Microbial strain, protein, and method for producing gallic acid using microbial strain or protein

Abstract

The purpose of the present invention is to provide: a protein which is capable of efficiently producing gallic acid from protocatechuic acid and has protocatechuic acid 5-position oxidation activity; and a microbial strain which expresses the protein. According to the present invention, gallic acid can be efficiently produced from protocatechuic acid by using a protocatechuic acid 5-position oxidase derived from a microorganism of the genus Comamonas, or a protein having 70% or more identity with the amino acid sequence of the protocatechuic acid 5-position oxidase. Further, gallic acid can be produced from protocatechuic acid by fermentation using a microbial strain into which a gene encoding the enzyme has been introduced.

Description

Technical Field

[0001] The present technology relates to a microbial strain into which a gene related to a specific aromatic hydroxylase is introduced, a protein serving as the specific aromatic hydroxylase, and a method for producing gallic acid using the microbial strain or the protein. Background Art

[0002] Gallic acid (GA) is a phenolic compound contained in many plants such as gallnut, gallic acid, witch hazel, tea leaves, and oak bark. Since gallic acid has three hydroxyl groups in the molecule, it shows a high antioxidant effect and is used as an antioxidant. In addition, derivatives of gallic acid are used as adhesives, coating agents, and electronic component-related products. In addition, esters of gallic acid are also widely used as additives for many food purposes. It is estimated that more than 8,000 tons of gallic acid will be needed each year, and the demand is increasing.

[0003] At present, gallic acid is industrially produced from plant raw materials represented by gallnut and gall. Therefore, the supply of raw materials harvested each time is unstable, which is the main reason for the price increase. In order to cope with the increasing demand for gallic acid, gallic acid is stably and cheaply produced, and a production method based on microbial fermentation using sugar as a substrate is expected.

[0004] The manufacturing method based on microbial fermentation does not utilize organic solvents, heavy metals, strong acids, and strong bases, and can utilize sustainable plant biomass such as starch and cellulose, so the environmental load is small compared with the manufacturing method using chemical synthesis. In addition, stable production can be achieved by controlling the culture conditions, so it can be expected that the object can be provided cheaply compared with the production based on plant cells.

[0005] However, in Figure 3 In the pathway for producing gallic acid from sugar (glucose) using microorganisms shown, p-hydroxybenzoate hydroxylase (PobA) that produces protocatechuic acid (PCA) from p-hydroxybenzoic acid (pHBA) is known, but an enzyme that can selectively produce gallic acid from protocatechuic acid, which is a gallic acid precursor, is unknown.

[0006] For example, the following Patent Document 1 discloses a method for producing gallic acid from protocatechuic acid using a microorganism expressing p-hydroxybenzoate hydroxylase (PobA) derived from Pseudomonas putida KT2440 and from Neosphingobacterium aromaticum DSM12444 (i.e., an enzyme that produces protocatechuic acid from p-hydroxybenzoic acid), but the reaction of producing protocatechuic acid from p-hydroxybenzoic acid takes precedence over the reaction of producing gallic acid from protocatechuic acid, so the ratio of gallic acid to protocatechuic acid is not sufficiently increased.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2009-065839 Summary of the invention

[0010] Problems to be solved by the invention

[0011] Therefore, the main object of the present technology is to provide a protein having protocatechuic acid 5-position oxidation activity and capable of efficiently generating gallic acid from protocatechuic acid, and a microbial strain expressing the protein.

[0012] Means for solving problems

[0013] The inventors of the present application have conducted intensive studies and found that the use of protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae or a protein having an amino acid sequence identity of 70% or more to the protocatechuate 5-oxidase is useful for efficiently producing gallic acid from protocatechuic acid.

[0014] That is, the present technology provides the following contents.

[0015] [1] A microbial strain into which a gene encoding a protocatechuate 5-position oxidase derived from a microorganism of the family Comamonadaceae or a protein having an amino acid sequence identity of 70% or more to the protocatechuate 5-position oxidase is introduced.

[0016] [2] The microbial strain according to [1], wherein the microorganism of the family Comamonadaceae is a microorganism selected from the genera Hylemonella, Polaromonas, and Hydrogenophaga.

[0017] [3] A microbial strain as described in [1] or [2], wherein the aforementioned microbial strain is a microbial strain selected from the genus Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Novosphingobium or Ralstonia.

[0018] [4] A method for producing gallic acid, comprising producing gallic acid from protocatechuic acid by fermentation using the microbial strain described in any one of [1] to [3].

[0019] [5] A method for producing gallic acid, comprising producing gallic acid by fermentation using the microbial strain described in any one of [1] to [3] in a medium containing glucose.

[0020] [6] The method for producing gallic acid according to [4] or [5], wherein the fermentation is carried out using a culture medium at a pH of 6.0 or less.

[0021] [7] The method for producing gallic acid according to any one of [4] to [6], wherein the fermentation is carried out at a temperature of 15°C to 45°C.

[0022] [8] A method for producing gallic acid from protocatechuic acid, wherein the method comprises using a protocatechuic acid 5-position oxidase derived from a microorganism of the family Comamonadaceae, or a protein having an amino acid sequence identity of 70% or more to the protocatechuic acid 5-position oxidase.

[0023] [9] A protein having an amino acid sequence in which leucine at positions 199 to 209 of a protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae, or a protein having an amino acid sequence identity of 70% or more to the protocatechuate 5-oxidase, is substituted with valine or glycine.

[0024]

[10] A protein having an amino acid sequence in which threonine at positions 294 to 304 of a protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae, or a protein having an amino acid sequence identity of 70% or more to the protocatechuate 5-oxidase, is substituted with alanine.

[0025]

[11] The protein according to [9] or

[10] , wherein the tyrosine at positions 385 to 395 is further substituted with phenylalanine, valine or alanine.

[0026] Effects of the Invention

[0027] The microbial strain according to the present technology has an improved yield of gallic acid derived from protocatechuic acid, and can efficiently produce gallic acid.

[0028] By fermentation using the microbial strain according to the present technique, the yield of gallic acid from protocatechuic acid is improved, and gallic acid can be efficiently produced.

[0029] By using the protein having protocatechuic acid 5-position oxidation activity according to the present technique, the yield of gallic acid from protocatechuic acid is improved, and gallic acid can be efficiently produced.

[0030] It should be noted that the effects of the present technology are not limited to the effects described here, and may be any effect described in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] [ Figure 1 ] is a diagram showing the results of molecular systematic analysis of PobA protein.

[0032] [ Figure 2 ] is a graph showing the gallic acid-producing activity of the protocatechuate 5-oxidase involved in the present technology.

[0033] [ Figure 3 ] is a diagram showing a pathway for producing gallic acid from sugar using microorganisms. DETAILED DESCRIPTION

[0034] Hereinafter, preferred embodiments of the present technology will be described. However, the present technology is not limited to the following preferred embodiments, and can be freely changed within the scope of the present technology.

[0035] The microbial strain of the present technology is a microbial strain into which a gene encoding a protocatechuate 5-position oxidase derived from a microorganism of the family Comamonadaceae or a protein having an amino acid sequence identity of 70% or more to the protocatechuate 5-position oxidase is introduced.

[0036] based on Figure 2 The microorganism of the Comamonadaceae family from which the protocatechuate 5-oxidase of the present technology is derived will be described. By using the protocatechuate 5-oxidase derived from the microorganism of the Comamonadaceae family, protocatechuic acid can be efficiently converted into gallic acid.

[0037] The microorganism of the Comamonadaceae family is not particularly limited as long as it satisfies the above characteristics. Figure 1 Among the genera listed in the systematic analysis results, preferred are Helemonella, Limnohabitans, Rhodoferax, Xylophilus, Ramlibacter, Polarmonas, and Hydrogenophage, and particularly preferred are Helemonella, Polarmonas, and Hydrogenophage.

[0038] Amino acid sequence information of PobA (HgPobA) from Hylemonella gracilis (SEQ ID NO: 20)

[0039] Amino acid sequence information of PobA from Limnohabitans sp. (SEQ ID NO: 23)

[0040] Amino acid sequence information of PobA from Rhodoferax sediminis (SEQ ID NO: 24)

[0041] Amino acid sequence information of PobA from Xylophilus ampelinus (SEQ ID NO: 25)

[0042] Amino acid sequence information of PobA from Ramlibacter henchirensis (SEQ ID NO: 26)

[0043] Amino acid sequence information of PobA from Comamonas phosphati (SEQ ID NO: 27)

[0044] Amino acid sequence information of PobA (HyPobA) from Limnohabitans sp. (SEQ ID NO: 21)

[0045] Amino acid sequence information of PobA (PoPobA) from Polaromonas sp. (SEQ ID NO: 22)

[0046] A gene encoding a protein having the same amino acid sequence as the protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae expressed in the microorganism strain of the present technology may be introduced, or a gene encoding a protein whose amino acid sequence is changed within the range in which the protocatechuate 5-oxidase can maintain the activity of hydroxylating protocatechuate to generate gallic acid may be introduced. A protein having an identity of preferably 70% or more, more preferably 80% or more, further preferably 90% or more, further preferably 95% or more, further preferably 97% or more, further preferably 98% or more, further preferably 99% or more, further preferably 99.5% or more, further preferably 99.7% or more to the amino acid sequence of the protocatechuate 5-oxidase can maintain the activity of hydroxylating protocatechuate to generate gallic acid, and therefore it is considered that the homology with the protocatechuate 5-oxidase involved in the present technology is high.

[0047] In the present technology, the form of the gene introduced into the microbial strain is not particularly limited, and for example, a polynucleotide encoding the aforementioned protocatechuate 5-position oxidase or protein, or a vector containing the polynucleotide in its sequence, etc. can be used.

[0048] As a polynucleotide encoding a protein such as protocatechuate 5-oxidase in the present technology, it is preferred to include a control region operably connected to an upstream region (a region on the 5' end side of the polynucleotide chain) relative to a region encoding a protein such as protocatechuate 5-oxidase. The control region preferably includes a promoter, and may also include a cis-acting element, a terminator, etc. that improve the transcriptional activity of the promoter. In addition, by making the polynucleotide also include a screening marker gene such as a drug resistance gene and an auxotrophic marker gene, it is possible to efficiently screen a microbial strain into which a gene has been introduced.

[0049] Furthermore, by providing restriction endonuclease recognition sequences at both ends of a polynucleotide encoding a protein such as protocatechuate 5-oxidase in the present technology, the polynucleotide can also be introduced into a vector by the restriction endonuclease method.

[0050] The type of vector that can be used in the present technology is not particularly limited, and can be any vector such as plasmid, cosmid, phasmid, phage, transposon, BAC vector, etc. In addition, the vector can be a vector for introducing into the chromosome in the cell of the microbial strain for gene introduction, or a vector that is retained outside the chromosome, preferably a vector that can be amplified in the cell of the microbial strain. The vector that can be used in the present technology is not limited, preferably a bacterial vector, and more preferably a vector for the microbial strain for gene introduction.

[0051] In the present technology, there is no particular limitation on the microbial strain for gene introduction, but preferably the microbial strain belongs to the genus Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Novosphingobium or Ralstonia, and particularly preferably to the genus Escherichia and Corynebacterium.

[0052] The method for introducing the aforementioned gene into a microbial strain is not particularly limited, and conventional transformation methods can be applied, for example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], electroporation [Nucleic Acids Res., 16, 6127 (1988)], transformation, transfection, conjugation, protoplast method, gene gun method, Agrobacterium method and other well-known transformation techniques.

[0053] Gallic acid can be suitably produced from protocatechuic acid by fermentation using the microbial strain of the present technology. Fermentation using the microbial strain of the present technology is not particularly limited, and can be suitably performed, for example, by adding a culture solution obtained by culturing the microbial strain of the present technology as a pre-culture to the medium for fermentation.

[0054] The culture medium for the pre-culture of the microbial strain of the present technology can suitably use the culture medium of the microbial strain conventionally used for gene introduction.For example, in the case where the microbial strain for gene introduction is Escherichia, LB culture medium, M9 culture medium, etc. can be used to suitably culture, and the conditions such as culture temperature of 15 ° C ~ 45 ° C and culture time of 1 day to 7 days can be cited.In the case where the microbial strain for gene introduction is Corynebacterium, LB culture medium, CGXII culture medium (Journal of Bacteriology, 1993, 175: 5595-5603) etc. can be used to suitably culture, and the conditions such as culture temperature of 15 ° C ~ 45 ° C and culture time of 1 day to 7 days can be cited.

[0055] By performing pre-culture before the fermentation-related steps, gallic acid can be efficiently and appropriately produced from protocatechuic acid using a microbial strain in a logarithmic growth phase.

[0056] The culture medium for fermentation of the microbial strains used in the present technology only needs to contain Figure 3 The compound that becomes gallic acid precursor in the gallic acid production pathway shown in the above-mentioned method can be used as a substrate without any particular limitation. It should be noted that the compound that becomes gallic acid precursor contained in the culture medium can directly contain Figure 3 The compounds shown in the gallic acid production pathway shown, for example, may also include Figure 3 Compounds shown in the gallic acid production pathway shown are terephthalic acid, phthalic acid, isophthalic acid and the like, which are precursors of protocatechuic acid and the like.

[0057] As a substrate contained in the culture medium for fermentation of the microbial strain used in the present technology, from the viewpoint of reducing the production cost of gallic acid, a culture medium containing glucose is preferably used. As a culture medium containing glucose, it can be prepared by adding 1 to 10%, preferably about 3 to 7%, of glucose to the culture medium of the conventional microbial strain used for gene introduction.

[0058] For example, when the microbial strain for gene introduction is Corynebacterium, a medium in which glucose is added to CGXII medium or LB medium can be suitably fermented under conditions such as a culture temperature of 15°C to 45°C and a culture time of 1 to 7 days. In addition, when the microbial strain for gene introduction is Escherichia, an LB medium or a fermentation production medium (Scientific reports, 2016, 6.1: 1-9.) with glucose added can be suitably fermented under conditions such as a culture temperature of 28°C to 37°C and a culture time of 1 to 7 days.

[0059] By lowering the pH of the culture medium used for the fermentation of the microbial strain of the present technology, the amount of acid such as sulfuric acid and hydrochloric acid used to crystallize gallic acid can be reduced in the process of recovering gallic acid from the fermentation liquid. In addition, by lowering the pH of the culture medium used for the fermentation of the microbial strain of the present technology, the risk of hindering the target fermentation due to the mixing of strains other than the microbial strain of the present technology can be reduced.

[0060] For example, the pH of the typical fermentation liquid during culture is 6.5 to 7.5, and the amount of sulfuric acid used at this time is assumed to be 13 to 16 g / L. However, when the pH of the culture liquid is 5.0, the amount of sulfuric acid used can be assumed to be 5 to 8 g / L, which can reduce the amount of sulfuric acid used.

[0061] The pH of the culture medium used for fermentation of the microbial strain of the present technique is, for example, preferably 6.0 or less, and more preferably 5.0 or less.

[0062] In addition, the protocatechuate 5-oxidase derived from the microorganism of the Comamonadaceae family used in the present technology can be expected to maintain enzyme activity even in a temperature range above 37°C, so the temperature conditions in the fermentation of the microbial strain using the present technology can also be set to a high temperature range of preferably 35°C to 45°C, and more preferably 37°C to 40°C.

[0063] As described above, the protocatechuate 5-oxidase derived from a microorganism of the Comamonadaceae family used in the present technology can also be expressed in a system using a microbial strain into which a gene encoding the protocatechuate 5-oxidase is introduced, but reagents such as amino acids can also be added to the gene encoding the protocatechuate 5-oxidase or its transcription product to express it in an in vitro (invitro) system.

[0064] In the present technology, protocatechuate 5-oxidase derived from a microorganism of the Comamonadaceae family, or a protein having an amino acid sequence identity of 70% or more to the protocatechuate 5-oxidase, expressed in a system using the aforementioned microbial strain or in vitro system, can be used to produce gallic acid from protocatechuic acid.

[0065] In this case, protocatechuate 5-oxidase, or a protein having an amino acid sequence identity of 70% or more with that of protocatechuate 5-oxidase, can be suitably recovered from a culture medium of a microbial strain, a cell disrupted medium, a reaction solution of an in vitro system, etc., by using conventional methods used for protein purification, such as cell disruption, centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination.

[0066] The protocatechuate 5-oxidase of the present technology may use an enzyme having the same amino acid sequence as the protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae, or an enzyme obtained by mutating the amino acid sequence.

[0067] For example, the activity of producing gallic acid from protocatechuic acid may be improved by substituting leucine located at positions 199 to 209, preferably 199 to 200, starting from the N-terminal side of the polypeptide chain involved in the enzyme with valine or glycine.

[0068] In addition, the activity of producing gallic acid from protocatechuic acid may be improved by substituting threonine at positions 294 to 304, preferably at position 294 or 302, starting from the N-terminal side of the polypeptide chain involved in the enzyme with alanine.

[0069] Furthermore, the activity of producing gallic acid from protocatechuic acid may be improved by substituting tyrosine at positions 385 to 395, preferably at position 385 or 393, starting from the N-terminal side of the polypeptide chain involved in the enzyme with phenylalanine, valine or alanine.

[0070] In the present technology, with respect to the mutation site of the amino acid sequence of protocatechuate 5-oxidase, the mutation sites exemplified in the above description can be introduced alone or in appropriate combination, but are not limited to the above examples. An enzyme having a mutated amino acid sequence can be designed by substituting any amino acid residue in the polypeptide chain with another amino acid, deleting any amino acid residue in the polypeptide chain, or inserting a new amino acid residue at any position in the polypeptide chain.

[0071] For the enzyme obtained by mutating the amino acid sequence, for example, the known site-specific mutagenesis method can be used to implement the gene encoding the protocatechuate 5-position oxidase of the present technology. As a known site-specific mutagenesis method, for example, a primer for mutation containing the nucleotide mutation to be introduced can be used to implement it by any method such as the inverse PCR method and the annealing method.

[0072] The aforementioned mutation primers can be designed in the following manner: annealing is performed to a region of the target gene containing a nucleotide sequence encoding an amino acid residue to be mutated, and the nucleotide sequence (codon) containing a nucleotide sequence encoding the mutated amino acid residue is replaced by a nucleotide sequence (codon) encoding the amino acid residue to be mutated.

[0073] In the present technology, when gallic acid is produced from protocatechuic acid by fermentation using a microbial strain, gallic acid is recovered from the fermentation solution, and when gallic acid is produced from protocatechuic acid using protocatechuic acid 5-oxidase, gallic acid is recovered from the reaction solution.

[0074] The method for recovering gallic acid is not particularly limited. For example, the pH of the fermentation liquid or reaction liquid is adjusted to below 4.0 by an acid such as sulfuric acid or hydrochloric acid. Insoluble proteins produced by the pH adjustment are removed by methods such as centrifugation or filtration. Gallic acid crystals are obtained by cooling the removed liquid to below 4°C while stirring it appropriately, and the gallic acid crystals can be more appropriately recovered by centrifugation or filtration. In addition, instead of adjusting the pH to below 4.0 as mentioned above, the organic solvent layer is recovered and evaporated after mixing the fermentation liquid or reaction liquid with an organic solvent such as benzene, toluene, dichloromethane, chloroform, diethyl ether, ethyl acetate. This also allows for the appropriate recovery of gallic acid crystals.

[0075] Example

[0076] Hereinafter, the present technology will be described in more detail using examples. It should be noted that the present technology is not limited in any way to the contents of the examples shown below.

[0077] Example 1 Preparation and Activity Determination of Protocatechuate 5-Oxidase

[0078] (1) Construction of plasmid for expression of protocatechuate 5-oxidase

[0079] <1> Construction of pRSF-HgPobA plasmid

[0080] Based on the gene sequence information of PobA (HgPobA) from Helemonella gracilis (SEQ ID NO: 1), a polynucleotide encoding PobA from Helemonella gracilis was artificially synthesized. Using this polynucleotide as a template, primers 1 and 2 having the base sequences shown below, KOD PCR Master Mix (Toyobo Co., Ltd.) was used to obtain polynucleotide fragments by PCR.

[0081] Primer 1 (SEQ ID NO: 2)

[0082] (5'-atcaccacagccaggatccgaattcAAGCTATCCCACAAGAACTCA GG-3')

[0083] Primer 2 (SEQ ID NO: 3)

[0084] (5'-ttaagcattatgcggccgcaagcttAGGCACCGAAGTCGAGGG-3')

[0085] The polynucleotide fragment and pRSFDuet-1 were digested with restriction endonucleases EcoRI and HindIII, respectively, and then reacted and ligated using Gibson Assembly Master Mix (New England Biolabs) to obtain pRSF-HgPobA plasmid.

[0086] <2> Construction of pRSF-HyPobA plasmid

[0087] The target pRSF-PoPobA plasmid was obtained by the same procedure based on the gene sequence information of PobA (HyPobA) derived from the genus Hydrogenophage (SEQ ID NO: 4). The base sequences of primers 3 and 4 used are shown below.

[0088] Primer 3 (SEQ ID NO: 5)

[0089] (5'-atcaccacagccaggatccgaattcGCGGACCCAGGTCGGCAT-3')

[0090] Primer 4 (SEQ ID NO: 6)

[0091] (5'-ttaagcattatgcggccgcaagcTTATTCAATCGGAAGTCCAGTGA AATTCTCTGAAAACG-3')

[0092] <3> Construction of pRSF-PoPobA plasmid

[0093] The target pRSF-PoPobA plasmid was obtained by the same procedure based on the gene sequence information of PobA (PoPobA) derived from the genus Polarmonas (SEQ ID NO: 7). The base sequences of primers 5 and 6 used are shown below.

[0094] Primer 5 (SEQ ID NO: 8)

[0095] (5'-atcaccacagccaggatccgaattcGCGCACCCAGGTTGGTATC-3')

[0096] Primer 6 (SEQ ID NO: 9)

[0097] (5'-ttaagcattatgcggccgcaagcTTAGTCGATGGGCAAGCC-3')

[0098] <4> Construction of pRSF-PaPobA plasmid (Comparison object 1)

[0099] Based on the gene sequence information of PobA (PaPobA) from Pseudomonas aeruginosa (SEQ ID NO: 14), a polynucleotide encoding PobA from Pseudomonas aeruginosa was artificially synthesized. Using this polynucleotide as a template, a primer set having the base sequence shown below, KOD PCR Master Mix (Toyobo Co., Ltd.) was used to obtain polynucleotide fragments by PCR.

[0100] Primer set for construction of pRSF-PaPobA plasmid

[0101] 5'-TTCGTCGGTCTGCCGTATGAGG-3' (SEQ ID NO: 15)

[0102] 5'-GTTTTCCGCAATTGTCGCCAG-3' (SEQ ID NO: 16)

[0103] The polynucleotide fragment and pRSFDuet-1 were digested with restriction endonucleases EcoRI and HindIII, respectively, and then reacted and ligated using Gibson Assembly Master Mix (New England Biolabs) to obtain pRSF-PaPobA plasmid.

[0104] <5> Construction of pRSF-CgPobA plasmid (Comparison object 2)

[0105] Based on the gene sequence information of PobA (CgPobA) from Corynebacterium glutamicum (SEQ ID NO: 17), a polynucleotide encoding PobA from Corynebacterium glutamicum was artificially synthesized. PCR Master Mix (Toyobo Co., Ltd.) was used to obtain polynucleotide fragments by PCR.

[0106] Primer set for construction of pRSF-CgPobA plasmid

[0107] 5'-atcaccacagccaggatccgaattcAAATCACGTACCTGTGGC-3' (serial number 18)

[0108] 5'-ttaagcattatgcggccgcaagcTTACACTTCAAACCGCGG-3' (SEQ ID NO: 19)

[0109] The polynucleotide fragment and pRSFDuet-1 were digested with restriction endonucleases EcoRI and HindIII, respectively, and then reacted and ligated using Gibson Assembly Master Mix (New England Biolabs) to obtain pRSF-CgPobA plasmid.

[0110] (2) Construction of plasmid for expression of mutant protein of protocatechuate 5-oxidase

[0111] <1> Construction of pRSF-HgPobA-T302A / Y393F plasmid

[0112] The aforementioned pRSF-HgPobA plasmid was used as a template and the Y393F introduction primer set and KOD PCR Master Mix (Toyobo Co., Ltd.) was used to obtain polynucleotide fragments by PCR.

[0113] Y393F introduction primer set

[0114] 5'-CTGTGGCGGAAAACTACGTGGGACTGC-3' (SEQ ID NO: 10)

[0115] 5'-GCAGTCCCACGAAGTTTTCCGCCACAG-3' (SEQ ID NO: 11)

[0116] The obtained polynucleotide fragment was treated with restriction endonuclease DpnI and then ligated into a circular form using T4 Polynucleotide Kinase (Takara Bio Co., Ltd.) and Ligation high Ver.2 (Toyobo Co., Ltd.). The obtained circular polynucleotide was used as pRSF-HgPobA-Y393F plasmid.

[0117] Then, the obtained plasmid pRSF-HgPobA-Y393F was used as a template and the primer set T302A and KOD were introduced with the base sequence shown below. PCR Master Mix (toyobo), to obtain polynucleotide fragments by PCR.

[0118] T302A introduction primer set

[0119] 5'-CATCGTTCCTCCGGCTGGCGCAAAGGG-3' (SEQ ID NO: 12)

[0120] 5'-CCCTTTGCGCCAGCCGGAGGAACGATG-3' (SEQ ID NO: 13)

[0121] The obtained polynucleotide fragment was treated with restriction endonuclease DpnI, and then ligated into a circle using T4 polynucleotide kinase (Takara Bio Co., Ltd.) and Ligation high Ver.2 (Toyobo Co., Ltd.). The obtained circular polynucleotide was used as pRSF-HgPobA-T302A / Y393F plasmid (pRSF-HgPobA-TY plasmid).

[0122] (3) Production of protocatechuate 5-oxidase

[0123] The aforementioned pRSF-HgPobA plasmid, pRSF-HyPobA plasmid, pRSF-PoPobA plasmid, pRSF-HgPobA-TY plasmid, and pRSF-PaPobA plasmid were respectively introduced into Escherichia coli BL21 Star (DE3) strain (Merck KGaA), and the Escherichia coli strain introduced with the target plasmid was selected by culturing in an agar medium containing kanamycin.

[0124] The obtained Escherichia coli strains were added to test tubes containing 5 mL of liquid LB medium, respectively, and cultured overnight at 37° C. and 180 rpm to prepare pre-culture solutions.

[0125] 100 mL of LB liquid medium was prepared and sterilized in a 500 mL baffled flask, and kanamycin was added to give a final concentration of 30 μg / L. The pre-culture solution was added to give a final concentration of 1%, and shake culture was performed at 37°C and 120 rpm.

[0126] When the bacterial turbidity (OD600) reached 0.6, IPTG was added to a final concentration of 0.1 mM, and the culture was cultured at 20°C and 120 rpm for 12 to 18 hours with shaking to induce the production of protocatechuate 5-oxidase.

[0127] (4) Purification of protocatechuate 5-oxidase

[0128] After protein induction, the aforementioned E. coli strain was collected, suspended in a buffer solution (20 mM Tris-HCl buffer solution (pH 7.9)), and ultrasonically disrupted. The disrupted solution was then centrifuged (8000 rpm, 10 minutes, 4°C) to recover the supernatant (cell-free extract).

[0129] The cell-free extract was applied to a HisTrap HP chromatography column (GE healthcare) equilibrated with an equilibration buffer (20 mM Tris-HCl buffer (pH 7.9), 20 mM imidazole), and the fraction eluted with an eluent (20 mM Tris-HCl buffer (pH 7.9), 500 mM imidazole) was recovered.

[0130] Then, the eluted fractions were supplied to Ultrafiltration centrifuge tube ( Ultra Centrifugal Filters) 0.5 mL 10k (Merck Milipore) were washed with buffer to remove imidazole from the protein solution.

[0131] The protein concentration of the purified enzyme solution was measured using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, USA), and diluted to 1 mg / mL using buffer to prepare purified enzyme solutions of protocatechuate 5-oxidase of HgPobA, HyPobA, PoPobA, HgPobA-TY and PaPobA.

[0132] (5) Confirmation of amino acid sequence identity between protocatechuate 5-oxidase and existing PobA

[0133] Using the protocatechuate 5-oxidases described above, HgPobA (SEQ ID NO: 20), HyPobA (SEQ ID NO: 21), and PoPobA (SEQ ID NO: 22), the amino acid sequence identity with p-hydroxybenzoate hydroxylase (PobA) of Pseudomonas and Corynebacterium was confirmed.

[0134] The results are shown in the following Table 1. It was confirmed that the amino acid sequence identity of the protocatechuate 5-oxidase derived from the aforementioned microorganisms according to the present technology and the p-hydroxybenzoate hydroxylase of Pseudomonas and Corynebacterium was 60% or less.

[0135] [Table 1]

[0136]

[0137] The amino acid sequence identity between the protocatechuate 5-oxidase involved in the present technology and the existing PobA

[0138] Amino acid sequence information of PobA (PaPobA) from Pseudomonas aeruginosa (SEQ ID NO: 28)

[0139] Amino acid sequence information of PobA (CgPobA) from Corynebacterium glutamicum (SEQ ID NO: 29)

[0140] Amino acid sequence information of PobA from Corynebacterium ammoniagenes (SEQ ID NO: 30)

[0141] Amino acid sequence information of PobA from Corynebacterium callunae (SEQ ID NO: 31)

[0142] Amino acid sequence information of PobA from Corynebacterium efficiens (SEQ ID NO: 32)

[0143] (6) Determination of protocatechuate 5-oxidase activity

[0144] <1> Reaction kinetics analysis

[0145] Reaction kinetics analysis was performed using the purified enzyme solutions of HgPobA, HgPobA-TY, PoPobA, HyPobA, and PaPobA described above.

[0146] For the enzyme activity measurement, 100 μL of a reaction solution containing 10 μM FAD, 200 μM NADPH, 1 mM pHBA or 2 mM PCA, and 1 μg of a purified enzyme solution in 20 mM Tris-HCl buffer (pH 7.9) was used.

[0147] The reaction was initiated by adding pHBA or PCA, and the absorbance at 340 nm was measured at 25° C. for 5 minutes using a spectrophotometer U3900 (HITACHI).

[0148] The results are shown in Table 2. When PCA was used as a substrate, HgPobA, PoPobA, and HyPobA showed higher k values ​​than PaPobA. cat The value was confirmed to be effective in efficiently producing gallic acid from PCA.

[0149] In addition, HgPobA-TY, which was obtained by introducing two mutations into HgPobA, showed a k cat The value is 3.6 times larger than that of PaPobA, and K m The value was also one-third or less, indicating that the excellent properties of PCA hydroxylase that hydroxylates PCA can be further improved by introducing mutations.

[0150] k of PoPobA and HyPobA against pHBA cat The value is one tenth to one twentieth of that of PaPobA. cat Since the value is 6 to 12 times larger than that of PaPobA, the reaction of generating gallic acid from PCA can proceed preferentially over the reaction of generating PCA from pHBA, and the improvement of the yield of gallic acid in the reaction solution can be expected.

[0151] [Table 2]

[0152]

[0153] Reaction kinetics analysis of a novel protocatechuate 5-hydroxylase

[0154] <2>Analysis of gallic acid generating activity

[0155] The gallic acid production activity was analyzed using the purified enzyme solutions of HyPobA, PoPobA, HgPobA-TY, and PaPobA described above.

[0156] 10 μM FAD, 0.5 mM NADPH, 2 mM PCA, and 10 μg of purified enzyme solution were added to 20 mM Tris-HCl buffer (pH 7.9) to prepare 100 μL of reaction solution, which was reacted at 30° C. for 30 minutes, and then the concentration of generated gallic acid was measured using HPLC (1200 Infinity Series: Hewlett Packer).

[0157] As the results, the activity of each enzyme in producing gallic acid from PCA was expressed as a relative value when the activity of PaPobA in producing gallic acid from PCA was set to 1, and the results are shown in Table 2.

[0158] As shown, HgPobA-TY was able to produce 8 times more gallic acid, and HyPobA and PoPobA were able to produce 20 times more gallic acid, compared to the case of using PaPobA, a well-known enzyme known to have high PCA hydroxylation activity.

[0159] Example 2 Production of Gallic Acid Based on Fermentation Using Microbial Strain

[0160] (1) Preparation of a Corynebacterium strain into which a gene encoding protocatechuate 5-position oxidase is introduced

[0161] Genes encoding HyPobA, PoPobA, HgPobA-TY, and CgPobA were introduced into the CT07 strain by the following procedure. The CT07 strain was a strain genetically modified by the following procedure based on Corynebacterium glutamicum ATCC13032, which is known as a strain producing PCA from glucose.

[0162] <1> Construction of CT07 strain

[0163] tkt (cg1774), ppsA (cg0644), aroF fbr The promoters of aroG (cg1129), aroG (cg2391) and qsuABCD operons (cg0501, cg0502, cg0503, cg0504) were replaced with the promoter of the constitutive elongation factor Tu (cg0587) gene that has strong gene expression ability by the method described below.

[0164] A DNA containing the promoter region of the above gene in the gene of Corynebacterium glutamicum ATCC13032 was synthesized and replaced with the promoter of elongation factor Tu.

[0165] For tkt (cg1774), the base sequence of the region from cg1773 to cg1774 registered in public databases such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes) was used. Similarly, for ppsA (cg0644), the base sequence of the region including cg0643 to cg0644 to cg0645 was used, and for aroF fbr (cg1129) used the base sequence of the region containing cg1127~cg1128~cg1129, for aroG (cg2391), the base sequence of the region containing cg2393~cg2392~cg2391 was used, and for the qsuABCD operon (cg0501, cg0502, cg0503, cg0504), the base sequence of the region containing cg0500~cg0501 was used.

[0166] The following DNA was synthesized: the DNA contained a sequence obtained by replacing the promoter region of each of the above genes with the promoter of the gene of elongation factor Tu, and SbfI recognition sequences were given to both ends of the DNA. Each of the obtained DNAs was digested with SbfI and inserted into the SbfI site of pHG0 as shown below to construct a plasmid for homologous recombination. The plasmid was introduced into Corynebacterium glutamicum ATCC13032 to construct a strain in which the promoter of each gene was replaced with the promoter of elongation factor Tu.

[0167] In addition, for the aroF gene, a P155L mutation was introduced during gene synthesis to construct aroF fbr . In addition, the base sequence of the cg2629-cg2630-cg2631-cg2633 region containing the protocatechuic acid decomposition gene pcaHG (cg2631, cg2630) was obtained from the aforementioned public database, and a base sequence that only lacked the pcaHG (cg2631, cg2630) portion was designed from the base sequence, and a gene with SbfI sequence attached to both ends of the base sequence was synthesized. The synthesized gene was digested with SbfI and inserted into the SbfI site of pHG0 constructed below, thereby constructing a plasmid for homologous recombination. The plasmid was introduced into the Corynebacterium glutamicum ATCC13032 strain into which the promoter of the aforementioned elongation factor Tu had been introduced, and homologous recombination was carried out with the gene of pcaHG on the chromosome, thereby deleting the pcaHG gene on the chromosome of the Corynebacterium glutamicum ATCC13032 strain. The strain thus obtained was used as the CT07 strain.

[0168] <2> Construction of genome editing vector

[0169] The levansucrase gene (sacB) derived from Bacillus subtilis and artificially synthesized by Eurofins Genomics was introduced into the FspI site of the commercially available vector pHSG298 (Takara Bio Co., Ltd.) to construct the vector pHG0 for producing microbial lethal compounds on a sucrose medium.

[0170] In order to introduce the target gene into the CT07 strain by homologous recombination, the polynucleotide fragment of cg0658 was prepared from the genome of Corynebacterium glutamicum ATCC13032 by PCR using the following primers with a SacI sequence and a primer with an XbaI sequence for the cg0658 gene (SEQ ID NO: 33), and the polynucleotide fragment was treated with restriction endonucleases using SacI and XbaI. Then, the polynucleotide fragment of cg0658 was introduced into the SacI and XbaI sites of the vector pHG0 that had been treated with restriction endonucleases using SacI and XbaI. The vector was named pHG0_0658.

[0171] SacI sequence introduction cg0658 primer

[0172] 5'-GAGCTCATGCGTTTTGATCTCCATTC-3' (SEQ ID NO: 34)

[0173] XbaI sequence introduced into cg0658 primer

[0174] 5'-GAGCTCCTCTAGATTAGTTAGGGACCGTATGCG-3' (SEQ ID NO: 35)

[0175] <3>Construction of enzyme expression unit

[0176] In order to express the genes of HyPobA, PoPobA, HgPobA-TY, and CgPobA in Corynebacterium glutamicum, a kanamycin resistance gene promoter derived from pHSG298 (SEQ ID NO: 36) was used as a known promoter.

[0177] Polynucleotide fragments having sequences obtained by connecting the kanamycin resistance gene promoter (SEQ ID NO: 36) with HypobA (SEQ ID NO: 4), PopobA (SEQ ID NO: 7), HgPobA_TY (SEQ ID NO: 37) or CgPobA (SEQ ID NO: 17) were obtained by artificial gene synthesis. The four polynucleotide fragments obtained were each used as a template, and a primer set with an XbaI sequence attached to the 5' end and a SalI sequence attached to the 3' end was used to prepare the four polynucleotide fragments by PCR. The combination of template and primer at this time is as follows.

[0178] Using the polynucleotide fragment containing HyPobA connected to the kanamycin resistance gene promoter as a template, the XbaI sequence was used to introduce the kanamycin resistance gene promoter amplification Fw primer (sequence number 38) and the HyPobASalI sequence was used to introduce the Rv primer (sequence number 39), and PCR amplification was performed to obtain the polynucleotide fragment.

[0179] XbaI sequence was introduced into the promoter of kanamycin resistance gene and amplified using Fw primer

[0180] 5'-GAGCTCATGCGCACCCAGGTTGGTATC-3' (SEQ ID NO: 38)

[0181] HyPobASalI sequence introduced into Rv primer

[0182] 5'-GTCGACTTAGTCGATGGGCAAGCCCG-3' (SEQ ID NO: 39)

[0183] Using a polynucleotide fragment containing PoPobA connected to the kanamycin resistance gene promoter as a template, the XbaI sequence was used to introduce the kanamycin resistance gene promoter amplification Fw primer (sequence number 38) and the PoPobASalI sequence was used to introduce the Rv primer (sequence number 40), and PCR amplification was performed to obtain a polynucleotide fragment.

[0184] PoPobASalI sequence introduced into Rv primer

[0185] 5'-GTCGACTTATTCAATCGGAAGTCCAG-3' (SEQ ID NO: 40)

[0186] Using the polynucleotide fragment containing HgPobA-TY connected to the kanamycin resistance gene promoter as a template, the XbaI sequence was used to introduce the kanamycin resistance gene promoter amplification Fw primer (sequence number 38) and the HgPobA_SalI sequence was used to introduce the Rv primer (sequence number 41), and PCR amplification was performed to obtain the polynucleotide fragment.

[0187] HgPobA_SalI sequence introduced into Rv primer

[0188] 5'-GTCGACTTAGGCACCGAAGTCGAGGG-3' (SEQ ID NO: 41)

[0189] Using the polynucleotide fragment containing CgPobA connected to the kanamycin resistance gene promoter as a template, the XbaI sequence was used to introduce the kanamycin resistance gene promoter amplification Fw primer (sequence number 38) and the CgPobA_SalI sequence was used to introduce the Rv primer (sequence number 42), and PCR amplification was performed to obtain the polynucleotide fragment.

[0190] CgPobA_SalI sequence introduced into Rv primer

[0191] 5'-GTCGACTTACACTTCAAACCGCGGG-3' (SEQ ID NO: 42)

[0192] The four polynucleotide fragments obtained by PCR were digested with XbaI and SalI, and the digested DNA fragments involving protocatechuate 5-oxidase were recovered using a DNA recovery column (Gel / PCR Extraction Kit manufactured by Nippon Genetics).

[0193] Similarly, the genome editing vector pHG0_0658 was digested with XbaI and SalI, and the digested pHG0_0658 was recovered using a DNA recovery column (Gel / PCR Extraction Kit manufactured by Nippon Genetics). The digested pHG0_0658 was ligated with the digested DNA fragments involving the protocatechuate 5-position oxidase described above using Gibson Assembly Master Mix (New England Biolabs) to obtain the following four vectors into which the enzyme expression unit was introduced.

[0194] pHG0_0658_Pkm_HyPobA

[0195] pHG0_0658_Pkm_PoPobA

[0196] pHG0_0658_Pkm_HgPobA-TY

[0197] pHG0_0658_Pkm_CgPobA

[0198] <4> Genomic introduction of enzyme expression unit

[0199] Electroporation was used to introduce the above-mentioned 4 kinds of plasmids into Corynebacterium glutamicum CT07 strains respectively, and the target gene was introduced by homologous recombination with cg0658 gene as the homologous recombination region, and the microbial strain obtained by culturing in LB medium containing 20 μg / L of kanamycin was obtained. These strains are gene-introduced microbial strains of Corynebacterium glutamicum CT07 strains with only 1 copy of each PobA gene per chromosome. They were named as CG07-HY strain, CG07-PO strain, CG07-HG strain and CG07-CG strain respectively.

[0200] <5>Cultivation test

[0201] The CG07-HY strain, CG07-PO strain, CG07-HG strain, and CG07-CG strain were cultured in LB liquid medium containing 20 μg / L of kanamycin at 30°C overnight with shaking to prepare a pre-culture solution. In addition, the CT07 strain without the introduction of the plasmid was cultured in LB liquid medium without antibiotics at 30°C overnight with shaking to prepare a pre-culture solution.

[0202] These pre-culture solutions were inoculated into CGXII medium (pH 7.0) supplemented with 5% glucose at 1%, and cultured with shaking at 30° C. for 72 hours. The culture solution was appropriately diluted, and the concentrations of produced protocatechuic acid and gallic acid were measured using HPLC (1200 Infinity series: Hewlett Packer).

[0203] The results of measuring the concentrations of protocatechuic acid and gallic acid in the culture medium of the CG07-HY strain, CG07-PO strain, CG07-HG strain, CG07-CG strain, and CT07 strain are shown in Table 3 below.

[0204] The CT07 strain produced 7.3 g / L of protocatechuic acid, but did not express PobA, so it did not produce gallic acid (GA). On the other hand, the CG07-CG strain expressing CgPobA produced 7.5 g / L of protocatechuic acid and 0.0001 g / L of gallic acid. In addition, CG07-HG expressing HgPobA-TY produced 5.5 g / L of protocatechuic acid and 0.65 g / L of gallic acid. The CG07-HY strain that retained HyPobA produced 7.3 g / L of protocatechuic acid and 0.03 g / L of gallic acid. The CG07-PO strain that retained PoPobA produced 7.3 g / L of protocatechuic acid and 0.0004 g / L of gallic acid.

[0205] The production ratio of gallic acid when CG07-CG having the known enzyme CgPobA is set to 1 is shown in the following table. By using HgPobA-TY, 6500 times more gallic acid (GA) can be produced than before.

[0206] [Table 3]

[0207] Strain name PCA(g / L) GA(g / L) GA (relative value) CT07 strain 7.3 0.00 0 CG07-CG strain 7.5 0.0001 1 CG07-HG strain 5.5 0.65 6500 CG07-HY strain 7.3 0.03 300 CG07-PO strain 7.2 0.0004 4

[0208] Among the natural enzymes developed so far, the enzymes with the largest production of gallic acid by fermentation are CgPobA or PaPobA, and it is reported that the production of gallic acid is equivalent when using the two. By using the protocatechuate 5-oxidase derived from a microorganism of the Comamonadaceae family involved in the present invention, it can be expected that the yield of gallic acid can be greatly improved compared to when CgPobA is used. In addition, by modifying the sequence of the protocatechuate 5-oxidase involved in the present invention, the yield of gallic acid in the fermentation broth can be further improved.

[0209] Furthermore, since gallic acid can be produced using a culture medium containing glucose as a substrate, it can be expected that the production cost of gallic acid can be reduced.

[0210] In Example 2, the use of HgPobA-TY enabled the production of 6500 times more gallic acid than the technique using the CG07-CG strain introduced with CgPobA. This indicates that mutations known so far for increasing gallic acid production by introduction into PobA are also effective for HgPobA.

[0211] [Low pH culture test]

[0212] The CG07-HY strain, CG07-PO strain and CG07-HG strain were cultured overnight at 30°C with shaking in an LB liquid medium containing 20 μg / L of kanamycin to prepare a pre-culture solution. These pre-culture solutions were inoculated into a CGXII medium (pH 4.0) supplemented with 5% glucose in a 1% manner and cultured at 30°C with shaking for 72 hours. The culture solution was appropriately diluted, and the concentrations of the generated protocatechuic acid and gallic acid were measured using HPLC (1200 Infinity Series: Hewlett Packerd). Thus, for the CG07-HY strain, CG07-PO strain and CG07-HG strain, it was confirmed that gallic acid was also generated under the culture condition of pH 4.0. By setting this culture condition, the use of acids such as sulfuric acid and hydrochloric acid can be reduced, and the risk of contamination of strains other than the microbial strains of the present technology can also be reduced.

[0213] [High temperature culture test]

[0214] The CG07-HY strain, CG07-PO strain and CG07-HG strain were cultured overnight at 30°C with shaking in an LB liquid medium containing 20 μg / L of kanamycin to prepare a pre-culture solution. These pre-culture solutions were inoculated into a CGXII medium (pH 7.0) supplemented with 5% glucose in a 1% manner and cultured with shaking at 40°C for 72 hours. The culture solution was appropriately diluted, and the concentrations of the generated protocatechuic acid and gallic acid were measured using HPLC (1200 Infinity Series: Hewlett Packerd). Thus, for the CG07-HY strain, CG07-PO strain and CG07-HG strain, it was confirmed that gallic acid was also generated under the culture condition of 40°C. By setting the culture conditions to this level, it can be expected that the culture efficiency will be improved and the production efficiency of gallic acid will be improved.

[0215] Example 3 Gallic acid production based on scale-up

[0216] The CG07-HG strain expressing HgPobA-TY was cultured in an LB liquid medium containing 20 μg / L of kanamycin at 30° C. with shaking overnight to prepare a preculture solution.

[0217] In a fermenter (Marubishi Bioengineering Co., Ltd., product name MDL-8C), CGXII medium with 5% glucose added was prepared, the aforementioned pre-culture solution was inoculated, and cultured at 30°C, 56 hours, and pH 6.8. As a result, 0.7 g / L of protocatechuic acid and 15.1 g / L of gallic acid were produced at the 56th hour of culture. Judging from this production level, the developed fermentation production system can be applied to actual production. In addition, by optimizing the culture conditions of the fermentation production system, it is expected to further increase the production of gallic acid.

[0218] After the culture solution is sterilized by heat, the bacterial cells are removed by centrifugation or filtration. The culture filtrate is adjusted to a pH of 4.0 or less by sulfuric acid or the like. Insoluble proteins generated in the process are removed by centrifugation, and the obtained liquid is cooled while being appropriately stirred to obtain crystals of gallic acid. The crystals are recovered by centrifugation.

[0219] [Sequence Listing]

[0220] <110> National University Corporation University of Tsukuba

[0221] <120> Microbial strain, protein, and method for producing gallic acid using the microbial strain or protein

[0222] <130> FPCT1388

[0223] <160> 42

[0224] <210> Sequence Number 1

[0225] <211> Length 1209

[0226] <212> Type DNA

[0227] <213> Species Helemonella gracilis

[0228] <400> Sequence 1

[0229] ATG

[0230] <210> Sequence number 2

[0231] <211> Length 48

[0232] <212> Type DNA

[0233] <213> Species Helemonella gracilis

[0234] <400> Sequence 2

[0235] ATCACCACAGCCAGGATCCGAATTCAAGCTATCCCACAAGAACTCAGG

[0236] <210> Sequence number 3

[0237] <211> Length 43

[0238] <212> Type DNA

[0239] <213> Species Helemonella gracilis

[0240] <400> Sequence 3

[0241] TTAAGCATTATGCGGCCGCAAGCTTAGGCACCGAAGTCGAGGG

[0242] <210> Sequence Number 4

[0243] <211> Length 1176

[0244] <212> Type DNA

[0245] <213> Species Hydrogenophage

[0246] <400> Sequence 4

[0247] ATG

[0248] <210> Sequence number 5

[0249] <211> Length 43

[0250] <212> Type DNA

[0251] <213> Species Hydrogenophage

[0252] <400> Sequence 5

[0253] ATCACCACAGCCAGGATCCGAATTCGCGGACCCAGGTCGGCAT

[0254] <210> Sequence Number 6

[0255] <211> Length 61

[0256] <212> Type DNA

[0257] <213> Species Hydrogenophage

[0258] <400> Sequence 6

[0259] TTAAGCATTATGCGGCCGCAAGCTTATTCAATCGGAAGTCCAGTGAAATTCTCTGAAAACG

[0260] <210> Sequence Number 7

[0261] <211> Length 1176

[0262] <212> Type DNA

[0263] <213> Species: Polarmonas

[0264] <400> Sequence 7

[0265]

[0266] <210> Sequence number 8

[0267] <211> Length 44

[0268] <212> Type DNA

[0269] <213> Species: Polarmonas

[0270] <400> Sequence 8

[0271] ATCACCACAGCCAGGATCCGAATTCGCGCACCCAGGTTGGTATC

[0272] <210> Sequence Number 9

[0273] <211> Length 41

[0274] <212> Type DNA

[0275] <213> Species: Polarmonas

[0276] <400> Sequence 9

[0277] TTAAGCATTATGCGGCCGCAAGCTTAGTCGATGGGCAAGCC

[0278] <210> Sequence number 10

[0279] <211> Length 27

[0280] <212> Type DNA

[0281] <213> Species Helemonella gracilis

[0282] <400> Sequence 10

[0283] CTGTGGCGGAAAACTACGTGGGACTGC

[0284] <210> Sequence Number 11

[0285] <211> 27

[0286] <212> Type DNA

[0287] <213> Species Helemonella gracilis

[0288] <400> Sequence 11

[0289] GCAGTCCCACGAAGTTTTCCGCCACAG

[0290] <210> Sequence Number 12

[0291] <211> Length 27

[0292] <212> Type DNA

[0293] <213> Species Helemonella gracilis

[0294] <400> Sequence 12

[0295] CATCGTTCCTCCGGCTGGCGCAAAGGG

[0296] <210> Sequence Number 13

[0297] <211> Length 27

[0298] <212> Type DNA

[0299] <213> Species Helemonella gracilis

[0300] <400> Sequence 13

[0301] CCCTTTGCGCCAGCCGGAGGAACGATG

[0302] <210> Sequence Number 14

[0303] <211> Length 1185

[0304] <212> Type DNA

[0305] <213> Species Pseudomonas aeruginosa

[0306] <400> Sequence 14

[0307] ATG

[0308] <210> Sequence Number 15

[0309] <211> Length 22

[0310] <212> Type DNA

[0311] <213> Species Pseudomonas aeruginosa

[0312] <400> Sequence 15

[0313] TTCGTCGGTCTGCCGTATGAGG

[0314] <210> Sequence Number 16

[0315] <211> Length 21

[0316] <212> Type DNA

[0317] <213> Species Pseudomonas aeruginosa

[0318] <400> Sequence 16

[0319] GTTTTCCGCAATTGTCGCCAG

[0320] <210> Sequence Number 17

[0321] <211> Length 1188

[0322] <212> Type DNA

[0323] <213> Species Corynebacterium glutamicum

[0324] <400> Sequence 17

[0325] ATG

[0326] <210> Sequence Number 18

[0327] <211> Length 43

[0328] <212> Type DNA

[0329] <213> Species Corynebacterium glutamicum

[0330] <400> Sequence 18

[0331] ATCACCACAGCCAGGATCCGAATTCAAATCACGTACCTGTGGC

[0332] <210> Sequence Number 19

[0333] <211> Length 41

[0334] <212> Type DNA

[0335] <213> Species Corynebacterium glutamicum

[0336] <400> Sequence 19

[0337] TTAAGCATTATGCGGCCGCAAGCTTACACTTCAAACCGCGG

[0338] <210> Sequence number 20

[0339] <211> Length 402

[0340] <212> Type PRT

[0341] <213> Species Helemonella gracilis

[0342] <400> Sequence 20MSYPTRTQVAIIGAGPSGLMLGALLHKAGIDTVVLERQSGDYVLGRIRAGVLEQVTMDLMDEIGVGARMHQEGLVHGGFDMLFQGQRHRIDMNRLTG GQNVMVYGQTEVTRDLMDARQQAGLTTIYEAANVAIHDFGSGKPRVTYEKDGKTHELQCDFIAGCDGFHGVCRDTVKKSASASAIREYEKVYNFGWLGLLSD TPPVHHELIYVNSERGFALCSQRSATRSRYYLQVPLTDKVEQWSDQAFWDELKLRLDPQAREHLVTGPSIEKSIAPLRSFVTEPLRFGRLFLAGDAGHIVPPAGAKGLNLAATDVKYLSAALTEFYQGKSEAGINHYSERCLKRIWRAERFSWWFTSLMHRFPENGEIGQKLQEAELDYIVHSETGARSVAENFVGLPLDFGA

[0343] <210> Sequence Number 21

[0344] <211> Length 391

[0345] <212> Type PRT

[0346] <213> Species Hydrogenophage

[0347] <400> Sequence 21

[0348] MRTQVGIIGAGPAGLMLAHMLHLEGIESVIIERSKREHVQNRLRAGVLEQGTVEMMRELGLGERIGQLGLEQHRIDFRFGGESHPIDFHEATGGRSAWVYPQHEVVKDLMAARTAAGAQMLFETPVTAIEGIDGTRPVVRFEKDGVAGELHCDFVVGCDGFRGISRRAIPAREGQVHDRIYPFGWLGILAEAPPATNEVTWGCHEDGFAMMSIRTPSVTRLYLQCEPDENPDHWSDDRIWSALHKRLDVPGLPPINEGRITQKGVTAMRSFLFEPMQHGRLFLAGDAAHIVPPTGAKGLNSAMADVKVLARALEQHYQRGSQDLLARYSKTCLKRMWLVQRFSAGLCTMVHSFAGDSSFVRRLQRADLDYMTGTTAGRLQFSENFTGLPID

[0349] <210>Sequence number 22

[0350] <211>Length 391

[0351] <212>Type PRT

[0352] <213>Species Polaromonas sp.

[0353] <400>Sequence 22

[0354] MRTQVGIVGAGPAGLMLSHMLHLEGIESVIIERASREHVQSRLRAGVLEHGTVDMLRELGLGERIGKIGLEQHAIDFRFGGESHRLDFHQATNGRSAWVYPQHELVTDLMNARMQAGAEILFETPVTRIDGLHTSKPTVHFEQGGEPRELVCDFVVGCDGFRGVCRDAIPDTVLKTYDRVYPFGWLGILAEAPAPTREITWGCHEEGFAMLSIRSPSVTRLYLQCEPDDDPDHWSDDHIWAELHKRLDVPGMPPVNEGKIMQKGVTAMRSFLAEPMQYGKLFLAGDAAHIVPPTGAKGLNSAMADVKVLGRGLAEKYRRGADHLLERYSQICLKRMWLVQRFSAGLCTMVHQFPGDNAFVRRLQRADLDYMTGTAAGRLAFSENFTGLPIE

[0355] <210> Sequence Number 23

[0356] <211> Length 397

[0357] <212> Type PRT

[0358] <213> Species: Lactotrichum

[0359] <400> Sequence 23

[0360] MSTTRTQVAIVGAGPSGLLLGQLLFKAGIDAIIVERQSPDYVLSRIRAGVLEQVTMDLLDEAGVGQRMHKEGLVHTGFDLLFKGARHRIDMDHLTGGKKVIVYGQTEVTRDLMDTRQAEGLPTVYEAGNVQVHDFDSQKPRVTYEKNGQLHTIECDFIAGCDGFHGVCRASAPKNAIKEYEKVYPFGWLGVLSDTPPV HHELIYANSTRGFALCSQRSATRSRYYLQVPTTDKVEQWSDDAFWAELRNRLDPEAREHLVTGPSIEKSIAPLRSFVTEPMRFGRMFLAGDAAHIVPPTGAKGLNLAATDVKYLSTAFIEFYADQTEAGIDAYSERCLRRVWRAERFSWWFTSLMHHFPENGDIGQKFQDAELDYLIHSEAGSRTMAENYVGLPLNFGE

[0361] <210> Sequence Number 24

[0362] <211> Length 391

[0363] <212> Type PRT

[0364] <213> SpeciesRhodoferax sediminis

[0365] <400> Sequence 24

[0366] MRTQVGIVGAGPAGLMLSHMLHLEGIESVIIERASREHVQSRLRAGVLEHGTVDMLKELGLGERISTLGLEQHAIDFRFGGESHRLDFHQATNGRRTWVYPQHEVVTDLMKARMQAGAQILFETSVTRIEGLQTRKPTIHFEQGGEPRELQCDFIVGCDGFRGVCRDAIPDSVLKIYDRVYPFGWLGILAEAPAPTNEITWGCHEDGFAMLSIRSPSVTRLYLQCEPNEDADRWSDGRIWAELHKRLDVPGMPPVNEGKITQKGVTAMRSFLAEPMQYGRLFLAGDAAHIVPPTGAKGLNSSMADVKVLGRGLVEQYKRGVADILDRYSQICLKRMWLVQRFSAGLCTMVHQIPGDNAFVRRLQRADLDYMTGTVAGRLAFSENFTGLPIE

[0367] <210>Sequence number 25

[0368] <211>Length 391

[0369] <212>Type PRT

[0370] <213>Species Xylophilus ampelinus

[0371] <400>Sequence 25

[0372] MRTQVGIVGAGPAGLMLAHLLRREGIDAVVIERAAREHVRTRLRAGVLEQGTVEMLREAGVGGRIDAVGMEMHAIDFRFGGRSHRLDFHEASGGRRAWVYPQHEVVTDLMSACDAGDVPILYEAPVERIEGLEDDRARIVFGQDGAAGEITCDFVAGCDGFRGVSRGSMPAGIARGYDRIYPFGWLGILADAPPASPDVTWGCSDRGFAMMSMRSPTVTRLYLQCEPDEDPDAWSDDRIWSELHRRLDVEGMPSLREGPIRDKGVTAMRSFLSEPMQHGRLFLAGDAAHIVPPTGAKGLNSAMADIKVLAAALVDHYRHGRSDRLATYSERCLRRMWLVQRFSAALCTMVHQFPGQNEFVRRLQRADLDYMTGTHAGRLQFAENFTGLPIE

[0373] <210> Sequence Number 26

[0374] <211> Length 390

[0375] <212> Type PRT

[0376] <213> Species Agrobacterium hanzileri

[0377] <400> Sequence 26

[0378] MRTQVGIVGAGPAGLMLAQILHLQGIDSVLIERASQEHVRSRLRAGVLEQGTVEMMREFGVGERVMKVGLRQRAIDFRFNGESHPIDFESVCGRNTWVYPQHEVVSDLMAARERSGLPVVYDAPVSRIEGLDGGRPVIHYERDGEPLRLECDYVAGCDGFRGISRSWIPADRLKVYDRIYPFGWLGILADAKPAI QDITWGCHEDGFAMSIRSPTVTRLYLQCEPDENPDDWPDDRIWAELHKRLDVPGLPRLEEGRITQKGVTAMRSFLAEPMQYGRLFLAGDAAHIVPPTGAKGLNSALADVKVLARALTEQYRNENGQWLERYSDICLKRMWLVQRFSSGLCTMTHQFRGDEPFVRRLQRADLEYMTMHAPGRLEFSENFTGLPVE

[0379] <210> Sequence Number 27

[0380] <211> Length 374

[0381] <212> Type PRT

[0382] <213> Species Comamonas phosphate

[0383] <400> Sequence 27

[0384] MSHMLHLAGIESVVLERAGREHVRTRLRAGVLEQGVVEMLRELGLGERLDQLALKQSALDFRFDGASRRIDFEAEIGRNVWVYPQHEVVTDLMNARQKAGAAIFYEAPVQRIDGLEGERPVLHFEHQGESCRLECDYVAGCDGFRGVSRSAIPADKLKVFDRIYPFGWLGILAEAPAATNEIVWGCHDGGFAMQSIRSPSVTRLYLQCAPDENPDHWSDDRIWSELHKRLDVEGMPPVNEGRVTQKGVTAMRSFLAEPMQWGRLFLAGDAAHIVPPTGAKGLNSALADIKVLARALVAHYREGADAHLARYSDTCLRRMWLVQRFSAGLCTMTHQFPGQNPFVQRLQRTDLDYMTGTRAGRLQFSENFTGLPVE

[0385] <210>Sequence number 28

[0386] <211>Length 394

[0387] <212>Type PRT

[0388] <213>Pseudomonas aeruginosa

[0389] <400>Sequence 28

[0390] MKTQVAIIGAGPSGLLLGQLLHKAGIDNVILERQTPDYVLGRIRAGVLEQGMVDLLREAGVDRRMARDGLVHEGVEIAFAGQRRRIDLKRLSGGKTVTVYGQTEVTRDLMEAREACGATTVYQAAEVRLHDLQGERPYVTFERDGERLRLDCDYIAGCDGFHGISRQSIPAERLKVFERVYPFGWLGLLADTPPVSHELIYANHPRGFALCSQRSATRSRYYVQVPLSEKVEDWSDERFWTELKARLPSEVAEKLVTGPSLEKSIAPLRSFVVEPMQHGRLFLAGDAAHIVPPTGAKGLNLAASDVSTLYRLLLKAYREGRGELLERYSAICLRRIWKAERFSWWMTSVLHRFPDTDAFSQRIQQTELEYYLGSEAGLATIAENYVGLPYEEIE

[0391] <210> Sequence Number 29

[0392] <211> Length 395

[0393] <212> Type PRT

[0394] <213> Species Corynebacterium glutamicum

[0395] <400> Sequence 29

[0396] MNHVPVAIIGAGPAGLTLAHLLHLQGVESIVFESRTRKDVEETVRAGILEQGTLNLMRETGVGARMEAEADHDEAIDISINNERTRIPLTELTGHKVAIYPQHEYLKDFIAKRIEDGGELLFTTTVDSVENYEGDLAKVTYTEADGSSTTITADYVIAADGSNSPYRKLITEDGGVRARHEYPYAWFGILVEAPKTQ KELIYATHPEGFALISTRTDEIQRYYLQCNPDDTPDMWPDDRIWEQLHLRADSPGITVSEGRIFDKAVLRFRSAVTEPMQKGRLFLAGDAAHTVPPTGAKGLNLAVADVSVLAPALVRALKKKDTGLLDSYTSLAVPRIWKAQHFSYWMSSMLHAVPGEDHFATQRRFAELRSVLESQSGQRYLAEQYVGRDLPRFEV

[0397] <210> Sequence number 30

[0398] <211> Length 408

[0399] <212> Type PRT

[0400] <213> Species Corynebacterium ammoniagenes

[0401] <400> Sequence 30

[0402] MGTPTNSDSSHTPVAIIGAGPAGLMLSHLLHLEGVESVVIEKQSREEVESTVRAGILEQGTIDLLRKTGVGERLDREAEIDEGISISIAGESHRIDFAKYTGKQVAVYPQHEVLIDLIAKRLSDDGELWFDTEVVSIADHESDNPKVHYRTADGTEGVLSADFVVGADGSKSLARKLITDDGGLRMKHEYPFAWFGIMVNAPQTAPEVIYATHPEGFALISTRSENVQRYYLQCNPDDTPDMWSDDRIWEQLHLRADSDTVTVSEGEITDKAVLRFRSAVTDPMQRGRLFIAGDAAHTVPPTGAKGLNLAMADVCALAPSLVRAVKKSDTSLLDTYSERALPRVWKTQHFSYWMSSMLHSVPAENEMSTLFQTNRRLAELSTVVTSDAGRQLLAQQFVGWDFPSIEAL

[0403] <210>Sequence number 31

[0404] <211>Length 404

[0405] <212>Type PRT

[0406] <213>Species Phaeocytophaga scopiformis

[0407] <400>Sequence 31

[0408] MNHIPVAIIGAGPAGLTLAHLLHLQGIESIVFEKRTRKEVEETVRAGILEQGTLNLMRETGVGARMEAEADHDEAIDISINGERIRIPLTEITGHKVAIYPQHEYLKDFIAKRLEDGGELLFSTTVDAVEGTGGTGGTEGFDNDKVKVVYTEADGSSTTISADYVIAADGSSSPYRKIITEGSGVRARHEYPYAWFGILVEAAKTQKEVIYATHPEGFALISTRTDTVQRYYLQCDPNDTPDMWSDDRIWEQLHLRADSPGITVSEGKIFDKAVLRFRSAVTEPMQKGRLFLAGDAAHTVPPTGAKGLNLAVADVSVLAPGLVRALKKKDSALLDDYTKLALPRVWKAQHFSYWMSSMLHAVPDEDYFATQRRFAELHSVLGSEAGQRYLAEQFVGRDLPRFEV

[0409] <210>Sequence number 32

[0410] <211>Length 399

[0411] <212>Type PRT

[0412] <213>Species Corynebacterium efficiens

[0413] <400>Sequence 32

[0414] MNHVPVAIIGAGPAGLTLAHLLHLQGIESVVFESRSRQDVEETVRAGILEQGTLNLMRETGVGERMEREADHDEAIDIAINNENIRIPLTELTGHKVAIYPQHEYLKDFIAKRIEDGGELLFETTVDSVENFEGDAQENKATITYTGVDGTTRQLTADYVVAADGSNSPYRKIITDDGGIRARHEYPYAWFGILVEAPKTQKEVIYATHPEGFALISTRTDTVQRYYLQCDPNDTPEMWSDDRIWEQLHLRADSPGITVSEGKIFDKAVLRFRSAVTEPMQKGRLFIAGDAAHTVPPTGAKGLNLAVADVSVLAPGLTRAIKRNDTGLLDDYTRLTVPRIWKAQHFSYWMSSMLHAVPGEDHFATQRRFAELRSVLESEAGQRYLAEQFVGRDLPRFDY

[0415] <210>Sequence number 33

[0416] <211>Length 2241

[0417] <212>Type DNA

[0418] <213>Species Corynebacterium glutamicum

[0419] <400>Sequence 33

[0420]

[0421] <210> Serial Number 34

[0422] <211> Length 26

[0423] <212> Type DNA

[0424] <213> Species Corynebacterium glutamicum

[0425] <400> Sequence 34

[0426] GAGCTCATGCGTTTTGATCTCCATTC

[0427] <210> Sequence Number 35

[0428] <211> Length 33

[0429] <212> Type DNA

[0430] <213> Species Corynebacterium glutamicum

[0431] <400> Sequence 35

[0432] GAGCTCCTCTAGATTAGTTAGGGACCGTATGCG

[0433] <210> Serial Number 36

[0434] <211> Length 125

[0435] <212> Type DNA

[0436] <213> Species Escherichia coli

[0437] <400> Sequence 36

[0438] TTCGATTTATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTT

[0439] <210> Sequence number 37 (sequence of HgPobA_TY)

[0440] <211> Length 1209

[0441] <212> Type DNA

[0442] <213> Species Helemonella gracilis

[0443] <400> Sequence 37

[0444]

[0445] <210> Serial Number 38

[0446] <211> Length 27

[0447] <212> Type DNA

[0448] <213> Species Escherichia coli

[0449] <400> Sequence 38

[0450] GAGCTCATGCGCACCCAGGTTGGTATC

[0451] <210> Serial Number 39

[0452] <211> Length 26

[0453] <212> Type DNA

[0454] <213> Species Hydrogenophage

[0455] <400> Sequence 39

[0456] GTCGACTTAGTCGATGGGCAAGCCCG

[0457] <210> Serial Number 40

[0458] <211> Length 26

[0459] <212> Type DNA

[0460] <213> Species: Polarmonas

[0461] <400> Sequence 40

[0462] GTCGACTTATTCAATCGGAAGTCCAG

[0463] <210> Serial Number 41

[0464] <211> Length 26

[0465] <212> Type DNA

[0466] <213> Species Helemonella gracilis

[0467] <400> Sequence 41

[0468] GTCGACTTAGGCACCGAAGTCGAGGG

[0469] <210> Serial Number 42

[0470] <211> Length 25

[0471] <212> Type DNA

[0472] <213> Species Corynebacterium glutamicum

[0473] <400> Sequence 42

[0474] GTCGACTTACACTTCAAACCGCGGG

Claims

1. A microbial strain into which a gene encoding a protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae or a protein having an amino acid sequence identity of 70% or more to the protocatechuate 5-oxidase is introduced.

2. The microbial strain according to claim 1, wherein The microorganism of the Comamonadaceae family is a microorganism selected from the genera Helemonella, Polarmonas, and Hydrogenophage.

3. The microbial strain according to claim 1, wherein The microbial strain is a microbial strain selected from the genus Escherichia, Rhodococcus, Acinetobacter, Bradyrhizobium, Corynebacterium, Pseudomonas, Rhodopseudomonas, Sinorhizobium, Brevibacterium, Neosphingobacterium or Ralstonia.

4. A method for producing gallic acid, wherein: Gallic acid is produced from protocatechuic acid by fermentation using the microbial strain according to claim 1.

5. A method for producing gallic acid, wherein: Gallic acid is produced by fermentation using the microbial strain according to claim 1 in a medium containing glucose.

6. The method for producing gallic acid according to claim 4 or 5, wherein: The fermentation was carried out using a medium with a pH below 6.

0.

7. The method for producing gallic acid according to claim 4 or 5, wherein: Fermentation is carried out at a temperature of 15°C to 45°C.

8. A method for producing gallic acid from protocatechuic acid, wherein: A protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae or a protein having an amino acid sequence identity of 70% or more to the protocatechuate 5-oxidase is used.

9. A protein having an amino acid sequence in which leucine at positions 199 to 209 of a protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae or a protein having 70% or more identity with the amino acid sequence of the protocatechuate 5-oxidase is substituted with valine or glycine.

10. A protein having an amino acid sequence in which threonine at positions 294 to 304 of a protocatechuate 5-oxidase derived from a microorganism of the family Comamonadaceae or a protein having 70% or more identity with the amino acid sequence of the protocatechuate 5-oxidase is substituted with alanine.

11. The protein according to claim 9 or 10, wherein The tyrosine at positions 385 to 395 is further substituted with phenylalanine, valine or alanine.

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