Hydrogenophilus bacterium transformant capable of producing protocatechuic acid

Abstract

A transformant produced by introducing a chorismate pyruvate-lyase gene comprising DNA (a), (b), (c), (d) or (e) and a 4-hydroxybenzoate hydroxylase gene comprising DNA (f), (g), (h), (i) or (j) into a Hydrogenophilus bacterium can utilize carbon dioxide as a sole carbon source to produce protocatechuic acid with high efficiency. (a) DNA that comprises the nucleotide sequence represented by SEQ ID NO:1; (b) DNA that comprises a nucleotide sequence having 90% or higher identity with SEQ ID NO:1 and encodes a polypeptide having a chorismate pyruvate-lyase activity; (c) DNA that encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2; (d) DNA that encodes a polypeptide comprising an amino acid sequence having 90% or higher identity with SEQ ID NO:2 and having a chorismate pyruvate-lyase activity; (e) DNA that encodes a polypeptide comprising an amino acid sequence having the deletion, substitution, insertion or addition of 1 to 10 amino acid residues in the amino acid sequence represented by SEQ ID NO:2 and having a chorismate pyruvate-lyase activity; (f) DNA that comprises the nucleotide sequence represented by SEQ ID NO:3; (g) DNA that comprises a nucleotide sequence having 90% or higher identity with SEQ ID NO:3 and encodes a polypeptide having a 4-hydroxybenzoate hydroxylase activity; (h) DNA that encodes a polypeptide comprising the amino acid sequence represented by SEQ ID NO:4; (i) DNA that encodes a polypeptide comprising an amino acid sequence having 90% or higher identity with SEQ ID NO:4 and having a chorismate pyruvate-lyase activity; and (j) DNA that encodes a polypeptide comprising an amino acid sequence having the deletion, substitution, insertion or addition of 1 to 30 amino acid residues in the amino acid sequence represented by SEQ ID NO:4 and having a 4-hydroxybenzoate hydroxylase activity.

Description

Hydrogenophilus transformants producing protocatechuic acid

[0001] The present invention relates to a transformant of a bacterium belonging to the genus Hydrogenophilus having the ability to produce protocatechuic acid, and a method for producing protocatechuic acid 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 a reduction of global anthropogenic CO2 emissions by approximately 45% compared to 2010 levels by 2030, reaching zero by around 2050, achieving carbon neutrality. In accordance with this report, drastically reducing greenhouse gas emissions, such as carbon dioxide and methane, has become a global challenge, and there is an increasing need for technological development 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 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 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 atmospheric gases 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 is great expectation for fixing and effectively utilizing carbon dioxide, which contributes greatly to global warming.

[0005] Protocatechuic acid is a useful compound that can be used as an ingredient or raw material for pharmaceuticals, cosmetics, pesticides, feed, fragrances, etc., and, because it is a polyphenol with antioxidant properties, can also be used as an ingredient in functional foods. It is also useful as a precursor for producing useful chemicals such as catechol and cis-cis-muconic acid through bio- or chemical conversion, and as a raw material for high-performance polymers. Traditionally, protocatechuic acid has been produced by extraction from natural products such as medicinal plants, as well as by chemical synthesis from petroleum-derived raw materials. However, extraction from natural products is costly due to low yields, and chemical synthesis from petroleum-derived raw materials places a heavy burden on the environment.

[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 protocatechuic acid 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 need for complex processes, the disruption of stable food and feed supplies, and the large environmental impact. Therefore, a practical method for producing protocatechuic acid using microorganisms in 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 protocatechuic acid by fixing carbon dioxide is needed.

[0007] In many microorganisms, protocatechuate is produced from 4-hydroxybenzoate (4-HBA) by the catalytic action of 4-hydroxybenzoate hydroxylase, and also from 3-dehydroshikimate by the catalytic action of 3-dehydroshikimate dehydratase in the shikimate pathway involved in the synthesis of aromatic amino acids.

[0008] As techniques for producing protocatechuic acid by fermentation using genetically modified microorganisms, Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2 disclose a method for producing protocatechuic acid using glucose as a carbon source by introducing into Corynebacterium bacteria the chorismate-pyruvate lyase (ubiC) gene, which produces 4-hydroxybenzoic acid from chorismate, the 4-hydroxybenzoate hydroxylase (pobA) gene, which produces protocatechuic acid from 4-hydroxybenzoic acid, and the 3-dehydroshikimate dehydratase (qsuB) gene, which produces protocatechuic acid from 3-dehydroshikimate. In the methods of these documents, a gene derived from Providencia rustigianii is used as the chorismate-pyruvate lyase gene, a gene derived from Corynebacterium glutamicum is used as the 4-hydroxybenzoate hydroxylase gene, and numerous genes derived from bacteria such as Corynebacterium glutamicum are used as the 3-dehydroshikimate dehydratase gene. Non-Patent Document 2 teaches a method of producing protocatechuic acid using glucose and D-xylose as carbon sources by introducing the qsuB gene derived from Corynebacterium glutamicum into Corynebacterium glutamicum.

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

[0010] Re-table 2017 / 169399

[0011] Metab Eng., 65:232-242(2021)Biotechnol Bioeng., 118(11):4414-4427(2021)

[0012] An objective of the present invention is to provide a Hydrogenophilus bacterium transformant (in other words, a transformed Hydrogenophilus bacterium) that can efficiently produce protocatechuic acid using carbon dioxide as a sole carbon source, and a method for efficiently producing protocatechuic acid using the transformant.

[0013] 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 the enzyme required to produce protocatechuic acid as a metabolic intermediate. Therefore, in order to impart the ability to produce protocatechuic acid on an industrial scale to Hydrogenophilus bacteria, it is necessary to introduce a gene encoding an enzyme that catalyzes the reaction that produces protocatechuic acid.

[0014] 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.

[0015] Under these circumstances, in order to obtain a chorismate-pyruvate lyase that can be expressed in bacteria of the genus Hydrogenophilus, the present inventors examined genes predicted to be chorismate-pyruvate lyase genes present in the genomes of other microorganisms and found that the chorismate-pyruvate lyase gene from Shewanella algae can express a chorismate-pyruvate lyase that functions in bacteria of the genus Hydrogenophilus. Furthermore, in order to obtain a 4-hydroxybenzoate hydroxylase gene that can be expressed in bacteria of the genus Hydrogenophilus, the present inventors examined genes predicted to be 4-hydroxybenzoate hydroxylase genes present in the genomes of other microorganisms and found that the 4-hydroxybenzoate hydroxylase gene from Corynebacterium glutamicum can express a 4-hydroxybenzoate hydroxylase that functions in bacteria of the genus Hydrogenophilus. They also found that transformants obtained by introducing these genes into Hydrogenophilus bacteria were able to efficiently produce protocatechuic acid using carbon dioxide as the sole carbon source.

[0016] The present invention was completed based on the above findings and provides the following transformant and method for producing protocatechuic acid: [1] A transformant (transformed bacterium) obtainable by introducing into a bacterium belonging to the genus Hydrogenophilus the following chorismate-pyruvate lyase gene (a), (b), (c), (d), or (e) and the following 4-hydroxybenzoate hydroxylase gene (f), (g), (h), (i), or (j): (a) DNA comprising the nucleotide sequence of SEQ ID NO: 1; (b) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 1 and encoding a polypeptide having chorismate-pyruvate lyase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (d) DNA comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 2 and encoding a polypeptide having chorismate-pyruvate lyase activity; (e) DNA consisting of 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: 2 and encoding a polypeptide having chorismate-pyruvate lyase activity; (f) DNA comprising the nucleotide sequence of SEQ ID NO: 3; (g) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 3 and encoding a polypeptide having 4-hydroxybenzoate hydroxylase activity; (h) DNA encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4; (i) DNA comprising an amino acid sequence having 90% or more identity with SEQ ID NO: 4 and encoding a polypeptide having 4-hydroxybenzoate hydroxylase activity; (j) [1] A DNA encoding a polypeptide having 4-hydroxybenzoate hydroxylase activity, the polypeptide comprising an amino acid sequence of SEQ ID NO: 4 in which 1 to 30 amino acids have been deleted, substituted, inserted, or added. [2] The transformant according to [1], wherein the bacterium of the genus Hydrogenophilus is Hydrogenophilus thermorteolus. [3] A method for producing protocatechuic acid, the method comprising culturing the transformant according to [1] or [2] using carbon dioxide as substantially the only carbon source.

[0017] 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.

[0018] Protocatechuic acid is an aromatic compound of industrial importance. However, protocatechuic acid extracted from natural sources is expensive due to its low yield. Furthermore, chemical synthesis from petroleum feedstocks is undesirable from an environmental perspective. In contrast, the production of protocatechuic acid using carbon dioxide-fixing microorganisms can simultaneously address global warming caused by increased carbon dioxide emissions and ensure the industrial supply of protocatechuic acid.

[0019] 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."

[0020] According to the present invention, by introducing specific chorismate-pyruvate lyase genes and 4-hydroxybenzoate hydroxylase genes into Hydrogenophilus bacteria, chorismate-pyruvate lyase and 4-hydroxybenzoate hydroxylase that function in the Hydrogenophilus bacteria are expressed, enabling industrial-scale production of protocatechuic 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 protocatechuic acid using carbon dioxide.

[0021] The present invention is described in detail below. (1) Transformant Ability to Produce Protocatechuic Acid The transformant of the present invention is a transformant obtainable by introducing a Shewanella algae chorismate-pyruvate lyase gene (hereinafter sometimes abbreviated as the "ubiC gene") or a homolog thereof, and a Corynebacterium glutamicum 4-hydroxybenzoate hydroxylase gene (hereinafter sometimes abbreviated as the "pobA 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 ubiC gene and an exogenous pobA gene, such as the Shewanella algae ubiC gene or a homolog thereof, and the Corynebacterium glutamicum pobA gene or a homolog thereof. The ubiC gene and pobA gene used in the present invention do not necessarily have to be known to have been identified as the ubiC gene and the pobA gene, respectively. They may be DNAs encoding a polypeptide having chorismate-pyruvate lyase activity and DNAs encoding a polypeptide having 4-hydroxybenzoate hydroxylase activity. Chorismate-pyruvate lyase activity refers to the enzyme activity that produces 4-hydroxybenzoate from chorismate, and 4-hydroxybenzoate hydroxylase activity refers to the enzyme activity that produces protocatechuate from 4-hydroxybenzoate. Furthermore, in the present invention, the ubiC gene and pobA gene may be DNAs of the ubiC gene and pobA gene isolated from naturally occurring bacteria, or may be DNAs artificially synthesized using techniques known to those skilled in the art.

[0022] The transformant of the present invention may be a Hydrogenophilus bacterium into which the Shewanella algae ubiC gene or a homolog thereof and the Corynebacterium glutamicum pobA gene or a homolog thereof have been introduced, and two or more Shewanella algae ubiC genes or homologs thereof may be introduced, or two or more Corynebacterium glutamicum pobA genes or homologs thereof may be introduced. Furthermore, transformants of the present invention also include Hydrogenophilus bacterium into which other genes have been introduced in addition to these genes.

[0023] Chorismate-pyruvate lyase gene (ubiC gene) In the present invention, DNA containing the nucleotide sequence of SEQ ID NO: 1 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 1) can be used as the ubiC gene. SEQ ID NO: 1 is the nucleotide sequence of the ubiC gene of Shewanella algae. Also usable is DNA containing a nucleotide sequence that is 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identical to SEQ ID NO: 1 (particularly, consisting of a nucleotide sequence that is 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more identical to SEQ ID NO: 1) and that encodes a polypeptide having chorismate-pyruvate lyase activity.

[0024] Furthermore, in the present invention, a DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2 (particularly, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2) can be used as the ubiC gene. SEQ ID NO: 2 is the amino acid sequence of chorismate-pyruvate lyase from Shewanella algae.

[0025] DNA can also be used that contains 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: 2 (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 SEQ ID NO: 2) and encodes a polypeptide having chorismate-pyruvate lyase activity. Furthermore, DNA can also be used that contains an amino acid sequence in which 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: 2 (particularly, consisting of an amino acid sequence in which 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: 2) and encodes a polypeptide having chorismate-pyruvate lyase activity.

[0026] In the present invention, whether a test polypeptide has chorismate-pyruvate lyase activity is confirmed by reacting the test polypeptide with barium chorismate and detecting an increase in absorbance at 246 nm resulting from the product, 4-hydroxybenzoic acid.

[0027] 4-Hydroxybenzoate Hydroxylase Gene (pobA Gene) In the present invention, DNA containing the nucleotide sequence of SEQ ID NO: 3 (particularly, DNA consisting of the nucleotide sequence of SEQ ID NO: 3) can be used as the pobA gene. SEQ ID NO: 3 is the nucleotide sequence of the pobA gene of Corynebacterium glutamicum. In addition, 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: 3 (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: 3) and encoding a polypeptide having 4-hydroxybenzoate hydroxylase activity can also be used.

[0028] Furthermore, in the present invention, a DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 4 (particularly, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4) can be used as the pobA gene. SEQ ID NO: 4 is the amino acid sequence of 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum.

[0029] 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 SEQ ID NO: 4 (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 SEQ ID NO: 4) and that encode a polypeptide having 4-hydroxybenzoate hydroxylase activity can also be used. Furthermore, DNAs that contain an amino acid sequence in which 1 to 30, 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: 4 (particularly, consisting of an amino acid sequence in which 1 to 30, 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: 4) and that encode a polypeptide having 4-hydroxybenzoate hydroxylase activity can also be used.

[0030] In the present invention, whether a test polypeptide has 4-hydroxybenzoate hydroxylase activity is confirmed by reacting the test polypeptide with 4-hydroxybenzoic acid in the presence of NADPH and detecting a decrease in absorbance at 340 nm. Because 4-hydroxybenzoate hydroxylase consumes NADPH when producing protocatechuic acid from 4-hydroxybenzoic acid, a decrease in the amount of NADPH is detected using a decrease in absorbance at 340 nm as an indicator.

[0031] A DNA sequence encoding a protein containing the amino acid sequence of SEQ ID NO: 2 or 4 may have various base substitutions in the coding region, taking into account codon degeneracy or preferred codons in Hydrogenophilus bacteria, as long as the amino acid sequence of the protein expressed from the coding region is not changed.

[0032] In the present invention, the identities of base sequences and amino acid sequences are values ​​calculated using GENETYX ver. 17 (GENETYX).

[0033] 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 that encodes a polypeptide with the same type of activity is referred to as a "homolog" of that DNA. A polypeptide that contains an amino acid sequence that is 90% to 100% identical to the amino acid sequence of a certain polypeptide and has the same type of activity is referred to as a homolog of that polypeptide. A DNA that contains an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added to the amino acid sequence of a certain polypeptide and encodes a polypeptide with the same type of activity is referred to as a homolog of that DNA.

[0034] 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.

[0035] 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 chorismate-pyruvate lyase gene and 4-hydroxybenzoate hydroxylase gene. Such modifications can be achieved by, for example, removing (curing) the endogenous plasmid in the Hydrogenophilus bacterium. Well-known methods for removing endogenous plasmids include the use of chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide; destabilizing the plasmid by introducing a plasmid with the same replication origin as the endogenous plasmid; and utilizing plasmid incompatibility, such as destabilizing the plasmid by disrupting factors involved in the plasmid partition system.

[0036] Method for Preparing Transformants A method for obtaining transformants by introducing the ubiC gene and the pobA gene into a bacterium belonging to the genus Hydrogenophilus is described below. The ubiC gene and the pobA gene can be introduced into a bacterium belonging to the genus Hydrogenophilus using a general method for introducing foreign genes into bacteria. The ubiC gene and the pobA gene may be introduced directly into the bacterium belonging to the genus Hydrogenophilus, or a vector for transformation (e.g., a plasmid vector, a viral vector, a cosmid, a fosmid, a BAC, a YAC, etc.) may be used. When a vector is used, a vector incorporating the ubiC gene and the pobA gene may be introduced into the bacterium belonging to the genus Hydrogenophilus, or a vector incorporating the ubiC gene and a vector incorporating the pobA gene may be introduced into the 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 is preferred. pCAMO-6 can be prepared by those skilled in the art according to the Examples. Examples of promoters contained in the vector include the tac promoter, lac promoter, trc promoter, and each of the Oxford Genetics OXB1, OXB11 to OXB20 promoters. 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 ubiC and pobA genes can be introduced (transformed) into Hydrogenophilus bacteria using common techniques, such as the calcium chloride method, calcium phosphate method, rubidium chloride method, and electric pulse method (electroporation).

[0037] (2) Method for Protocatechuic Acid The present invention provides a method for producing protocatechuic acid using the transformant of the present invention described above. This method includes culturing the transformant of the present invention in an inorganic or organic medium while supplying a carbon dioxide-containing gas, preferably a mixed gas containing hydrogen, oxygen, and carbon dioxide. The gas supplied is preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide, but other gases may be mixed in as long as protocatechuic acid can be efficiently produced.

[0038] Hydrogenophilus bacteria can grow using hydrogen as an energy source and carbon dioxide as the sole carbon source, and therefore, by culturing the transformant of the present invention using substantially only carbon dioxide as a carbon source (particularly, using only carbon dioxide), carbon dioxide can be efficiently fixed. 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 (particularly, 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.

[0039] 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 protocatechuic acid. When performing batch culture, the mixed gas can be sealed in a sealed culture vessel and cultured statically or with shaking. When performing continuous culture, the mixed gas can be continuously supplied to a sealed culture vessel while being cultured with shaking, or the transformant can be cultured in a sealed culture vessel while introducing the mixed gas into the culture medium by bubbling. Shaking culture is preferred because it improves the dissolution of the mixed gas into the culture medium. The volume ratio of hydrogen, oxygen, and carbon dioxide in the supply gas (hydrogen:oxygen:carbon dioxide) 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 protocatechuic acid 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, protocatechuic acid can be efficiently produced, and waste of the mixed gas is reduced. The culture temperature is preferably 35 to 55°C, more preferably 37 to 52°C, and even more preferably 50 to 52°C. Within this range, the growth of the transformant is favorable and protocatechuic acid can be efficiently produced.

[0040] By culturing as described above, protocatechuic acid or a salt thereof is produced in the culture medium.

[0041] 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.

[0042] (1) Construction of Plasmid Vector (pCAMO-6) The construction method of the plasmid vector pCAMO-6 used to introduce the ubiC and qsuB genes is described below. (1-1) Preparation of the tac Promoter DNA Fragment To amplify the tac promoter DNA fragment, PCR was performed using the following pair of primers, using the New England BioLabs plasmid pMAL-c5X as a template. PCR was performed using a Thermo Fisher Scientific 2720 Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as the reaction reagent, according to standard procedures. Primers for tac promoter amplification: (a-1) 5'-TTTTATAACCCGGGCCATCGACTGCACGGTGCACC-3' (SEQ ID NO: 5) (b-1) 5'-TGCTAGCACTGTTTCCTGTGTGAAATTGTTATCCG-3' (SEQ ID NO: 6) Primer (a-1) contained an SmaI restriction enzyme site, as underlined. The reaction mixture prepared above was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of approximately 0.3 kbp corresponding to the tac promoter was detected.

[0043] (1-2) Preparation of DNA Fragments of the Multicloning Region To prepare the DNA fragments of the multicloning region, PCR was performed using the following pairs of primers to generate the first and second halves of the fragments. PCR was performed using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as reaction reagents, according to standard methods. This method involves annealing and elongating the 3' ends of each pair of primers to form double-stranded DNA without using template DNA. Primers for preparing the first half of the multicloning region: (a-2) 5'-ggaaacagtgctagcagatctggaggagaaaggcatat-3' (SEQ ID NO: 7) (b-2) 5'-CAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGATATCAGCATATGCGTTTCTCCTCCAGA-3' (SEQ ID NO: 8) The 3' base sequences of primers (a-2) and (b-2) are complementary to each other. Primers for preparing the second half of the multicloning region: (a-3) 5'-ACAAGCTTGCGGCCGCACTGCAGCACCATCACCACCATCATTGATAAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCT-3' (SEQ ID NO: 9) (b-3) 5'-TATTTGAATCGAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGT-3' (SEQ ID NO: 10). The 3'-terminal nucleotide sequences of primers (a-3) and (b-3) are complementary to each other. The reaction mixture prepared above was subjected to electrophoresis on a 1% agarose gel, and approximately 0.1 kbp DNA fragments corresponding to the first and second halves of the multicloning region were detected. Each DNA fragment was excised from the agarose gel, and the DNA was recovered from the gel by freezing and thawing.

[0044] Overlap extension PCR was performed using the recovered DNA fragments corresponding to the first and second halves of the multicloning region as templates. The 5'-terminal base sequences of primers (b-2) and (a-3) used to amplify the template DNA fragments were complementary to each other. For overlap extension PCR, a combination of primers (a-2) and (b-3) was used to generate DNA for the multicloning region. PCR was performed using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as reaction reagents, according to standard procedures. The resulting reaction mixture was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 0.2 kbp corresponding to the multicloning region was detected.

[0045] (1-3) Preparation of a DNA fragment containing the rrnB terminator and the replication origin of pUC19 Using the plasmid pMAL-c5X (New England BioLabs) as a template, PCR was performed with the following pair of primers to amplify a DNA fragment of the rrnB terminator. Primers for rrnB terminator amplification: (a-4) 5'-TAACTCGATTCAAATAAAACGAAAGGCTCAGTCGA-3' (SEQ ID NO: 11) (b-4) 5'-CCTAGATCCGCGGAGTTTGTAGAAACGCAAAAAGG-3' (SEQ ID NO: 12) Furthermore, PCR was performed with the following pair of primers to amplify a DNA fragment of the DNA replication origin using the plasmid pUC19 as a template. Primers for amplifying the DNA replication origin of pUC19: (a-5) 5'-ACAAACTCCGCGGATCTAGGTGAAGATCCTTTTTG-3' (SEQ ID NO: 13); (b-5) 5'-CGTCCGCGGCCAGCAAAAGGCCAGGAACCGTAAAA-3' (SEQ ID NO: 14). PCR was performed using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as reaction reagents according to standard procedures. The resulting reaction mixture was subjected to electrophoresis on a 1% agarose gel, detecting a DNA fragment of approximately 0.3 kbp for the rrnB terminator and approximately 0.8 kbp for the DNA replication origin of pUC19. Each DNA fragment was excised from the agarose gel, and the DNA was recovered from the gel by freezing and thawing.

[0046] Overlap extension PCR was performed using the recovered rrnB terminator and a DNA fragment corresponding to the pUC19 DNA replication origin as templates. The 5'-terminal sequences of primers (b-4) and (a-5) used to amplify these template DNA fragments were complementary to each other. For overlap extension PCR, a combination of primers (a-4) and (b-5) was used to generate a DNA fragment linking the rrnB terminator and the pUC19 replication origin. PCR was performed according to standard procedures using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as reaction reagents. The resulting reaction solution was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 1.0 kbp corresponding to the DNA linking the rrnB terminator and the pUC19 replication origin was detected.

[0047] (1-4) Preparation of DNA fragment of neomycin / kanamycin resistance gene PCR was performed by standard methods using the plasmid pK18mobsacB (GenBank: FJ437239.1) [Gene, 145, 69-73 (1994)] containing the neomycin / kanamycin resistance gene (hereinafter sometimes referred to as "nptII") sequence as a template. PCR was performed using the following pair of primers to amplify a DNA fragment containing the nptII gene sequence. PCR was performed by standard methods using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as a reaction reagent. Primers for amplifying the nptII gene: (a-6) 5'-TTGCTGGCCGCGGACGTAGAAAGCCTGTCCGCAGA-3' (SEQ ID NO: 15) (b-6) 5'-GGCCCGGGTTATAAAAGCCAGTCATTAGGCCTATC-3' (SEQ ID NO: 16) Primer (b-6) contains an SmaI restriction enzyme site, as underlined. The resulting reaction solution was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 1.0 kbp corresponding to the nptII gene was detected.

[0048] (1-5) Construction of Circular Plasmids: The DNA fragments prepared in steps (1-1) to (1-4) above share 16 bp of homologous sequence with each other: the end of DNA fragment (1-1) shares 16 bp with the ends of DNA fragments (1-4) and (1-2); the end of DNA fragment (1-2) shares 16 bp with the ends of DNA fragments (1-1) and (1-3); the end of DNA fragment (1-3) shares 16 bp with the ends of DNA fragments (1-2) and (1-4); and the end of DNA fragment (1-4) shares 16 bp with the ends of DNA fragments (1-3) and (1-1). Therefore, the DNA fragments (1-1), (1-2), (1-3), and (1-4) can be ligated into a circular DNA by Gibson assembly. To ligate the DNA fragments prepared in steps (1-1) to (1-4) into a circular DNA, Gibson assembly was performed using Gibson Assembly Master Mix (New England BioLabs). The resulting reaction mixture was used to transform Escherichia coli JM109 using the calcium chloride method and then spread onto LB solid medium containing 50 μg / mL kanamycin. The strain growing on the medium was cultured in liquid culture using standard methods, and plasmid DNA was extracted from the culture medium and reacted with the restriction enzyme SmaI. Electrophoresis of this reaction mixture using a 1% agarose gel revealed a DNA fragment of approximately 2.3 kbp, corresponding to the size of the single DNA fragment prepared in (1-1) to (1-4), confirming that they had been ligated. This circular plasmid DNA, which replicates in E. coli, was designated pCAMO-1.

[0049] (1-6) Construction of Plasmid Vector (pCAMO-4) The Hydrogenophilus thermorteolus TH-1 strain was cultured in liquid medium A [(NH4)2SO4 3.0 g, KH2PO4 1.0 g, K2HPO4 2.0 g, NaCl 0.25 g, FeSO4 7H2O 0.014 g, MgSO4 7H2O 0.5 g, CaCl2 0.03 g, MoO3 4.0 mg, ZnSO4 7H2O 28 mg, CuSO4 5H2O 2.0 mg, H3BO3 4.0 mg, MnSO4 5H2O 4.0 mg, and CoCl2 6H2O 4.0 mg dissolved in 1 L of distilled water (pH 7.0). The bacteria were inoculated into a test tube containing 5 mL of ethanol using a platinum loop. The tube was then filled with a 7.5:1:1.5 H2:O2:CO2 gas mixture and incubated at 50°C. The endogenous plasmid pTH1 [Microbiol Resour Announcement. 2018 Aug 16;7(6)] was extracted from the culture medium using the standard alkaline SDS method. The approximately 66 kbp pTH-1 plasmid was digested with the restriction enzymes ScaI and PvuII, and the pCAMO-1 plasmid was digested with the restriction enzyme SmaI. The two plasmids were then ligated together using T4 DNA (Takara Bio Inc.).

[0050] The resulting ligation solution was used to transform the Hydrogenophilus thermorteolus TH-1 strain (NBRC 14978) by electroporation. The transformants were grown on solid medium A containing 50 μg / mL 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, CoCl2 6H2O 4.0 mg, and 15 g of agar dissolved in 1 L of distilled water (pH 7.0). 7.0) and cultured for 60 hours at 50°C in a chamber filled with a gas mixture of H2:O2:CO2 = 7.5:1:1.5. Seven strains grown on solid medium A were inoculated using a platinum loop into test tubes containing 5 mL of liquid medium A containing 50 μg / mL kanamycin. The test tubes were filled with a gas mixture of H2:O2:CO2 = 7.5:1:1.5 and cultured with shaking at 50°C. Plasmid DNA was extracted from the culture medium and electrophoresed on a 1% agarose gel. This confirmed that all seven strains contained the same plasmid DNA of approximately 5.5 kbp.

[0051] Using the extracted plasmid as a template, PCR was performed using the following pair of primers, which correspond to both sides of the SmaI restriction enzyme site of pCAMO-1, to amplify the DNA fragment of the endogenous plasmid pTH1 inserted into the SmaI restriction enzyme site of pCAMO-1. PCR was performed using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as reaction reagents, according to standard procedures. Primers for amplifying the inserted pTH1 DNA fragment: (a-7) 5'-AATTGTCAGATAGGCCTAATGACTGGCTTTTATAA-3' (SEQ ID NO: 17) (b-7) 5'-TGACGCCAGAAGCATTGGTGCACCGTGCAGTCGAT-3' (SEQ ID NO: 18). The resulting reaction solution was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 3.2 kbp was detected. In other words, the resulting plasmid has a 3.2 kbp DNA fragment, a part of pTH1, inserted into the SmaI restriction enzyme site of the pCAMO-1 plasmid. Because this 3.2 kbp DNA fragment contains a replication origin that functions in Hydrogenophilus thermorteolus cells, the resulting plasmid replicates in Hydrogenophilus thermorteolus cells. The constructed plasmid was named pCAMO-4.

[0052] (1-7) Construction of Plasmid Vector (pCAMO-5) The following pair of oligonucleotides were mixed in equimolar amounts and slowly cooled from 98°C to 20°C to prepare a double-stranded DNA fragment to which the oligonucleotides were annealed. The ends of this DNA fragment were equivalent to the protruding ends obtained by cleavage with the restriction enzymes BglII and NdeI. This DNA fragment and a fragment obtained by cleavage of pCAMO-4 with the restriction enzymes BglII and NdeI were ligated together using T4 DNA ligase (Takara Bio Inc.). (a-8) 5'-GATCTGGAGGAGAAACGCA-3' (SEQ ID NO: 19) (b-8) 5'-TATGCGTTTCTCCTCCA-3' (SEQ ID NO: 20) The constructed plasmid was named pCAMO-5.

[0053] (1-8) Construction of Plasmid Vector (pCAMO-6) Using the endogenous plasmid pTH-1 as a template, PCR was performed with the following pair of primers to amplify a DNA fragment of the mazF gene encoding a plasmid stabilization factor. PCR was performed by standard methods using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as reaction reagents. Primers for amplifying the mazF gene: (a-9) 5'-GAGGCCATCTAGGCCATGAGTAAGTCTGACGGAAC-3' (SEQ ID NO: 21) (b-9) 5'-ATCCGGCACCCATATCTGAACCGGACGCAAACCCG-3' (SEQ ID NO: 22)

[0054] To amplify a DNA fragment of the mazE gene encoding a plasmid stabilization factor, PCR was performed using the endogenous plasmid pTH-1 as a template and the following pair of primers. PCR was performed by standard methods using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as reaction reagents. Primers for amplifying the mazE gene: (a-10) 5'-TTCAGATATGGGTGCCGGATACCCGCCGCCCGGGC-3' (SEQ ID NO: 23) (b-10) 5'-AAGGCCTTCATGGCCTTATTTCGCGATTCCCAAGA-3' (SEQ ID NO: 24)

[0055] The 3' end of the mazF DNA fragment shares a 20-bp homologous sequence with the 5' end of the mazE DNA fragment. Therefore, the mazF and mazE DNA fragments can be ligated by PCR. The DNA fragments prepared above were mixed, and the ligated DNA fragment was amplified using primers (a-9) and (b-10) as a template. PCR was performed using a Life Technologies DNA Thermal Cycler and KOD FX Neo (Toyobo Co., Ltd.) as reaction reagents according to standard methods. The resulting reaction mixture was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 0.7 kbp corresponding to the ligated DNA of the mazF and mazE DNA fragments was detected.

[0056] The prepared DNA fragment of approximately 0.7 kbp was digested with the restriction enzyme SfiI, and the plasmid pCAMO-5 was digested with the restriction enzyme SfiI and ligated to each other using T4 DNA ligase (Takara Bio Inc.). The constructed plasmid was named pCAMO-6.

[0057] (2) Construction of the ubiC gene expression plasmid The ubiC gene expression plasmid was constructed by seamless cloning, a method for cloning the target DNA into a vector using recombination between homologous sequences. Here, the DNA fragment of the ubiC gene derived from Shewanella algae (SEQ ID NO: 1) was amplified by PCR.

[0058] The following primers were used for PCR. 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 procedures. Primers for amplifying the Shewanella algae ubiC gene: (a-11) 5'-GCAGATCTGGAGGAGAAACGCATATGAGTGTGACCAGTTTAAGC-3' (SEQ ID NO: 25) (b-11) 5'-CTTGTCGACGGAGCTCGAATTCTCAAGGCGTAAGTTGGCCTTG-3' (SEQ ID NO: 26). Primers (a-11) and (b-11) contain sequences homologous to the pCAMO-6 vector. The resulting reaction mixture was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 0.7 kbp was detected for the ubiC gene from each strain. The DNA fragment corresponding to the ubiC gene was excised from the agarose gel, and the DNA fragment of the ubiC gene was recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0059] To perform seamless cloning, the plasmid vector pCAMO-6 was amplified by PCR. The following primers were used for PCR. 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. Primers used for amplification of plasmid vector pCAMO-6: (a-12) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 27) (b-12) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 28). The DNA fragment corresponding to the vector gene was excised from the agarose gel, and the DNA fragment of the vector gene was recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0060] The DNA fragment of the pCAMO-6 vector synthesized above and the DNA fragment of the ubiC gene were ligated using recombinase extracted from Escherichia coli JM109. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the resulting transformants were plated onto LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured with shaking at 37°C. Plasmid DNA was extracted from the culture medium. The sequence of the ubiC gene inserted into each plasmid was analyzed by Sanger analysis at Eurofins Genomics, and confirmed to match the sequence in the database.

[0061] (3) Construction of pobA gene expression plasmid The pobA gene expression plasmid was constructed by seamless cloning, a method of cloning the target DNA into a vector using recombination between homologous sequences. DNA fragments of the pobA gene from each of the following bacteria were amplified by PCR: Corynebacterium glutamicum (SEQ ID NO: 3), Meiothermus timidus (SEQ ID NO: 29), and Amycolatopsis thermoflava (SEQ ID NO: 30).

[0062] The following primers were used for 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. Primers for amplifying the Corynebacterium glutamicum pobA gene: (a-13) 5'-CAGATCTGGAGGAGAAACGCATATGAACCACGTACCAGTGGC-3' (SEQ ID NO: 31) (b-13) 5'-CTTGTCGACGGAGCTCGAATTCTTATACCTCGAAGCGTGGTAG-3' (SEQ ID NO: 32) Primers (a-13) and (b-13) contain sequences homologous to the vector pCAMO-6. Primers for amplifying the pobA gene of Meiothermus timidus: (a-14) 5'-CAGATCTGGAGGAGAAACGCATATGGCTAGGGTGGGCGTGGTG-3' (SEQ ID NO: 33) (b-14) 5'-GTCGACGGAGCTCGAATTCCTAGGCCGAGACCCCCTCCCCCA-3' (SEQ ID NO: 34) Primers (a-13) and (b-13) contain sequences homologous to that of the vector pCAMO-6. Primers for amplifying the Amycolatopsis thermoflava pobA gene: (a-15) 5'-GATCTGGAGGAGAAACGCATATGACGCGCACACAGGTCGGGATC-3' (SEQ ID NO: 35) (b-15) 5'-CTTGTCGACGGAGCTCGAATTCTTACGCATTACTCGTCCAGGCG-3' (SEQ ID NO: 36). Primers (a-13) and (b-13) contain sequences homologous to the pCAMO-6 vector. The resulting reaction mixture was subjected to electrophoresis on a 1% agarose gel, and a DNA fragment of approximately 1.2 kbp was detected for the pobA gene from each strain. The DNA fragment corresponding to the pobA gene was excised from the agarose gel and recovered from the agarose gel using the GEL / PCR Purification Mini Kit (FAVORGEN).

[0063] To perform seamless cloning, the plasmid vector pCAMO-6 was amplified by PCR. The following primers were used for PCR. 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 procedures. Primers for amplifying the plasmid vector pCAMO-6: (a-12) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 27) (b-12) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 28). The DNA fragment corresponding to the vector gene was excised from the agarose gel and recovered from the agarose gel using the GEL / PCR Purification Mini Kit (FAVORGEN).

[0064] The DNA fragment of the pCAMO-6 vector synthesized above and the DNA fragment of the pobA gene were ligated using recombinase extracted from Escherichia coli JM109. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, and the resulting mixture was plated on LB medium containing 50 μg / mL kanamycin and cultured at 37°C for 24 hours. 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 kanamycin, and cultured with shaking at 37°C. Plasmid DNA was extracted from the culture medium. The sequence of the pobA gene inserted into each plasmid was analyzed by Sanger analysis at Eurofins Genomics, and confirmed to match the sequence in the database.

[0065] (4) Construction of a Plasmid for Protocatechuic Acid Production The plasmid for protocatechuic acid production was constructed by seamless cloning, a method of cloning the target DNA into a vector using recombination between homologous sequences. The expression plasmid containing the Shewanella algae-derived ubiC gene described above was amplified by PCR. The following primers were used for PCR. 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. Primers for amplifying the expression plasmid containing the Shewanella algae-derived ubiC gene: (a-16) 5'-CCGCGGATCTAGGTGAAGATC-3' (SEQ ID NO: 37) (b-16) 5'-AGTTTGTAGAAACGCAAAAAGGCCATC-3' (SEQ ID NO: 38) Primers (a-13) and (b-13) contained sequences homologous to the vector pCAMO-6. A DNA fragment corresponding to the vector gene containing the ubiC gene derived from Shewanella algae was excised from the agarose gel, and the DNA fragment was recovered using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0066] In addition, the pobA gene containing the trc promoter and rrnB terminator was amplified by PCR from the expression plasmid containing the Corynebacterium glutamicum-derived pobA gene described above. The following primers were used for PCR. 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 procedures. Primers for amplifying the pobA gene containing the trc promoter and rrnB terminator: (a-17) 5'-GCCTTTTTGCGTTTCTACAAACTCCCATCGACTGCACGGTGCAC-3' (SEQ ID NO: 39) (b-17) 5'-GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCGCG-3' (SEQ ID NO: 40). The DNA fragment corresponding to the pobA gene containing the trc promoter and rrnB terminator was excised from an agarose gel and recovered from the agarose gel using a GEL / PCR Purification Mini Kit (FAVORGEN).

[0067] Using recombinase extracted from Escherichia coli JM109, the DNA fragment of the vector containing the ubiC gene derived from Shewanella algae synthesized above was ligated to a DNA fragment of the pobA gene containing the trc promoter and rrnB terminator. The resulting reaction mixture was transformed into Escherichia coli JM109 by the heat shock method, plated onto LB medium containing 50 μg / mL kanamycin, and cultured at 37°C for 24 hours. Each strain growing on LB medium was inoculated into a test tube containing 5 mL of LB liquid medium containing 50 μg / mL kanamycin using a platinum loop and cultured at 37°C with shaking. Plasmid DNA was extracted from the culture medium. The sequences of the ubiC and pobA genes inserted into the plasmids were analyzed by Sanger analysis at Eurofins Genomics, and were confirmed to match the sequences listed in the database.

[0068] (5) Hydrogenophilus thermorteolus Transformants (5-1) Introduction of the pobA Gene Expression Plasmid The Hydrogenophilus thermorteolus TH-1 strain was transformed with the plasmid obtained in section "(3) Construction of the pobA Gene Expression Plasmid" 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 fragment 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. Amplification of a DNA fragment of approximately 1.2 kbp corresponding to the pobA gene was confirmed. The plasmids containing the pobA gene derived from each bacterium and the Hydrogenophilus thermorteolus TH-1 strain transformants transformed with the plasmids were named as shown in Table 1.

[0069] (5-2) Measurement of 4-hydroxybenzoate hydroxylase activity. The pobA gene-transfected strain of Hydrogenophilus thermorteolus 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 culture was incubated at 52°C for 24 hours with shaking. The cultured cells were harvested by centrifugation (4°C, 5,000 g, 10 minutes). The cells were disrupted by sonication, and then centrifuged (4°C, 20,000 g, 3 minutes) to obtain the cell lysate supernatant. This cell lysate supernatant was used as a crude enzyme solution to measure 4-hydroxybenzoate hydroxylase activity. The crude enzyme solution was mixed with 50 mM Tris-HCl (pH 7.5), 175 μM NADPH, 65 μM FAD, and 0.5 mM 4-hydroxybenzoate, and the reaction was carried out at 52°C. The initial reaction rate was analyzed by monitoring the decrease in absorbance at 340 nm, which was derived from NADPH. The specific activity of 4-hydroxybenzoate hydroxylase was calculated from the initial reaction rate (1 unit is the specific activity of the enzyme required to produce 1 μmol of protocatechuate per minute per 1 mg of total protein). As shown in Table 1, 4-hydroxybenzoate hydroxylase activity was detected only in the PobA01 strain, which had been transformed with the pobA gene from Corynebacterium glutamicum.

[0070]

[0071] (5-3) Introduction of a Plasmid for Protocatechuic Acid Production. The Hydrogenophilus thermorteolus TH-1 strain was transformed with the plasmid obtained in section "(4) Construction of a Plasmid for Protocatechuic Acid Production" by electroporation. The transformed strains 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 plasmid insert fragment 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. A DNA fragment of approximately 3.2 kbp in length corresponding to the ubiC and pobA genes, each linked to the tac promoter and rrnB terminator, was observed.

[0072] (5-4) Protocatechuic Acid Production: The strain of Hydrogenophilus thermorteolus carrying the protocatechuic acid-producing plasmid 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 strain was cultured with shaking at 52°C for 72 hours. A strain containing the empty vector pCAMO-6 was also cultured in the same manner. After the culture, the culture supernatants were obtained by centrifugation (4°C, 5,000 g, 10 minutes). The protocatechuic acid concentration in the culture supernatants was measured using high-performance liquid chromatography (Shimadzu Corporation) under the following conditions. Mobile phase A: 0.1% phosphoric acid in water. Mobile phase B: 50% acetonitrile. Flow rate: 1 mL / min. Column: 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 a result, 0.41 mM protocatechuic acid was detected in the culture supernatant. On the other hand, no protocatechuic acid was detected in the strain lacking the ubiC and pobA genes. Introducing the ubiC gene from Shewanella algae and the pobA gene from Corynebacterium glutamicum conferred protocatechuic acid production to Hydrogenophilus bacteria.

[0073] 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.

[0074] The transformant of the present invention can produce protocatechuic acid with high efficiency using carbon dioxide as the sole carbon source, and therefore contributes to the industrial production of protocatechuic acid and chemical products using protocatechuic acid as a raw material with high efficiency while resolving global warming caused by increased carbon dioxide.

Claims

1. A transformant obtained by introducing into a Hydrogenophilus bacterium the chorismate-pyruvate lyase gene shown in (a), (b), (c), (d), or (e) below and the 4-hydroxybenzoate hydroxylase gene shown in (f), (g), (h), (i), or (j) below. (a) DNA comprising the nucleotide sequence of SEQ ID NO:1; (b) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO:1 and encoding a polypeptide having chorismate-pyruvate lyase activity; (c) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2; (d) DNA comprising an amino acid sequence having 90% or more identity with SEQ ID NO:2 and encoding a polypeptide having chorismate-pyruvate lyase activity; (e) DNA comprising 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:2 and encoding a polypeptide having chorismate-pyruvate lyase activity; (f) DNA comprising the nucleotide sequence of SEQ ID NO:3; (g) DNA comprising a nucleotide sequence having 90% or more identity with SEQ ID NO:3 and encoding a polypeptide having 4-hydroxybenzoate hydroxylase activity; (h) DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:4; (i) DNA comprising an amino acid sequence having 90% or more identity with SEQ ID NO:4 and encoding a polypeptide having chorismate-pyruvate lyase activity; (j) A DNA encoding a polypeptide having 4-hydroxybenzoate hydroxylase activity and containing an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO:

4.

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

3. A method for producing protocatechuic acid, comprising the step of culturing the transformant according to claim 1 or 2 using carbon dioxide as substantially the only carbon source.

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