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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/15—Corynebacterium

Definitions

• the present invention relates to a Hydrogenophilus bacterial transformant capable of producing protocatechuic acid, and a method for producing protocatechuic acid using this transformant.
• gases such as carbon dioxide, methane, and carbon monoxide are attracting attention as carbon raw materials with a higher degree of sustainability. If these gases present in the atmosphere could be fixed by microorganisms and used industrially, it would contribute greatly to carbon recycling and lead to the achievement of the target of carbon neutrality. For this reason, there is growing interest in technologies that use microorganisms that grow on these gases to produce valuable chemicals and biofuels. In particular, there are high hopes for fixing and effectively using carbon dioxide, which contributes greatly to global warming.
• Protocatechuic acid is a useful compound that can be used as an ingredient or raw material for medicines, cosmetics, pesticides, feed, fragrances, etc., and as an ingredient of functional foods because it is a polyphenol with antioxidant properties. It is also useful as a precursor for producing useful chemicals such as catechol and cis-cis-muconic acid by bio- or chemical conversion, and as a raw material for high-performance polymers.
• protocatechuic acid has been produced by extraction from natural products such as medicinal plants, as well as by chemical synthesis from petroleum feedstocks. However, extraction from natural products is costly due to low yields, and chemical synthesis from petroleum feedstocks places a heavy burden on the environment.
• protocatechuate is produced from 3-dehydroshikimate by the catalytic action of 3-dehydroshikimate dehydratase in the shikimate pathway, which is involved in the synthesis of aromatic amino acids.
• Protocatechuate can also be produced from 4-hydroxybenzoate by the catalytic action of 4-hydroxybenzoate hydroxylase.
• Patent Document 1 discloses a method for producing protocatechuic acid by introducing the gene for 3-dehydroshikimate dehydratase (qsuB gene), which produces protocatechuic acid from 3-dehydroshikimic acid, into Escherichia coli or Klebsiella bacteria and using glucose as a carbon source.
• qsuB gene 3-dehydroshikimate dehydratase
• Patent Document 2 and Non-Patent Document 1 disclose a method for producing protocatechuic acid using glucose as a carbon source by introducing the 3-dehydroshikimate dehydratase (qsuB) gene, the chorismate-pyruvate lyase (ubiC) gene, and the 4-hydroxybenzoate hydroxylase (pobA) gene into a bacterium belonging to the genus Corynebacterium.
• Chorismate-pyruvate lyase is an enzyme that catalyzes the conversion of chorismate to 4-hydroxybenzoic acid
• 4-hydroxybenzoate hydroxylase is an enzyme that catalyzes the conversion of 4-hydroxybenzoic acid to protocatechuic acid.
• Non-Patent Document 2 teaches a method for producing protocatechuic acid by introducing the qsuB gene derived from Corynebacterium glutamicum into Corynebacterium glutamicum using glucose and D-xylose as carbon sources.
• the objective of the present invention is to provide a Hydrogenophilus bacterium transformant (in other words, a transformed Hydrogenophilus bacterium) capable of efficiently producing protocatechuic acid using carbon dioxide as the sole carbon source, and a method for efficiently producing protocatechuic acid using this transformant.
• a Hydrogenophilus bacterium transformant in other words, a transformed Hydrogenophilus bacterium
• the present inventors have focused on bacteria of the genus Hydrogenophilus as a microorganism capable of fixing carbon dioxide on an industrial scale, with the aim of avoiding the use of high-cost raw materials such as sugars and contributing to global warming countermeasures.
• Hydrogenophilus bacteria grow by using hydrogen as an energy source to produce organic substances from carbon dioxide. Generally, such bacteria grow very slowly, but Hydrogenophilus bacteria grow quickly and have a much higher carbon dioxide fixation capacity than plants or photosynthetic bacteria.
• Hydrogenophilus bacteria do not have an enzyme for producing protocatechuic acid as a metabolic intermediate, and 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 for an enzyme that catalyzes the reaction for producing protocatechuic acid.
• the inventors have found that even heterologous genes that are expressed in bacteria other than Hydrogenophilus bacteria are often not expressed or are expressed insufficiently in Hydrogenophilus bacteria. Therefore, it is generally not useful to use genes that can be introduced into other bacteria to produce substances in Hydrogenophilus bacteria for substance production.
• the present inventors investigated genes predicted to be 3-dehydroshikimate dehydratase genes present in the genomes of other microorganisms in order to obtain a 3-dehydroshikimate dehydratase gene that is expressed in Hydrogenophilus bacteria, and found that the 3-dehydroshikimate dehydratase genes of Corynebacterium glutamicum and Pseudomonas thermotolerans express functional 3-dehydroshikimate dehydratase in Hydrogenophilus bacteria. They also found that transformants obtained by introducing these genes into Hydrogenophilus bacteria can efficiently produce protocatechuic acid using carbon dioxide as the sole carbon source.
• a transformant obtained by introducing the following 3-dehydroshikimate dehydratase gene (a), (b), (c), (d), or (e) into a Hydrogenophilus bacterium: (a) DNA containing the base sequence of SEQ ID NO: 1 or 3 (b) A DNA containing a base sequence having 90% or more identity to SEQ ID NO: 1 or 3 and encoding a polypeptide having 3-dehydroshikimate dehydratase activity.
• Measures to curb the increase in carbon dioxide include reducing carbon dioxide emissions and fixing the carbon dioxide that has been emitted.
• To reduce carbon dioxide emissions, solar, wind, geothermal, and other energies are being used in place of fossil energy.
• Carbon dioxide can be fixed physically or chemically, but if it is fixed using living organisms, it can produce organic matter that can be used as food, feed, fuel, etc. In other words, carbon dioxide itself can be directly converted into a valuable resource. This can solve two problems at the same time: global warming caused by an increase in carbon dioxide, and the difficulty in securing food, feed, and fuel. It can also produce chemical products that are in demand while suppressing global warming caused by an increase in carbon dioxide.
• Protocatechuic acid is an aromatic compound that is industrially important.
• protocatechuic acid extracted from natural products is expensive due to the low yield.
• chemical synthesis from petroleum raw materials is undesirable from an environmental standpoint.
• the production of protocatechuic acid using microorganisms that fix carbon dioxide can simultaneously solve the problem of global warming caused by increased carbon dioxide and ensure the supply of protocatechuic acid that is industrially necessary.
• such bacteria grow slowly, but hydrogen bacteria, the genus Hydrogenophilus, have an exceptionally fast growth rate.
• the Mitsubishi Research Institute Report No. 34 1999 evaluated Hydrogenophilus bacteria as "Their growth rate is so high that it cannot be compared to the carbon dioxide fixation ability of plants, and it clearly demonstrates the high carbon dioxide fixation ability of microorganisms.”
• the present invention by introducing a specific 3-dehydroshikimate dehydratase gene into Hydrogenophilus bacteria, 3-dehydroshikimate dehydratase that functions in the Hydrogenophilus bacteria can be expressed, and protocatechuic acid can be produced on an industrial scale.
• 3-dehydroshikimate dehydratase that functions in the Hydrogenophilus bacteria can be expressed, and protocatechuic acid can be produced on an industrial scale.
• Hydrogenophilus bacteria have a particularly excellent ability to fix carbon dioxide. Therefore, the present invention has opened up a way to produce protocatechuic acid on an industrial scale using carbon dioxide.
• the transformant of the present invention is a transformant obtained by introducing a 3-dehydroshikimate dehydratase gene (hereinafter sometimes abbreviated as "qsuB gene") of Corynebacterium glutamicum or Pseudomonas thermotolerans, or a homolog thereof, into a host Hydrogenophilus bacterium. That is, the transformant of the present invention is a Hydrogenophilus bacterium transformant having a foreign qsuB gene such as the qsuB gene of Corynebacterium glutamicum or Pseudomonas thermotolerans, or a homolog thereof.
• qsuB gene 3-dehydroshikimate dehydratase gene
• the qsuB gene used in the present invention does not need to have been identified as a qsuB gene, and may be any DNA encoding a polypeptide having 3-dehydroshikimate dehydratase activity, which refers to the enzyme activity of producing protocatechuic acid from 3-dehydroshikimic acid.
• the qsuB gene may be DNA of a qsuB gene isolated from a bacterium existing in nature, or may be DNA artificially synthesized using a technique known to those skilled in the art.
• the transformant of the present invention may be any bacterium of the genus Hydrogenophilus in which the qusB gene of Corynebacterium glutamicum, the qsuB gene of Pseudomonas thermotolerans, or a homologue thereof has been introduced, and includes transformants in which two or more of these genes have been introduced into a bacterium of the genus Hydrogenophilus, and transformants in which other genes have been introduced in addition to these genes into a bacterium of the genus Hydrogenophilus.
• a DNA containing the nucleotide sequence of SEQ ID NO: 1 or 3 (particularly, a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or 3) can be used as the qsuB gene.
• SEQ ID NO: 1 is the nucleotide sequence of the qsuB gene of Corynebacterium glutamicum
• SEQ ID NO: 3 is the nucleotide sequence of the qsuB gene of Pseudomonas thermotolerans.
• DNA can also be used that contains a base sequence that has an identity of 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more to SEQ ID NO: 1 or 3 (particularly, the base sequence has an identity of 90% or more, preferably 95% or more, preferably 98% or more, and preferably 99% or more to SEQ ID NO: 1 or 3) and encodes a polypeptide having 3-dehydroshikimate dehydratase activity.
• a DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2 or 4 can be used as the qsuB gene.
• SEQ ID NO: 2 is the amino acid sequence of 3-dehydroshikimate dehydratase from Corynebacterium glutamicum
• SEQ ID NO: 4 is the amino acid sequence of 3-dehydroshikimate dehydratase from Pseudomonas thermotolerans.
• DNA can also be used that contains an amino acid sequence that has an identity of 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more to SEQ ID NO: 2 or 4 (particularly, that consists of an amino acid sequence that has an identity of 90% or more, particularly 95% or more, particularly 98% or more, and particularly 99% or more to SEQ ID NO: 2 or 4) and that encodes a polypeptide having 3-dehydroshikimate dehydratase activity.
• DNAs that include an amino acid sequence in which 1 to 60, 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 in the amino acid sequence of SEQ ID NO: 2 or 4 (particularly, an amino acid sequence in which 1 to 60, 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 in the amino acid sequence of SEQ ID NO: 2 or 4) and that encode a polypeptide having 3-dehydroshikimate dehydratase activity can also be used.
• 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 substitutions do not change the amino acid sequence of the protein expressed from the coding region.
• the identity of base sequences and amino acid sequences is a value calculated using GENETYX ver.17 (GENETYX).
• test polypeptide has 3-dehydroshikimic acid dehydratase activity is confirmed by reacting the test polypeptide with 3-dehydroshikimic acid and detecting the resulting protocatechuic acid by high performance liquid chromatography.
• a DNA that contains a base sequence that is 90% to 100% identical to the base sequence of a certain DNA and that encodes a polypeptide with the same type of activity is called 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 called a homolog of that polypeptide.
• a DNA that contains an amino acid sequence in which 1 to 60 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 called a homolog of that DNA.
• Hydrogenophilus bacteria examples include Hydrogenophilus thermoluteolus, Hydrogenophilus halorhabdus, Hydrogenophilus denitrificans, Hydrogenophilus hirschii, Hydrogenophilus islandicus, Hydrogenophilus thiooxidans, Hydrogenophilus sp. Mar3, and Hydrogenophilus sp. Z1038.
• Hydrogenophilus thermoluteolus is preferred because it has a top-level growth rate and carbon dioxide fixation ability as a carbon dioxide fixation 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) strain shows the highest growth rate among carbon fixation microorganisms [Agricultural and Biological Chemistry, 41, 685-690 (1977)] (doubling in one hour). Hydrogenophilus thermorteolus NBRC 14978 strain has been internationally deposited under the Budapest Treaty and is publicly available.
• the host Hydrogenophilus bacterium may be a bacterium isolated from nature, or may be a bacterium obtained by genetically modifying a bacterium isolated from nature, for example, to enable high expression of the introduced 3-dehydroshikimate dehydratase gene. Such modification can be achieved by removing (curing) endogenous plasmids in Hydrogenophilus bacteria, etc.
• Methods for removing endogenous plasmids include, for example, methods that utilize plasmid incompatibility, such as a method of applying chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide, a method of destabilizing the endogenous plasmid by introducing a plasmid having the same replication origin as the endogenous plasmid, and a method of destabilizing the endogenous plasmid by destroying factors involved in the plasmid partition system.
• methods that utilize plasmid incompatibility such as a method of applying chemicals such as novobiocin, SDS, acriflavine, or ethidium bromide
• a method of destabilizing the endogenous plasmid by introducing a plasmid having the same replication origin as the endogenous plasmid such as a method of destabilizing the endogenous plasmid by destroying factors involved in the plasmid partition system.
• the qsuB gene can be introduced into the Hydrogenophilus bacterium by a general method for introducing a foreign gene into bacteria.
• the qsuB gene may be directly introduced into the Hydrogenophilus bacterium, or a vector for transformation (e.g., a plasmid vector, a virus vector, a cosmid, a fosmid, a BAC, a YAC, etc.) into which the qsuB gene has been incorporated may be introduced into the Hydrogenophilus bacterium.
• the vector for transformation may contain DNA capable of autonomously replicating in Hydrogenophilus bacteria, and examples of such vectors 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).
• broad-host-range vectors such as pRK415 (GenBank: EF437940.1), pBHR1 (GenBank: Y14439.1), pMMB67EH (ATCC 37622), pCAR1 (NCBI
• pCAMO-6 is preferable.
• pCAMO-6 can be prepared by those skilled in the art according to the Examples.
• promoters contained in the vector include tac promoter, lac promoter, trc promoter, and each of the Oxford Genetics OXB1, OXB11 to OXB20 promoters.
• terminators contained in the vector include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the bacteriophage ⁇ t0 transcription terminator, and the T7 terminator.
• the qsuB gene can be introduced (transformed) into a Hydrogenophilus bacterium by a general method, such as the calcium chloride method, the calcium phosphate method, the rubidium chloride method, or the electric pulse method (electroporation method).
• a general method such as the calcium chloride method, the calcium phosphate method, the rubidium chloride method, or the electric pulse method (electroporation method).
• the present invention provides a method for producing protocatechuic acid using the transformant of the present invention described above.
• This method includes a step of culturing the transformant of the present invention in an inorganic or organic medium while supplying a gas containing carbon dioxide, preferably a mixed gas containing hydrogen, oxygen, and carbon dioxide.
• the gas supplied is preferably a mixed gas consisting of hydrogen, oxygen, and carbon dioxide, but other gases may be mixed in as long as protocatechuic acid can be efficiently produced.
• the pH of the medium used for the culture is preferably 6.2 to 8, more preferably 6.4 to 7.4, and even more preferably 6.6 to 7. Within this range, the growth of the bacteria and the solubility of the mixed gas in the medium are high, and protocatechuic acid can be produced with high efficiency.
• the mixed gas can be sealed in a sealed culture vessel and cultured statically or with shaking, and in the case of continuous culture, the mixed gas can be continuously supplied to a sealed culture vessel while cultured with shaking, or the transformant can be cultured in a sealed culture vessel while introducing the mixed gas into the medium by bubbling. Shaking culture is preferred because it improves the dissolution of the mixed gas into the medium.
• the volume ratio of hydrogen, oxygen, and carbon dioxide in the feed 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.
• the supply rate of the mixed gas or raw gas may be 10 to 60 L/hour, preferably 10 to 40 L/hour, and more preferably 10 to 20 L/hour per L of medium.
• the growth of the transformant is favorable, protocatechuic acid can be efficiently produced, and waste of the mixed gas is suppressed.
• 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 transformant grows well and protocatechuic acid can be produced efficiently. By culturing as described above, protocatechuic acid or a salt thereof is produced in the culture medium.
• Plasmid Vector (pCAMO-6) The method for constructing the plasmid vector pCAMO-6 used for introducing the qsuB gene is described below.
• (1-1) Preparation of DNA fragment of tac promoter PCR was carried out using the following pair of primers to amplify the DNA fragment of tac promoter with plasmid pMAL-c5X (New England Biolabs) as a template. PCR was carried out by a conventional method using "2720 Thermal Cycler" (Thermo Fisher Scientific) and KOD FX Neo (Toyobo Co., Ltd.) as a reaction reagent.
• Primer for amplifying tac promoter (a-1): 5'-TTTTATAA CCCGGG CCATCGACTGCACGGTGCACC-3' (SEQ ID NO: 5) (b-1) 5'-TGCTAGCACTGTTTCCTGTGTGAAATTGTTATCCG-3' (SEQ ID NO: 6)
• a SmaI restriction enzyme site was added to primer (a-1) as underlined.
• the reaction solution prepared above was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 0.3 kbp corresponding to the tac promoter was detected.
• Primer 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.
• Primer for preparing the latter 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 base sequences of primers (a-3) and (b-3) are complementary to each other.
• the reaction mixture was subjected to electrophoresis using a 1% agarose gel, and DNA fragments of approximately 0.1 kbp each 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 the gel.
• 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'-side base sequences of primers (b-2) and (a-3) used to amplify the template DNA fragments are complementary to each other.
• a combination of primers (a-2) and (b-3) was used to prepare DNA of the multicloning region.
• PCR was performed in the usual manner using a Life Technologies "DNA Thermal Cycler" and KOD FX Neo (Toyobo Co., Ltd.) as a reaction reagent.
• the resulting reaction solution was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 0.2 kbp corresponding to the multicloning region was detected.
• Primer for amplifying rrnB terminator (a-4): 5'-TAACTCGATTCAAATAAAACGAAAGGCTCAGTCGA-3' (SEQ ID NO: 11) (b-4) 5'-CCTAGATCCGCGGAGTTTGTAGAAACGCAAAAAGG-3' (SEQ ID NO: 12)
• PCR was performed using the following pair of primers with the plasmid pUC19 as a template.
• the resulting reaction solution was subjected to electrophoresis using a 1% agarose gel, and a DNA fragment of about 0.3 kbp was detected for the rrnB terminator and about 0.8 kbp for the DNA replication origin region of pUC19. Each DNA fragment was excised from the agarose gel, and the DNA was recovered from the gel by freezing and thawing the gel.
• Overlap extension PCR was performed using the recovered rrnB terminator and the DNA fragment corresponding to the DNA replication origin of pUC19 as templates.
• the 5'-side base sequences of primers (b-4) and (a-5) used to amplify these template DNA fragments are complementary to each other.
• overlap extension PCR a combination of primers (a-4) and (b-5) was used to prepare a DNA fragment in which the rrnB terminator and the replication origin of pUC19 were linked.
• PCR was performed in the usual manner using a "DNA Thermal Cycler" manufactured by Life Technologies and KOD FX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.
• the resulting reaction solution was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of approximately 1.0 kbp corresponding to the DNA consisting of the rrnB terminator and the replication origin of pUC19 was detected.
• PCR was carried out by a standard method 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 carried out using the following pair of primers to amplify a DNA fragment containing the nptII gene sequence.
• PCR was carried out by a standard method using a "DNA Thermal Cycler” manufactured by Life Technologies and KOD FX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.
• Primer (a-6) for amplifying the nptII gene 5'-TTGCTGGCCGCGGACGTAGAAAGCCTGTCCGCAGA-3' (SEQ ID NO: 15)
• b-6) 5'-GG CCCGGG TTATAAAAGCCAGTCATTAGGCCTATC-3'
• the primer (b-6) has an SmaI restriction enzyme site added thereto as shown by the underline.
• the resulting reaction solution was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about 1.0 kbp corresponding to the nptII gene was detected.
• the end of DNA fragment (1-1) has a 16-bp homologous sequence to the ends of DNA fragments (1-4) and (1-2)
• the end of DNA fragment (1-2) has a 16-bp homologous sequence to the ends of DNA fragments (1-1) and (1-3)
• the end of DNA fragment (1-3) has a 16-bp homologous sequence to the ends of DNA fragments (1-2) and (1-4)
• the end of DNA fragment (1-4) has a 16-bp homologous sequence to the ends of DNA fragments (1-3) and (1-1).
• the DNA fragments (1-1), (1-2), (1-3), and (1-4) can be ligated to a circular DNA by Gibson Assembly.
• Gibson Assembly was performed using Gibson Assembly Master Mix (New England Biolabs).
• Escherichia coli JM109 was transformed by the calcium chloride method and spread on LB solid medium containing 50 ⁇ g/mL kanamycin.
• the strain grown on the medium was cultured in liquid by a conventional method, and plasmid DNA was extracted from the culture solution and reacted with the restriction enzyme SmaI.
• the prepared pTH-1 of about 66 kbp was cleaved with the restriction enzymes ScaI and PvuII, and the plasmid pCAMO-1 was cleaved with the restriction enzyme SmaI, and the two were ligated together using T4 DNA (Takara Bio Inc.).
• thermorteolus TH-1 (NBRC 14978) was transformed with the ligation solution by the electric pulse method (electroporation method) and the transformants were grown on solid medium A containing 50 ⁇ g/mL kanamycin [(NH 4 ) 2 SO 4 3.0 g, KH 2 PO 4 1.0 g, K 2 HPO 4 2.0 g, NaCl 0.25 g, FeSO 4 ⁇ 7H 2 O 0.014 g, MgSO 4 ⁇ 7H 2 O 0.5 g, CaCl 2 0.03 g, MoO 3 4.0 mg, ZnSO 4 ⁇ 7H 2 O 28 mg, CuSO 4 ⁇ 5H 2 O 2.0 mg, H 3 BO 3 4.0 mg, MnSO 4 ⁇ 5H 2 O 4.0 mg, CoCl 2 ⁇ 6H 2 O 5.0 mg, and 1.0 g of 1.0- ...
• PCR was performed using the following pair of primers corresponding 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 in a standard manner using a "DNA Thermal Cycler" manufactured by Life Technologies and KOD FX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.
• Primer (a-7) for amplifying the inserted DNA fragment of pTH1 5'-AATTGTCAGATAGGCCTAATGACTGGCTTTTATAA-3' (SEQ ID NO: 17)
• the resulting reaction solution was subjected to electrophoresis using a 1% agarose gel, and a DNA fragment of about 3.2 kbp was detected.
• the resulting plasmid has a DNA fragment of about 3.2 kbp, which is a part of pTH1, inserted into the SmaI restriction enzyme site of the pCAMO-1 plasmid.
• PCR was performed using the following pair of primers with the endogenous plasmid pTH-1 as a template. PCR was performed by standard methods using a DNA Thermal Cycler manufactured by Life Technologies and KOD FX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.
• Primer (a-9) for amplifying the mazF gene 5'-GAGGCCATCTAGGCCATGAGTAAGTCTGACGGAAC-3' (SEQ ID NO: 21)
• (b-9) 5'-ATCCGGCACCCATATCTGAACCGGACGCAAACCCG-3' (SEQ ID NO: 22)
• PCR was performed using the endogenous plasmid pTH-1 as a template and the following pair of primers: PCR was performed in a standard manner using a DNA Thermal Cycler manufactured by Life Technologies and KOD FX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.
• Primer for amplifying the mazE gene (a-10): 5'-TTCAGATATGGGTGCCGGATACCCGCCGCCCGGGC-3' (SEQ ID NO: 23) (b-10) 5'-AAGGCCTTCATGGCCTTATTTCGCGATTCCCAAGA-3' (SEQ ID NO: 24)
• the 3' end of the mazF DNA fragment has 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 in the usual manner using a DNA Thermal Cycler manufactured by Life Technologies and KOD FX Neo (manufactured by Toyobo Co., Ltd.) as a reaction reagent.
• the resulting reaction solution was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of approximately 0.7 kbp corresponding to the DNA formed by ligating the mazF and mazE DNA fragments was detected.
• the prepared DNA fragment of about 0.7 kbp was cleaved with the restriction enzyme SfiI, and the plasmid pCAMO-5 was cleaved with the restriction enzyme SfiI, and the fragments were ligated to each other using T4 DNA ligase (Takara Bio Inc.).
• the constructed plasmid was named pCAMO-6.
• the qsuB gene expression plasmid was constructed by a method of cloning a target DNA into a vector by utilizing recombination reaction between homologous sequences, that is, by so-called seamless cloning. DNA fragments of the qsuB genes derived from the following bacteria were amplified by PCR. Corynebacterium glutamicum (SEQ ID NO: 1) Pseudomonas thermotolerans (SEQ ID NO:3)
• the primers (a-11) and (b-11) contain sequences homologous to the vector pCAMO-6.
• Pseudomonas thermotolerans qsuB gene amplification primer (a-12) 5'-CTGGAGGAGAAACGCATATGCAGCGTTCGATCGCCACCGTCTC-3' (SEQ ID NO: 27)
• (b-12) 5'-CGACGGAGCTCGAATTCTCAGAGCCTGGGCTGACGAGCGGCGC-3' (SEQ ID NO: 28)
• Each of the primers (a-12) and (b-12) contains a sequence homologous to the vector pCAMO-6.
• the resulting reaction solution was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of approximately 1.9 kbp was detected for the 3-dehydroshikimate dehydratase gene derived from each strain.
• a DNA fragment corresponding to the qsuB gene was excised from the agarose gel, and a DNA fragment of the 3-dehydroshikimate dehydratase gene was recovered from the agarose gel using a GEL/PCR Purification Mini Kit (FAVORGEN).
• the plasmid vector pCAMO-6 was amplified by PCR.
• the following primers were used for PCR.
• PCR was performed in a standard manner using a DNA Thermal Cycler manufactured by Life Technologies and KOD One PCR Master Mix (manufactured by Toyobo Co., Ltd.) as a reaction reagent.
• Primer used for amplification of plasmid vector pCAMO-6 (a-13) 5'-GAATTCGAGCTCCGTCGACA-3' (SEQ ID NO: 29) (b-13) 5'-ATGCGTTTCTCCTCCAGATC-3' (SEQ ID NO: 30)
• a 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).
• the DNA fragment of the vector pCAMO-6 synthesized above and the DNA fragment of the qsuB gene were ligated to each other using recombinase extracted from the Escherichia coli JM109 strain.
• the resulting reaction solution was used to transform Escherichia coli JM109 strain by the heat shock method, which was then spread onto LB medium containing 50 ⁇ g/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 ⁇ g/mL kanamycin using a platinum loop, cultured with shaking at 37°C, and plasmid DNA was extracted from the culture medium.
• the sequence of the qsuB gene inserted into each plasmid was analyzed by Sanger method at Eurofins Genomics, Inc., and confirmed to match the sequence in the database.
• PCR was performed by the usual method using a "DNA Thermal Cycler” manufactured by Life Technologies and KOD One PCR Master Mix (manufactured by Toyobo Co., Ltd.) as a reaction reagent. As a result, amplification of a DNA fragment of approximately 1.9 kbp in length corresponding to the qsuB gene was confirmed.
• the plasmids containing the qsuB gene derived from each bacterium and the Hydrogenophilus thermorteolus TH-1 strain transformants transformed with the plasmids were named as shown in Table 1.
• Mobile phase A 0.1% phosphoric acid water
• Mobile phase B 50% acetonitrile Flow rate: 1mL/min
• Table 1 0.033 mM protocatechuic acid was detected in the culture supernatant of the QsuB01 strain, into which the qsuB gene of Corynebacterium glutamicum was introduced.
• nucleotide sequence of the qsuB gene of Corynebacterium glutamicum and that of the qsuB gene of Pseudomonas thermotolerans are significantly different, with only about 42% identity between the two nucleotide sequences. As such, it is clear that even those skilled in the art would have difficulty predicting the nucleotide sequence of a foreign gene capable of expressing a protein that functions in a Hydrogenophilus bacterium, since no consistent trend is observed.
• the qsuB gene of Corynebacterium glutamicum enhances the production of protocatechuic acid when introduced into microorganisms other than those of the genus Hydrogenophilus, but in the genus Hydrogenophilus, the qsuB gene of Pseudomonas thermotolerans produced protocatechuic acid more efficiently than the qsuB gene of Corynebacterium glutamicum.
• the expression efficiency of functional 3-dehydroshikimate dehydratase is not parallel between other microorganisms and Hydrogenophilus bacteria. Therefore, it is not easy to search for a qsuB gene that is expressed in Hydrogenophilus bacteria and is effective in producing protocatechuic acid.
• Hydrogenophilus bacterial transformants of the present invention can be prepared with reference to the description in the Examples.
• other strains described herein are either internationally deposited under the Budapest Treaty, held by an institution from which they may be obtained without conditions, commercially available, or can be prepared by one skilled in the art based on the present specification and are publicly available.
• the transformant of the present invention can produce protocatechuic acid with high efficiency using carbon dioxide as the sole carbon source, which contributes to the industrial production of protocatechuic acid and chemical products using it as a raw material with high efficiency while resolving global warming caused by increased carbon dioxide.

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