruvate lyase gene, which was then recovered from an agarose gel.

[0061] The DNA fragment of the vector pCAMO-6 and the DNA fragment of the isochorismate-pyruvate lyase gene were ligated together and used to transform Escherichia coli JM109. The plasmid was then extracted and the sequence of the isochorismate-pyruvate lyase gene was confirmed.

[0062] (5) Construction of Expression Plasmids for the Isochorismate Synthase Gene and the Isochorismate-Pyruvate Lyase Gene The isochorismate synthase gene of Alisicolobacillus contamins was amplified using the expression vector obtained in "(4) Construction of Expression Plasmid for the Isochorismate Synthase Gene" as a template with the following primers: (a-16) 5'-GGCTCGTATAATGTGTGGAATTGTGAGCGGATAAC-3' (SEQ ID NO: 19) (b-16) 5'-ATGCGATACTCCTCCTCACGGGCTGTACCTCCCTTCGAGCGGC-3' (SEQ ID NO: 20). Primer (b-16) contains a sequence homologous to the ribosome binding sequence and a sequence homologous to the isochorismate synthase gene. Electrophoresis detected a DNA fragment of approximately 1.5 kbp for the isochorismate synthase gene, and the DNA was recovered from an agarose gel.

[0063] The Pseudomonas aeruginosa isochorismate-pyruvate lyase gene was amplified using the expression vector obtained in "(5) Construction of an Expression Plasmid for the Isochorismate-Pyruvate Lyase Gene" as a template with the following primers: (a-17) 5'-CCGTGAGGAGGAGTATCGCATATGAAAACTCCCGAAGACTGCAC-3' (SEQ ID NO: 21) (b-17) 5'-CCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACC-3' (SEQ ID NO: 22). Primer (a-17) contains sequences homologous to the ribosome binding sequence and the isochorismate-pyruvate lyase gene. Electrophoresis detected a DNA fragment of approximately 0.3 kbp for the isochorismate-pyruvate lyase gene, and the DNA was recovered from an agarose gel.

[0064] The DNA fragments of the isochorismate synthase gene and the isochorismate-pyruvate lyase gene thus generated were mixed, ligated with the following primers, and amplified: (a-16) 5'-GGCTCGTATAATGTGTGGAATTGTGAGCGGATAAC -3' (SEQ ID NO: 19) (b-17) 5'-CCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACC-3' (SEQ ID NO: 22) Electrophoresis revealed a DNA fragment of approximately 1.8 kbp for the gene in which the isochorismate synthase gene and the isochorismate-pyruvate lyase gene were ligated, and the DNA was recovered from an agarose gel.

[0065] The DNA fragment of the vector pCAMO-6 and the DNA fragment of the ligated gene of the isochorismate synthase gene and the isochorismate-pyruvate lyase gene were ligated together, and the Escherichia coli JM109 strain was transformed with the ligated DNA fragment. The plasmid was then extracted, and the sequences of the isochorismate synthase gene and the isochorismate-pyruvate lyase gene were confirmed.

[0066] (6) Transformants of Hydrogenophilus thermorteolus (6-1) Gene Introduction. The Hydrogenophilus thermorteolus TH-1 strain was transformed by electroporation with the plasmids obtained in "(2) Construction of Expression Plasmids for the Salicylate Synthase Gene" and "(5) Construction of Expression Plasmids for the Isochorismate Synthase Gene and Isochorismate-Pyruvate Lyase Gene," respectively. 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 into LB solid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 52°C for 24 hours. Amplification of the insert fragment of each plasmid was confirmed for each strain grown on LB solid medium by PCR. PCR was performed by standard methods using a Life Technologies DNA Thermal Cycler and KOD One PCR Master Mix (Toyobo Co., Ltd.) as the reaction reagent. As a result, amplification of a DNA fragment of approximately 1.7 kbp in length corresponding to the salicylate synthase gene and a DNA fragment of approximately 1.8 kbp corresponding to the combined length of the isochorismate synthase gene and isochorismate-pyruvate lyase gene was confirmed for each plasmid. The resulting transformants were named as shown in Table 1.

[0067] (6-2) Salicylic Acid Production. Each Hydrogenophilus thermorteolus transformant prepared as described above was inoculated into liquid medium A containing 50 μg / mL kanamycin using a platinum loop. A gas mixture of H2:O2:CO2 (7.5:1:1.5) was supplied during the culture, and the transformants were cultured at 52°C for 72 hours with shaking. A strain in which the empty vector pCAMO-6 had been introduced into the Hydrogenophilus thermorteolus TH-1 strain was also cultured in the same manner. After the culture, the culture supernatant was obtained by centrifugation (4°C, 5,000 g, 10 minutes). The salicylic acid 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: 200-300 nm. Reference wavelength: 210 nm. As shown in Table 1, the SAS101 strain, which contained the Raceella tenchongensis salicylate synthase gene, detected 1.1 mM salicylic acid in the culture supernatant. The SAS103 strain, which contained the Alisicolobacillus contamins isochorismate synthase gene and the Pseudomonas aeruginosa isochorismate-pyruvate lyase gene, detected 0.9 mM salicylic acid in the culture supernatant. On the other hand, salicylic acid was not detected in the SAS102 strain into which the salicylate synthase gene of Klebsiella pneumoniae had been introduced, and in the strain into which the salicylate synthase gene had not been introduced.

[0068]

[0069] 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.

[0070] SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 10 are shown below.

[0071]

[0072] The transformant of the present invention can produce salicylic acid with high efficiency using carbon dioxide as the sole carbon source, and therefore contributes to the industrial production of chemical products with high efficiency while resolving global warming caused by increased carbon dioxide.

Claims

1. A transformant obtained by introducing into a Hydrogenophilus bacterium a salicylate synthase gene as shown in (a), (b), (c), (d), or (e) below, or by introducing into the bacterium a isochorismate synthase gene as shown in (f), (g), (h), (i), or (j) below and an isochorismate-pyruvate lyase gene as shown in (k), (l), (m), (n), or (o) below.(a) DNA comprising the nucleotide sequence of SEQ ID NO:2; (b) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO:2, and encoding a polypeptide having salicylic acid synthase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:3; (d) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO:3, and having salicylic acid synthase activity; (e) DNA encoding a polypeptide having an amino acid sequence in which 1 to 40 amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO:3, and having salicylic acid synthase activity; (f) DNA comprising the nucleotide sequence of SEQ ID NO:4; (g) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO:4, and encoding a polypeptide having isochorismate synthase activity; (h) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:5; (i) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO:5, and having isochorismate synthase activity; (j) (k) 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:5, and having isochorismate synthase activity. (l) DNA encoding a polypeptide having a nucleotide sequence having 90% or more identity with SEQ ID NO:6, and having isochorismate-pyruvate lyase activity. (m) DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO:

7. (n) DNA encoding a polypeptide having an amino acid sequence having 90% or more identity with SEQ ID NO:7, and having isochorismate-pyruvate lyase activity. (o) DNA encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO:7, and having isochorismate-pyruvate lyase activity.

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

3. A method for producing salicylic acid, comprising the step of culturing the transformant described in claim 1 or 2.

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