An enzyme that dehydroxylates the hydroxyl groups of urolithins

Abstract

The present disclosure addresses the problem of providing an enzyme capable of dehydroxylating a hydroxyl group located at a specific position in a urolithin compound that has a hydroxyl group at at least the specific position. The problem can be solved by an enzyme having the following properties (1) and (2): (1) the enzyme can dehydroxylate a hydroxyl group located at position-4 in a urolithin compound; and (2) the enzyme can be activated by one or more components selected from the group consisting of reduced-type nicotinamide adenine dinucleotide (NADH), reduced-type nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD) and flavin adenine mononucleotide (FMN) in the presence of methyl viologen (MV).

Description

[Technical field]

[0001] The present disclosure relates to enzymes that dehydroxylate hydroxyl groups of urolithins. [Background technology]

[0002] Urolithins, represented by urolithin A and urolithin C, are known as intestinal metabolites of ellagic acid derived from ellagitannins contained in berries such as pomegranates, raspberries, blackberries, cloudberries, strawberries, and walnuts.

[0003] One method reported for synthesizing these urolithins is to use 2-bromo-5-methoxybenzoic acid as a starting material, demethylate it to produce 2-bromo-5-hydroxybenzoic acid, and then react it with resorcinol to obtain urolithin A (Non-Patent Document 1). However, such chemical synthesis methods are not suitable for using urolithins as ingredients in functional foods (including beverages and supplements).

[0004] On the other hand, it is known that after ingesting ellagitannins and ellagic acid, they are metabolized by the intestinal microflora and converted into urolithins. In recent years, microorganisms belonging to Gordonibacter urolithinfaciens and Gordonibacter pamelaeae have been found to be intestinal bacteria that produce urolithin C, a type of urolithin, from ellagic acid, and methods have been reported for producing urolithin C by fermenting ellagic acid using these intestinal bacteria (Patent Document 1, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 147280 [Non-patent literature]

[0006] [Non-Patent Document 1] J. Agric. Food Chem., 56, 393-400 (2008) Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present disclosure is to provide at least an enzyme that dehydroxylates a hydroxyl group at a specific position in urolithins that have a hydroxyl group at that position. [Means for solving the problem]

[0008] Through proteomic analysis of a specific strain of Gordonibacter urolithinfaciens, the inventors discovered that there are proteins whose expression levels are significantly higher when the strain is cultured in a medium containing ellagic acid than when it is cultured in a medium lacking ellagic acid. Furthermore, an enzyme that catalyzes the dehydroxylation of the hydroxyl group at position 4 of urolithins having a hydroxyl group at position 4 was purified from a specific bacterial strain and subjected to proteomic analysis. As a result, it was found that the purified enzyme obtained was identical to a part of a protein whose expression level was significantly increased when the microorganism was cultured in a medium containing ellagic acid. By introducing the gene encoding the enzyme into a heterologous microorganism, the enzyme was successfully expressed and the function of the gene could be identified. Furthermore, the researchers found that, in the genome surrounding the gene encoding the enzyme, there are genes that are homologous to the gene and encode an enzyme that catalyzes the dehydroxylation of the hydroxyl group at position 10 of urolithins that have a hydroxyl group at position 10, as well as a gene encoding an ellagic acid lactonase that hydrolyzes the lactone of ellagic acid to produce urolithin M5, and a gene encoding a urolithin transporter. The present disclosure includes the following aspects.

[0009] [1] An enzyme having the following properties (1) and (2): (1) The hydroxyl group at position 4 of urolithins is dehydroxylated. (2) In the presence of methyl viologen (MV), it is activated with one or more selected from the group consisting of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN). [2] The enzyme according to [1], having the following properties (3) and (4): (3) The optimum pH is 5.5 or higher and 7.5 or lower. (4) The results of SDS-PAGE include a band showing a molecular weight of 81,000 or more and 99,000 or less. [3] The enzyme according to [1] or [2], which has the following property (5): (5) The optimum temperature is 37°C or higher and 50°C or lower. [4] The enzyme according to any one of [1] to [3], which is derived from a microorganism belonging to the genus Gordonibacter. [5] The enzyme according to [4], wherein the microorganism belonging to the genus Gordonibacter is one or more selected from the group consisting of microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and microorganisms belonging to Gordonibacter faecihominis. [6] The amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2, or The amino acid sequence represented by SEQ ID NO: 13 and the amino acid sequence represented by SEQ ID NO: 14 are included. The enzyme according to any one of [1] to [5]. [7] An amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 3, or Contains the amino acid sequence represented by SEQ ID NO: 13, contains the amino acid sequence represented by SEQ ID NO: 14, and contains the amino acid sequence represented by SEQ ID NO: 15. The enzyme according to any one of [1] to [6]. [8] A nucleic acid sequence comprising the base sequence represented by SEQ ID NO: 7 and the base sequence represented by SEQ ID NO: 8, or The base sequence represented by SEQ ID NO: 19 and the base sequence represented by SEQ ID NO: 20 are included. Polynucleotide. [9] A nucleic acid sequence comprising a base sequence represented by SEQ ID NO: 7, a base sequence represented by SEQ ID NO: 8, and a base sequence represented by SEQ ID NO: 9, or The base sequence represented by SEQ ID NO: 19, the base sequence represented by SEQ ID NO: 20, and the base sequence represented by SEQ ID NO: 21 are included. Polynucleotide.

[10] A recombinant vector comprising the polynucleotide described in [8] or [9].

[0010]

[11] A transformant which retains the polynucleotide according to [8] or [9] in an expressible manner, or which retains the vector according to

[10] in an expressible manner.

[12] The transformant according to

[11] , wherein the host is a microorganism belonging to the genus Rhodococcus.

[13] A method for producing a protein encoded by the polynucleotide according to [8] or [9], comprising the step of culturing the transformant according to

[11] or

[12] .

[14] A method for dehydroxylating a hydroxyl group at the 4-position of a urolithin, comprising the following step (I): Step (I): A step of contacting one or more selected from the following (i) to (iv) with a urolithin having a hydroxyl group at the 4-position to dehydroxylate the hydroxyl group at the 4-position: (i) The enzyme according to any one of [1] to [7]; (ii) a protein encoded by the polynucleotide according to [8] or [9]; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above.

[15] The urolithin is urolithin M5, urolithin D, or urolithin E; The products produced by dehydroxylation of the hydroxyl group at position 4 of the urolithins are urolithin M6, urolithin C, and urolithin M7, respectively. The method according to

[14] .

[16] An enzyme having the following properties (1) and (2): (1) The hydroxyl group at position 10 of urolithins is dehydroxylated. (2) In the presence of methyl viologen (MV), it is activated with one or more selected from the group consisting of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN).

[17] The enzyme according to

[16] , having the following properties (3) and (4): (3) The optimum pH is 5.0 or higher and 7.0 or lower. (4) The optimum temperature is 37°C or higher and 42°C or lower.

[18] The enzyme according to

[16] or

[17] , which is derived from a microorganism belonging to the genus Gordonibacter.

[19] The enzyme described in

[18] , wherein the microorganism belonging to the genus Gordonibacter is one or more selected from the group consisting of microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and microorganisms belonging to Gordonibacter faecihominis.

[20] The amino acid sequence represented by SEQ ID NO: 4 and the amino acid sequence represented by SEQ ID NO: 5, or The amino acid sequence represented by SEQ ID NO: 16 and the amino acid sequence represented by SEQ ID NO: 17 are included. The enzyme according to any one of

[16] to

[19] .

[0011]

[21] An amino acid sequence represented by SEQ ID NO: 4, an amino acid sequence represented by SEQ ID NO: 5, and an amino acid sequence represented by SEQ ID NO: 6, or The amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence represented by SEQ ID NO: 17, and the amino acid sequence represented by SEQ ID NO: 18 are included. The enzyme according to any one of

[16] to

[20] .

[22] A nucleic acid sequence comprising the base sequence represented by SEQ ID NO: 10 and the base sequence represented by SEQ ID NO: 11, or A polynucleotide comprising the base sequence represented by SEQ ID NO:22 and the base sequence represented by SEQ ID NO:23.

[23] A nucleic acid sequence comprising a base sequence represented by SEQ ID NO: 10, a base sequence represented by SEQ ID NO: 11, and a base sequence represented by SEQ ID NO: 12, or The base sequence represented by SEQ ID NO: 22, the base sequence represented by SEQ ID NO: 23, and the base sequence represented by SEQ ID NO: 24 are included. The polynucleotide described in

[22] .

[24] A recombinant vector comprising the polynucleotide described in

[22] or 23.

[25] A transformant which retains the polynucleotide according to

[22] or

[23] in an expressible manner, or which retains the vector according to

[24] in an expressible manner.

[26] The transformant according to

[25] , wherein the host is a microorganism belonging to the genus Rhodococcus.

[27] A method for producing a protein encoded by the polynucleotide according to

[22] or

[23] , comprising the step of culturing the transformant according to

[25] or

[26] .

[28] A method for producing the enzyme according to any one of

[16] to

[21] , comprising a step of culturing a microorganism belonging to the genus Gordonibacter.

[29] The method according to

[28] , wherein the microorganism belonging to the genus Gordonibacter is one or more selected from the group consisting of microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and microorganisms belonging to Gordonibacter faecihominis.

[30] A method for dehydroxylating a hydroxyl group at position 10 of a urolithin, comprising the following step (I): Step (I): A step of contacting one or more selected from the following (i) to (iv) with a urolithin having a hydroxyl group at the 10-position to dehydroxylate the hydroxyl group at the 10-position: (i) The enzyme according to any one of

[16] to

[21] ; (ii) a protein encoded by the polynucleotide according to

[22] or

[23] ; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above.

[0012]

[31] The urolithin is urolithin M5, urolithin M6, or urolithin M7; The products produced by dehydroxylation of the hydroxyl group at position 10 of the urolithins are urolithin D, urolithin C, and urolithin A, respectively. The method according to

[30] .

[32] A method for producing urolithin C, comprising the following steps (I) to (III): Step (I): Allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin M6. (i) The enzyme according to any one of [1] to [7]; (ii) a protein encoded by the polynucleotide according to [8] or [9]; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with the urolithin M6 to produce urolithin C. (v) the enzyme according to any one of

[16] to

[21] ; (vi) a protein encoded by the polynucleotide according to

[22] or

[23] ; (vii) a microorganism producing the enzyme described in (v) or the protein described in (vi); (viii) A treated product of the microorganism described in (vii) above.

[33] A method for producing urolithin C, comprising the following steps (I) to (III): Step (I): Allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin D. (i) The enzyme according to any one of

[16] to

[21] ; (ii) a protein encoded by the polynucleotide according to

[22] or

[23] ; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with urolithin D to produce urolithin C: (v) the enzyme according to any one of [1] to [7]; (vi) a protein encoded by the polynucleotide according to [8] or [9]; (vii) a microorganism producing the enzyme described in (v) or the protein described in (vi); (viii) A treated product of the microorganism described in (vii) above.

[34] A method for producing urolithin A, comprising the following step (I): Step (I): A step of allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from urolithin C produced by the production method described in

[32] or

[33] .

[35] A protein comprising the amino acid sequence represented by SEQ ID NO: 25 or the amino acid sequence represented by SEQ ID NO: 27.

[36] A polynucleotide comprising the base sequence represented by SEQ ID NO:26 or the base sequence represented by SEQ ID NO:28.

[37] A recombinant vector comprising the polynucleotide described in

[36] .

[38] A transformant which retains the polynucleotide according to

[36] in an expressible manner, or which retains the vector according to

[37] in an expressible manner.

[39] A method for producing a protein encoded by the polynucleotide of

[36] , comprising the step of culturing the transformant of

[38] .

[40] A method for promoting the uptake of ellagic acid from outside the cell into the cell of the transformant described in

[38] , comprising the following step (I): Step (I): A step of contacting the transformant described in

[38] with ellagic acid.

[0013]

[41] A method for producing urolithin M5, comprising the following step (I): Step (I): A step of contacting the transformant described in

[38] , whose host is a microorganism having the ability to produce urolithin M5 from ellagic acid, with ellagic acid to produce urolithin M5 from ellagic acid.

[42] A method for producing urolithin C, comprising the following step (I): Step (I): A step of contacting the transformant described in claim 38, whose host is a microorganism having the ability to produce urolithin C from ellagic acid, with ellagic acid to produce urolithin C from ellagic acid.

[43] A method for producing urolithin A, comprising the following steps (I) and (II): Step (I): A step of contacting the transformant described in

[38] , whose host is a microorganism having the ability to produce urolithin C from ellagic acid, with ellagic acid to produce urolithin C from ellagic acid. Step (II): Allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C.

[44] A protein comprising the amino acid sequence represented by SEQ ID NO: 29 or the amino acid sequence represented by SEQ ID NO: 31.

[45] A polynucleotide comprising the base sequence represented by SEQ ID NO: 30 or the base sequence represented by SEQ ID NO: 32.

[46] A recombinant vector comprising the polynucleotide described in

[45] .

[47] A transformant which retains the polynucleotide according to

[45] in an expressible manner, or which retains the vector according to

[46] in an expressible manner.

[48] ​​A method for producing a protein encoded by the polynucleotide of

[45] , comprising the step of culturing the transformant of

[47] .

[49] A method for producing urolithin M5, comprising the following step (I): Step (I): A step of contacting the transformant described in

[47] with ellagic acid to produce urolithin M5 from ellagic acid.

[0014]

[51] A method for producing urolithin A, comprising the following steps (I) and (II): Step (I): A step of producing urolithin M5 from urolithin M5 by contacting the transformant described in

[47] , whose host is a microorganism having the ability to produce urolithin C from urolithin M5, with ellagic acid, thereby producing urolithin M5 from ellagic acid, and producing urolithin C from the urolithin M5. Step (II): Allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C. Effect of the Invention

[0015] The present disclosure may have an effect of providing at least an enzyme that dehydroxylates a hydroxyl group at a predetermined position in urolithins having the hydroxyl group at that position. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows the results of native-PAGE of an enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins and an enzyme that dehydroxylates the hydroxyl group at position 10 of urolithins in one experimental example of the present disclosure (photograph in place of drawing). [Diagram 2] FIG. 1 shows the results of SDS-PAGE of an enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins and an enzyme that dehydroxylates the hydroxyl group at position 10 of urolithins in one experimental example of the present disclosure (photograph in place of drawing). [Diagram 3] 1 is a graph showing the temperature dependence of an enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins in an experimental example of the present disclosure. [Figure 4] 1 is a graph showing the pH dependence of an enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins in an experimental example of the present disclosure. [Diagram 5] 1 is a graph showing the temperature stability of an enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins in an experimental example of the present disclosure. [Figure 6] 1 is a graph showing the pH stability of an enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins in an experimental example of the present disclosure. [Figure 7] 1 is a graph showing the dehydroxylation activity of bacterial cells that dehydroxylate the hydroxyl group at position 10 of urolithins in one experimental example of the present disclosure. [Figure 8] 1 is a graph showing the temperature dependence of an enzyme that dehydroxylates the hydroxyl group at position 10 of urolithins in an experimental example of the present disclosure. [Figure 9] 1 is a graph showing the pH dependence of an enzyme that dehydroxylates the hydroxyl group at position 10 of urolithins in an experimental example of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure will be described in detail below. Each configuration and combination thereof in each aspect is merely an example, and addition, omission, substitution, and other modifications of the configuration are possible as appropriate without departing from the spirit of the present invention. The present disclosure is not limited by the aspects, but is limited only by the scope of the claims. Additionally, each feature disclosed herein may be combined with any other feature disclosed herein.

[0018] In this disclosure, the accession numbers of strains beginning with the word DSM are numbers assigned to microorganisms stored at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). In addition, the accession numbers of strains beginning with the words "JCM" are numbers assigned to microorganisms preserved at the RIKEN BioResource Center.

[0019] Urolithins are represented by the following general formula (1): [ka] (In the formula, R1 to R6 each independently represent a hydroxyl group, a hydrogen atom, or a methoxy group, and at least one of R1 to R6 is a hydroxyl group.) The hydroxyl group at the 4th position of urolithins described in this specification refers to the hydroxyl group of urolithins in which R2 in the above general formula (1) is a hydroxyl group, and the hydroxyl group at the 10th position refers to the hydroxyl group of urolithins in which R5 in the above general formula (1) is a hydroxyl group.

[0020] <1-1-1(A). Enzymes that dehydroxylate the 4-hydroxyl group of urolithins> One aspect of the present disclosure is an enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins. More specifically, it is an enzyme that catalyzes the reaction of dehydroxylating the hydroxyl group at position 4 of urolithins that have a hydroxyl group at position 4. The enzyme is presumed to be composed of one subunit, two subunits, or three subunits.

[0021] The enzyme according to this embodiment is an enzyme having the following properties (1) and (2). (1) The hydroxyl group at position 4 of urolithins is dehydroxylated. (2) In the presence of methyl viologen (MV), it is activated with one or more selected from the group consisting of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN).

[0022] The urolithins in this embodiment are preferably urolithin M5, urolithin D, or urolithin E. When the hydroxyl group at position 4 of these urolithins is dehydroxylated by the enzyme of this embodiment, urolithin M6, urolithin C, or urolithin M7 are produced, respectively.

[0023] The enzyme according to this embodiment is activated by a cofactor, such as methyl viologen (MV), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN). As the cofactor in this embodiment, since it increases the activity of the enzyme according to this embodiment to dehydroxylate the hydroxyl group at position 4 of urolithins, it is preferable to use MV and NADH, it is more preferable to use MV and NADPH, it is even more preferable to use MV, NADPH and FMN, and it is even more preferable to use MV, NADPH and FAD.

[0024] The enzyme according to this embodiment preferably has the following properties (3) and (4). (3) The optimum pH is preferably 5.5 or higher, more preferably 6.0 or higher, and on the other hand, is preferably 7.5 or lower, more preferably 7.0 or lower, and even more preferably 6.5 or lower.

[0025] (4) As a result of SDS-PAGE, the molecular weight is preferably 81,000 or more, more preferably 85,000 or more, and even more preferably 87,000 or more, while including a band showing a molecular weight of preferably 99,000 or less, more preferably 95,000 or less, and even more preferably 93,000 or less.

[0026] The enzyme according to this embodiment preferably has the following property (5). (5) The optimum temperature is preferably 37°C or higher, more preferably 38°C or higher, and even more preferably 40°C or higher, while it is preferably 50°C or lower, more preferably 46°C or lower, and even more preferably 44°C or lower.

[0027] The enzyme according to this embodiment is preferably derived from a microorganism belonging to the genus Gordonibacter. More preferably, the microorganism is one or more selected from the group consisting of microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and microorganisms belonging to Gordonibacter faecihominis. A preferred microorganism belonging to Gordonibacter urolithinfaciens is the Gordonibacter urolithinfaciens DSM 27213 strain. A preferred microorganism belonging to the genus Gordonibacter pamelaeae is the DSM 19378 strain. A preferred microorganism belonging to the genus Gordonibacter faecihominis is the JCM 16058 strain.

[0028] The Gordonibacter urolithinfaciens DSM 27213 strain is not limited to the same strain, and may be a bacterium substantially equivalent to the deposited strain. The substantially equivalent bacterium is a microorganism belonging to Gordonibacter urolithinfaciens, capable of exerting the effect of the present embodiment, such as expressing the enzyme of the present embodiment, and further having a base sequence of its 16S rRNA gene that is preferably 98% or more, more preferably 99% or more, and more preferably 100% homologous to the base sequence of the 16S rRNA gene of the deposited strain, and preferably having the same bacteriological properties as the deposited strain. In addition, the bacterium may be a strain bred from the deposited strain or a substantially equivalent strain by mutation treatment, genetic recombination, selection of natural mutants, or the like, so long as the effect of the present embodiment is not impaired. This also applies to Gordonibacter pamelaeae DSM 19378 and Gordonibacter faecihominis JCM 16058.

[0029] Microorganisms belonging to the genus Gordonibacter are cultured in common media used for culturing anaerobic bacteria, such as Anaerobe Basal Broth (ThermoFisher Scientific, CM0957), Wilkins-Chalgren Anaerobe Broth (ThermoFisher Scientific, CM0643), GAM medium (Nissui Pharmaceutical Co., Ltd.), and modified GAM medium (Nissui Pharmaceutical Co., Ltd.). The culture temperature is preferably 25°C or higher, more preferably 30°C or higher, and even more preferably 33°C or higher, while it is preferably 45°C or lower, more preferably 42°C or lower, and even more preferably 40°C or lower.

[0030] Furthermore, for example, water-soluble organic substances can be added as carbon sources, such as sugars, such as sorbose, fructose, glucose, dextrin, and soluble starch; alcohols, such as methanol; organic acids, such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, and succinic acid; and amino acids, such as arginine, methionine, phenylalanine, valine, and glutamic acid. The concentration of the organic matter added to the medium as a carbon source can be appropriately adjusted for efficient growth, and is usually selected within the range of 0.1 to 10 wt / vol %.

[0031] In addition to the above carbon sources, the medium may contain a nitrogen source, which may be any of various nitrogen compounds that can be used in normal culture or fermentation. Preferred inorganic nitrogen sources include ammonium salts and nitrates, etc. More preferred inorganic nitrogen sources include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, triammonium citrate, potassium nitrate, and sodium nitrate. On the other hand, preferred organic nitrogen sources include amino acids, yeast extract, peptones (milk-derived peptone, soybean-derived peptone, soybean-derived peptide, etc.), meat extracts (e.g., Lab-Remco powder, bonito extract, tuna extract, bonito extract, bouillon, shellfish extract, etc.), liver extract, digested serum powder, etc. More preferred organic nitrogen sources include arginine, cysteine, citrulline, lysine, tryptophan, yeast extract, peptones, etc.

[0032] In addition to carbon and nitrogen sources, microbial growth factors such as extracts, vitamins, metal salts, and inorganic compounds can be added to the medium. Extracts include hemin, heme iron, digested serum powder, liver extract, and blood digest. Vitamins include biotin, folic acid, pyridoxal, thiamine, riboflavin, nicotinic acid, nicotinamide, pantothenic acid, vitamin B12, thiooctoic acid, p-aminobenzoic acid, and vitamin K. Metal salts and inorganic compounds include potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, alum, sodium molybdate, potassium chloride, boric acid, nickel chloride, sodium tungstate, sodium selenate, sodium selenite, ferrous ammonium sulfate, iron(II) citrate, sodium acetate trihydrate, magnesium sulfate heptahydrate, and manganese sulfate tetrahydrate. These metals can also be added in the form of mineral yeast. Methods for producing a culture medium by adding growth cofactors derived from animals and plants, such as inorganic compounds and vitamins, are known. The medium can be liquid, semi-solid, or solid. The preferred form of the medium is a liquid medium.

[0033] The production of the enzyme according to this embodiment by a microorganism belonging to the genus Gordonibacter is induced by adding ellagic acid or a precursor of ellagic acid as a raw material (substrate) to the medium. Examples of precursors of ellagic acid include ellagitannins such as punicalagin and geraniin. The raw material (substrate) is preferably added so that the concentration in the medium is 0.01 g / L or more and 20 g / L or less.

[0034] To recover the enzyme according to the present embodiment produced by a microorganism belonging to the genus Gordonibacter, the culture is recovered after the production of the enzyme according to the present embodiment, and the microorganism is disrupted in a buffer containing a reducing agent such as cysteine, 2-mercaptoethanol, or dithiothreitol, or a protease inhibitor such as phenylmethanesulfonyl fluoride (PMFS), pepstatin A, or ethylenediaminetetraacetic acid to obtain a cell-free extract, which can then be purified from the cell-free extract by an appropriate combination of fractionation based on protein solubility, various types of chromatography, etc. Either method can be used according to the usual method.

[0035] The first, second, or third subunit presumed to constitute the enzyme of this embodiment contained in the Gordonibacter urolithinfaciens DSM 27213 strain is preferably selected from the combination of GuUroA1, GuUroA2, and GuUroA3. The amino acid sequences of GuUroA1, GuUroA2, and GuUroA3 are the sequences shown in SEQ ID NOs: 1 to 3, respectively. The nucleotide sequences of the genes encoding GuUroA1, GuUroA2, and GuUroA3 are the sequences shown in SEQ ID NOs: 7 to 9, respectively. As described above, the enzyme according to this embodiment preferably has the property (4). The molecular weight of 81,000 or more and 99,000 or less is presumed to be the molecular weight of the GuUroA1 subunit.

[0036] The first, second, or third subunit presumed to constitute the enzyme according to this embodiment contained in the Gordonibacter pamelaeae DSM 19378 strain is preferably selected from the combination of GpUroA1, GpUroA2, and GpUroA3. The amino acid sequences of GpUroA1, GpUroA2, and GpUroA3 are shown in SEQ ID NOs: 13 to 15, respectively. The nucleotide sequences of the genes encoding GpUroA1, GpUroA2, and GpUroA3 are shown in SEQ ID NOs: 19 to 21, respectively.

[0037] Therefore, the enzyme according to this embodiment is preferably or comprising one or more selected from the group consisting of the amino acid sequence represented by SEQ ID NO:1, the amino acid sequence represented by SEQ ID NO:2, and the amino acid sequence represented by SEQ ID NO:3; or The enzyme comprises one or more selected from the group consisting of the amino acid sequence represented by SEQ ID NO:13, the amino acid sequence represented by SEQ ID NO:14, and the amino acid sequence represented by SEQ ID NO:15.

[0038] Furthermore, GuUroA1 may be a protein consisting of amino acids in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added in the amino acid sequence shown in SEQ ID NO: 1, so long as it has the activity of dehydroxylating the hydroxyl group at position 4 of urolithins when it serves as a subunit by itself or when it forms a unit together with the other subunits GuUroA2 and / or GuUroA3. "One or more" preferably means 1 to 80, more preferably 1 to 50, even more preferably 1 to 30, and particularly preferably 1 to 10 amino acids, and the same applies when amino acids are added to the N-terminus and / or C-terminus. The same also applies to GpUroA1.

[0039] Conservative substitutions are preferred, and conservative substitutions refer to substitutions between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid, between Gln and Asn when the substitution site is a polar amino acid, between Lys, Arg, and His when the substitution site is a basic amino acid, between Asp and Glu when the substitution site is an acidic amino acid, and between Ser and Thr when the substitution site is an amino acid having a hydroxyl group. Specific examples of conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile with L Examples of such substitutions include substitutions of eu, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. This also applies to GpUroA1.

[0040] Furthermore, GuUroA1, either when it acts alone as a subunit or when it forms a unit together with the other subunits GuUroA2 and / or GuUroA3, may be a protein that has a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more to the entire amino acid sequence shown in SEQ ID NO: 1, so long as it has the activity of dehydroxylating the hydroxyl group at position 4 of urolithins. The same also applies to GpUroA1.

[0041] Furthermore, GuUroA1 may be a protein encoded by a polynucleotide that hybridizes under stringent conditions with the base sequence of SEQ ID NO:7, so long as it has the activity of dehydroxylating the hydroxyl group at position 4 of urolithins when GuUroA1 itself is a subunit or when it forms a unit together with other subunits, GuUroA2 and / or GuUroA3. Examples of "stringent conditions" include conditions under which polynucleotides having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, hybridize with each other, and polynucleotides having a lower homology than that do not hybridize with each other. This also applies to GpUroA1.

[0042] The above amino acid substitutions, protein homology, stringent conditions, and the like are also applied to GuUroA2. However, with respect to GuUroA2, the above "one to more than one" is preferably 1 to 12, more preferably 1 to 11, even more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3. This also applies to GpUroA2. The above amino acid substitutions, protein homology, stringent conditions, and the like are also applied to GuUroA3. However, with respect to GuUroA3, the above "one to several" is preferably 1 to 7, more preferably 1 to 6, even more preferably 1 to 5, still more preferably 1 to 3, and particularly preferably 1 to 2. This also applies to GpUroA3.

[0043] The activity of the enzyme according to this embodiment to dehydroxylate the hydroxyl group at position 4 of urolithins can be evaluated, for example, as described below in Experimental Example 5.

[0044] <1-1-2(A). Polynucleotide> Another aspect of the present disclosure is The nucleic acid sequence includes one or more selected from the group consisting of the nucleic acid sequence represented by SEQ ID NO: 7, the nucleic acid sequence represented by SEQ ID NO: 8, and the nucleic acid sequence represented by SEQ ID NO: 9, or It is a polynucleotide comprising one or more selected from the group consisting of the base sequence represented by SEQ ID NO:19, the base sequence represented by SEQ ID NO:20, and the base sequence represented by SEQ ID NO:21. Details of each base sequence are as described in the above section “1-1-1(A). Dehydroxylase of the 4-hydroxyl group of urolithins.”

[0045] The base sequence of the gene encoding GuUroA1 may be a base sequence that hybridizes under stringent conditions with the base sequence represented by SEQ ID NO: 7, so long as GuUroA1, either alone as a subunit or when it forms a unit together with the other subunits GuUroA2 and / or GuUroA3, has the activity of dehydroxylating the hydroxyl group at position 4 of urolithins. Examples of "stringent conditions" include conditions under which polynucleotides having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more hybridize to each other, but polynucleotides having a lower homology than that do not hybridize to each other. This also applies to the nucleotide sequence of the gene encoding GuUroA2 and the nucleotide sequence of the gene encoding GuUroA3, as well as the nucleotide sequence of the gene encoding GpUroA1, the nucleotide sequence of the gene encoding GpUroA2, and the nucleotide sequence of the gene encoding GpUroA3.

[0046] <1-1-3(A). Genetic Engineering Aspects> Another aspect of the present disclosure is a method for producing a protein encoded by the polynucleotide, the method comprising the steps of: a recombinant vector containing the polynucleotide; a transformant carrying the polynucleotide in an expressible state or carrying the vector in an expressible state; and culturing the transformant. The protein is the "enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins." The polynucleotide can be inserted into a known expression vector to produce an expression vector that expresses the enzyme. The expression vector can then be used to transform a microorganism or the like to obtain a transformant, which can then be cultured or otherwise used to produce the enzyme, thereby obtaining the enzyme.

[0047] Examples of host-vector systems include, for example, Lactococcus lactis subsp. Cremoris-vector pNZ8148 (MoBiTech), Lactococcus lactis-pGKV11 (Appl. Environ. Microbiol., 50, 540-542 (1985)), Lactococcus lactis / Streptococcus thermophilus / Streptococcus faecalis-pBE194 (JP Patent Publication 2004-253861), Lactococcus lactis subsp. lactis / Lactobacillus delbrueckii-pBE194 (JP Patent Publication 2004-253861), and Lactococcus lactis subsp. lactis / Lactobacillus delbrueckii-pBE194 (JP Patent Publication 2004-253861). Examples of the bifidobacteria include Bifidobacterium adolescentis-pKKT427 (Nucleic Acids Research, 2009, Vol. 37, No. 1 e3 doi:10.1093 / nar / gkn884), a wide range of bifidobacteria such as Bifidobacterium longum-pNC7 (Res. Microbiol., 147, 133-143 (1996)), and Bifidobacterium longum-pBS423 (Appl. Environ. Microbiol., 78, 4984-4994 (2012)). Examples of anaerobic bacteria include Clostridium acetobutylicum-pTY10 (Agric. Biol. Chem., 54 (2), 437-441 (1990)) / pMTL500E (Microbiol. Sci. 5:310-315 (1988)) and ACE vector (Anaerobe, 41, 104-112 (2016)). Although the expression of the enzyme requires an anaerobic environment, an anaerobic host-vector system is not necessarily required for heterologous expression of genes. For example, the Rhodococcus erythropolis-pTip, pNit, and pCpi Vector series (Hokkaido System Science, Biotechnol. Bioeng., 86, 136-148 (2004)) can be suitably used. An example of the host is the L88 strain, and an example of the vector is pTipQC1. In addition to microorganisms, various host and vector systems have been developed for plants and animals. For example, a system using silkworms (Nature 315, 592-594 (1985)) and systems for expressing large amounts of heterologous proteins in plants such as rapeseed, corn, and potato have been developed, and these may be used.

[0048] <1-2-1(A). Method for dehydroxylating the 4-hydroxyl group of urolithins> Another aspect of the present disclosure is a method for dehydroxylating the hydroxyl group at position 4 of urolithins. More specifically, it is a method for dehydroxylating the hydroxyl group at position 4 of urolithins having a hydroxyl group at position 4. The present embodiment is a method for dehydroxylating the hydroxyl group at position 4 of urolithins, comprising the following step (I): Step (I): A step of contacting one or more selected from the following (i) to (iv) with a urolithin having a hydroxyl group at the 4-position to dehydroxylate the hydroxyl group at the 4-position: (i) an enzyme described in the above section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins"; (ii) a protein encoded by a polynucleotide described in the above section "1-1-2(A). Polynucleotides"; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above.

[0049] (Urolithins) Details of urolithins in this embodiment are as described above in the section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins."

[0050] (Process (I)) In step (I), (i) the enzyme and (ii) the protein encoded by the polynucleotide each dehydroxylate the hydroxyl group at position 4 of urolithins upon contact with the urolithins having a hydroxyl group at position 4. In addition, in (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii), and (iv) a treated product of the microorganism described in (iii), the enzyme or protein contained in the microorganism or the treated product of the microorganism dehydroxylates the hydroxyl group at position 4 of urolithins upon contact with the urolithins having a hydroxyl group at position 4.

[0051] (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) the microorganism in the processed product of the microorganism described in (iii) may be a microorganism obtained by genetic engineering techniques. An example of the microorganism is a microorganism belonging to the genus Gordonibacter. Preferred examples of the microorganism belonging to the genus Gordonibacter are the same as those described in the above section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins."

[0052] (i) The enzyme and (ii) the protein encoded by the polynucleotide are not limited to purified ones, and may be partially purified ones. Furthermore, (iv) examples of the processed products of the microorganism include microorganisms whose cell membrane permeability has been changed by treatment with a surfactant or an organic solvent such as toluene, cell-free extracts obtained by disrupting the cells by treatment with glass beads or enzymes, and partially purified versions of such cell-free extracts.

[0053] When one or more selected from (i) to (iv) are contacted with urolithins having a hydroxyl group at position 4, the contact can be carried out in water; in an organic solvent that is poorly soluble in water, such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, or a two-phase mixture system of such an organic solvent and an aqueous medium such as ethanol or acetone. It is also possible to immobilize one or more selected from (i) to (iv) and to carry out the contact using a membrane reactor or the like. Furthermore, when one or more selected from (i) to (iv) are microorganisms that produce the enzyme described in (i) or the protein described in (ii), the culture may be carried out in a solution typically used for culturing the microorganism, such as a medium or a phosphate buffer solution.

[0054] The temperature in step (I) is preferably 25°C or higher, more preferably 30°C or higher, and even more preferably 33°C or higher, while it is preferably 55°C or lower, more preferably 50°C or lower, and even more preferably 42°C or lower. The pH in step (I) is preferably 4.0 or higher, more preferably 5.0 or higher, and even more preferably 5.5 or higher, while it is preferably 9.0 or lower, more preferably 8.0 or lower, and even more preferably 7.0 or lower. The concentration of urolithins having a hydroxyl group at position 4, which are the raw material (substrate) in step (I), in the reaction solution is 0.001 g / L or more, preferably 0.01 g / L or more, and more preferably 0.1 g / L or more, and is 100 g / L or less, preferably 20 g / L or less, and more preferably 10 g / L or less.

[0055] This embodiment may include a step of quantifying the obtained product (quantification step). The quantification method may follow a conventional method. For example, ethyl acetate, to which an acid such as formic acid is added as necessary, is added to the culture solution, and the mixture is vigorously stirred and centrifuged to remove the ethyl acetate layer. If necessary, the same operation is repeated several times, and the ethyl acetate layers are combined to obtain an extract of the product. This extract is concentrated and dried under reduced pressure using an evaporator or the like, and dissolved in methanol. This is filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane, and the insoluble matter is removed, and then quantified using high performance liquid chromatography.

[0056] This embodiment may also include a step of recovering the product obtained. The recovery step includes a purification step, a concentration step, and the like. Examples of purification treatments in the purification step include sterilization of microorganisms by heat or the like; sterilization by microfiltration (MF), ultrafiltration (UF), and the like; removal of solids and polymeric substances; extraction by organic solvents, ionic liquids, and the like; and adsorption and decolorization using hydrophobic adsorbents, ion exchange resins, activated carbon columns, and the like. Examples of concentration treatments in the concentration step include concentration using an evaporator, a reverse osmosis membrane, and the like. Furthermore, the solution containing the obtained product can be powdered by freeze-drying, spray-drying, etc. In the powdering, an excipient such as lactose, dextrin, corn starch, etc. can also be added.

[0057] <1-1-1(B). Enzymes that dehydroxylate the 10-hydroxyl group of urolithins> Another aspect of the present disclosure is an enzyme that dehydroxylates the hydroxyl group at position 10 of urolithins. More specifically, it is an enzyme that catalyzes the reaction of dehydroxylating the hydroxyl group at position 10 of urolithins that have a hydroxyl group at position 10. The enzyme is presumed to be composed of one subunit, two subunits, or three subunits.

[0058] For the enzyme according to this embodiment, the contents described in the section "1-1-1(A). Enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins" above are incorporated by reference; however, there are some differences, which will be described below.

[0059] The urolithins in this embodiment are preferably urolithin M5, urolithin M6, or urolithin M7. When the hydroxyl group at position 10 of these is dehydroxylated by the enzyme of this embodiment, urolithin D, urolithin C, or urolithin A is produced, respectively.

[0060] The enzyme according to this embodiment is activated by a cofactor, such as methyl viologen (MV), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN). In this embodiment, the cofactor used increases the activity of the enzyme of this embodiment to dehydroxylate the hydroxyl group at position 10 of urolithins, and therefore it is preferable to use MV and NADH, more preferable to use MV, NADPH and FMN, even more preferable to use MV and NADPH, even more preferable to use MV, NADH and FAD, particularly preferable to use MV, NADH and FMN, and particularly preferable to use MV, NADPH and FAD.

[0061] The enzyme according to this embodiment preferably has the following properties (3) and (4). (3) The optimum pH is preferably 5.0 or higher, more preferably 5.5 or higher, and on the other hand, is preferably 7.0 or lower, more preferably 6.5 or lower, and even more preferably 6.0 or lower. (4) The optimum temperature is preferably 37°C or higher, more preferably 42°C or higher, while it is preferably 50°C or lower, more preferably 42°C or lower.

[0062] The enzyme according to this embodiment preferably has the following property (5). (5) As a result of SDS-PAGE, the molecular weight is preferably 78,000 or more, more preferably 82,000 or more, and even more preferably 84,000 or more, while including a band showing preferably 96,000 or less, more preferably 92,000 or less, and even more preferably 90,000 or less.

[0063] The first, second, or third subunit presumed to constitute the enzyme of this embodiment contained in the Gordonibacter urolithinfaciens DSM 27213 strain is preferably selected from the combination of GuUroB1, GuUroB2, and GuUroB3. The amino acid sequences of GuUroB1, GuUroB2, and GuUroB3 are the sequences shown in SEQ ID NOs: 4 to 6, respectively. The nucleotide sequences of the genes encoding GuUroB1, GuUroB2, and GuUroB3 are the sequences shown in SEQ ID NOs: 10 to 12, respectively. As described above, the enzyme according to this embodiment preferably has the property (4) described in the section "1-1-1(A). Enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins." The molecular weight of 78,000 or more and 96,000 or less is presumed to be the molecular weight of GuUroB1 subunit.

[0064] The first, second, or third subunit presumed to constitute the enzyme according to this embodiment contained in the Gordonibacter pamelaeae DSM 19378 strain is preferably selected from the combination of GpUroB1, GpUroB2, and GpUroB3. The amino acid sequences of GpUroB1, GpUroB2, and GpUroB3 are shown in SEQ ID NOs: 16 to 18, respectively. The nucleotide sequences of the genes encoding GpUroB1, GpUroB2, and GpUroB3 are the sequences shown in SEQ ID NOs: 22 to 24, respectively.

[0065] Therefore, the enzyme according to this embodiment is preferably or comprising one or more selected from the group consisting of the amino acid sequence represented by SEQ ID NO:4, the amino acid sequence represented by SEQ ID NO:5, and the amino acid sequence represented by SEQ ID NO:6; or The enzyme comprises one or more selected from the group consisting of the amino acid sequence represented by SEQ ID NO:16, the amino acid sequence represented by SEQ ID NO:17, and the amino acid sequence represented by SEQ ID NO:18.

[0066] Furthermore, GuUroB1 may be a protein consisting of amino acids in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added in the amino acid sequence shown in SEQ ID NO:4, so long as it has the activity of dehydroxylating the hydroxyl group at position 10 of urolithins when GuUroB1 itself acts as a subunit or when it forms a unit together with the other subunits GuUroB2 and / or GuUroB3. "One or more" preferably means 1 to 80, more preferably 1 to 79, even more preferably 1 to 50, still more preferably 1 to 30, and particularly preferably 1 to 10 amino acids, and the same applies when amino acids are added to the N-terminus and / or C-terminus. The same also applies to GpUroB1. In addition, the amino acid substitutions, protein homology, stringent conditions, and the like are the same as those for the enzymes described in the section "1-1-1(A). Enzymes that dehydroxylate the 4-hydroxyl group of urolithins." The same also applies to GpUroB1.

[0067] The above amino acid substitutions, protein homology, stringent conditions, and the like also apply to GuUroB2. However, with respect to GuUroB2, the above "one to more than one" is preferably 1 to 12, more preferably 1 to 11, even more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3. This also applies to GpUroB2. The above amino acid substitutions, protein homology, stringent conditions, and the like also apply to GuUroB3. However, with respect to GuUroB3, the above "one to more than one" is preferably 1 to 20, more preferably 1 to 19, even more preferably 1 to 18, even more preferably 1 to 10, particularly preferably 1 to 5, and even more particularly preferably 1 to 3. This also applies to GpUroB3.

[0068] The activity of the enzyme according to this embodiment to dehydroxylate the hydroxyl group at position 10 of urolithins can be evaluated, for example, by quantifying urolithin C produced as a product from urolithin M6, in a manner similar to that described in Experimental Example 5 below.

[0069] <1-1-2(B). Polynucleotide> Another aspect of the present disclosure is The nucleic acid sequence includes one or more selected from the group consisting of the nucleic acid sequence represented by SEQ ID NO: 10, the nucleic acid sequence represented by SEQ ID NO: 11, and the nucleic acid sequence represented by SEQ ID NO: 12, or It is a polynucleotide comprising one or more selected from the group consisting of the base sequence represented by SEQ ID NO: 22, the base sequence represented by SEQ ID NO: 23, and the base sequence represented by SEQ ID NO: 24. Details of each base sequence are as described in the above section “1-1-1(B). Enzyme that dehydroxylates the 10-hydroxyl group of urolithins.”

[0070] The base sequence of the gene encoding GuUroB1 may be a base sequence that hybridizes under stringent conditions with the base sequence represented by SEQ ID NO: 10, so long as GuUroB1, when alone as a subunit or when it forms a unit together with the other subunits GuUroB2 and / or GuUroB3, has the activity of dehydroxylating the hydroxyl group at position 10 of urolithins. Examples of "stringent conditions" include conditions under which polynucleotides having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more hybridize to each other, but polynucleotides having a lower homology than that do not hybridize to each other. This also applies to the nucleotide sequence of the gene encoding GuUroB2 and the nucleotide sequence of the gene encoding GuUroB3, as well as the nucleotide sequence of the gene encoding GpUroB1, the nucleotide sequence of the gene encoding GpUroB2, and the nucleotide sequence of the gene encoding GpUroB3.

[0071] <1-1-3(B). Genetic Engineering Aspects> Another aspect of the present disclosure is a method for producing a protein encoded by the polynucleotide, the method comprising the steps of: a recombinant vector containing the polynucleotide; a transformant carrying the polynucleotide in an expressible state or carrying the vector in an expressible state; and culturing the transformant. The protein is the "enzyme that dehydroxylates the hydroxyl group at position 10 of urolithins." The polynucleotide can be inserted into a known expression vector to produce an expression vector that expresses the enzyme. The expression vector can then be used to transform a microorganism or the like to obtain a transformant, which can then be cultured or otherwise used to produce the enzyme, thereby obtaining the enzyme. For other details, the descriptions in the above section "1-1-3(A). Genetic Engineering Aspects" are incorporated by reference.

[0072] <1-2-1(B). Method for dehydroxylating the 10-hydroxyl group of urolithins> Another aspect of the present disclosure is a method for dehydroxylating the hydroxyl group at position 10 of urolithins. More specifically, it is a method for dehydroxylating the hydroxyl group at position 10 of urolithins having a hydroxyl group at the 10 position. The present embodiment is a method for dehydroxylating a hydroxyl group at position 10 of a urolithin, comprising the following step (I): Step (I): A step of contacting one or more selected from the following (i) to (iv) with a urolithin having a hydroxyl group at the 10-position to dehydroxylate the hydroxyl group at the 10-position: (i) an enzyme described in the above section "1-1-1(B). Enzymes that dehydroxylate the hydroxyl group at position 10 of urolithins"; (ii) a protein encoded by a polynucleotide described in the above section "1-1-2(B). Polynucleotides"; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above.

[0073] (Urolithins) For details of urolithins in this embodiment, the content described above in the section "1-1-1(B). Enzymes that dehydroxylate the hydroxyl group at position 10 of urolithins" is cited.

[0074] (Process (I)) In step (I), (i) the enzyme and (ii) the protein encoded by the polynucleotide each come into contact with urolithins having a hydroxyl group at position 10, thereby dehydroxylating the hydroxyl group at position 10 present in the urolithins. In addition, in (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii), and (iv) a treated product of the microorganism described in (iii), the enzyme or protein contained in the microorganism or the treated product of the microorganism comes into contact with urolithins having a hydroxyl group at position 10, thereby dehydroxylating the hydroxyl group at position 10 present in the urolithins.

[0075] (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) the microorganism in the processed product of the microorganism described in (iii) may be a microorganism obtained by genetic engineering techniques. Examples of such microorganisms include those belonging to the genus Gordonibacter and those belonging to the genus Eggerthella. Preferred specific examples of microorganisms belonging to the genus Gordonibacter are the same as those described in the section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins." The microorganism belonging to the genus Eggerthella is preferably Eggerthella sp. DC3563 (NITE BP-02376) strain which belongs to the genus Eggerthella but does not fall into any existing species. The Eggerthella sp. DC3563 (NITE BP-02376) strain was deposited internationally on November 11, 2016 at the NITE Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) based on the Budapest Treaty. The Eggerthella sp. DC3563 (NITE BP-02376) strain is not limited to the same strain, and may be a bacterium substantially equivalent to the deposited strain. The substantially equivalent bacterium is a microorganism belonging to the genus Eggerthella, capable of exerting the effect of the present embodiment, such as dehydroxylating the hydroxyl group at the 10th position of urolithins having a hydroxyl group at the 10th position, and further having a base sequence of its 16S rRNA gene that is preferably 98% or more, more preferably 99% or more, and more preferably 100% homologous to the base sequence of the 16S rRNA gene of the deposited strain, and preferably having the same bacteriological properties as the deposited strain. In addition, the bacterium may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, or the like, so long as the effect of the present embodiment is not impaired.

[0076] For other details, the description in the section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins" above is incorporated by reference.

[0077] <1-3-1. Method for producing urolithin C (first embodiment)> Another aspect of the present disclosure is a method for producing urolithin C (first aspect). This aspect is a method for producing urolithin C, comprising the following steps (I) to (III): Step (I): Allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin M6. (i) an enzyme described in the above section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins"; (ii) a protein encoded by a polynucleotide described in the above section "1-1-1(A). Polynucleotides"; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with the urolithin M6 to produce urolithin C. (v) an enzyme described in the above section "1-1-1(B). Enzymes that dehydroxylate the hydroxyl group at position 10 of urolithins"; (vi) a protein encoded by a polynucleotide described in the above section "1-1-1(B). Polynucleotides"; (vii) a microorganism producing the enzyme described in (v) or the protein described in (vi); (viii) A treated product of the microorganism described in (vii) above.

[0078] (Process (I)) In step (I), a microorganism capable of producing urolithin M5 from ellagic acid is allowed to produce urolithin M5 from ellagic acid.

[0079] Microorganisms capable of producing urolithin M5 from ellagic acid include the microorganisms described in step (I) of <1-2-1(B). Method for dehydroxylating the hydroxyl group at position 10 of urolithins> above.

[0080] Examples of methods for producing urolithin M5 from ellagic acid include an embodiment in which the subject that comes into contact with urolithins having a hydroxyl group at the 10th position in the section "1-2-1(B). Method for dehydroxylating the hydroxyl group at position 10 of urolithins" is a microorganism that produces an enzyme described in the section "1-1-1(B). Enzyme that dehydroxylates the hydroxyl group at position 10 of urolithins", or a microorganism that produces a protein encoded by a polynucleotide described in the section "1-1-1(B). Polynucleotide".

[0081] (Step (II)) In step (II), one or more selected from the following (i) to (iv) are contacted with the urolithin M5 to produce urolithin M6. (i) an enzyme described in the above section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins"; (ii) a protein encoded by a polynucleotide described in the above section "1-1-1(A). Polynucleotides"; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above.

[0082] In step (II), (i) the enzyme and (ii) the protein encoded by the polynucleotide each dehydroxylate the hydroxyl group at position 4 of urolithin M5 upon contact with the urolithin M5 having a hydroxyl group at position 4. In addition, in (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii), and (iv) a treated product of the microorganism described in (iii), the enzyme or protein contained in the microorganism or the treated product of the microorganism dehydroxylates the hydroxyl group at position 4 of urolithin M5 upon contact with the urolithin M5 having a hydroxyl group at position 4. The mode of this process is similar to the details described in the section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins."

[0083] (Process (III)) In step (III), one or more selected from the following (v) to (viii) are contacted with the urolithin M6 to produce urolithin C. (v) an enzyme described in the above section "1-1-1(B). Enzymes that dehydroxylate the hydroxyl group at position 10 of urolithins"; (vi) a protein encoded by a polynucleotide described in the above section "1-1-1(B). Polynucleotides"; (vii) a microorganism producing the enzyme described in (v) or the protein described in (vi); (viii) A treated product of the microorganism described in (vii) above.

[0084] In step (III), (v) the enzyme and (vi) the protein encoded by the polynucleotide each dehydroxylate the hydroxyl group at position 10 of urolithin M6 upon contact with the urolithin M6 having a hydroxyl group at position 10. In addition, in (vii) a microorganism that produces the enzyme described in (v) or the protein described in (vi), and (viii) a treated product of the microorganism described in (vii), the enzyme or protein contained in the microorganism or the treated product of the microorganism dehydroxylates the hydroxyl group at position 10 of urolithin M6 upon contact with the urolithin M6 having a hydroxyl group at position 10. The mode of this process is similar to the details described in the section "1-2-1(B). Method for dehydroxylating the hydroxyl group at position 10 of urolithins" above.

[0085] One or more selected from the group consisting of steps (I) to (III) may be carried out in the same system. The phrase "carried out in the same system" means that the urolithin M5 produced in step (I) is used as is as urolithin M5 in step (II), urolithin M6 is produced in step (II), and the urolithin M6 is used as is as urolithin M6 in step (III), to produce urolithin C in step (III), all of which are carried out continuously in the same system. In other words, it does not include, for example, a step of separating and / or purifying urolithin M5 produced in step (I). When microorganisms are used in each step, the microorganisms in each step may be the same or different.

[0086] This embodiment may include other steps. For example, it may include a step of quantifying the obtained urolithin C (quantification step), a step of purifying (purification step), or a step of concentrating (concentration step). The details thereof are similar to those described in the above section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins". Furthermore, it may include a step of powdering the solution containing urolithin C by freeze-drying, spray-drying, or the like. In powderization, excipients such as lactose, dextrin, and corn starch can also be added.

[0087] <1-3-2. Method for producing urolithin C (second embodiment)> Another aspect of the present disclosure is a method for producing urolithin C (second aspect). This aspect is a method for producing urolithin C, comprising the following steps (I) to (III): Step (I): Allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin D. (i) an enzyme described in the above section "1-1-1(B). Enzymes that dehydroxylate the hydroxyl group at position 10 of urolithins"; (ii) a protein encoded by a polynucleotide described in the above section "1-1-1(B). Polynucleotides"; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with urolithin D to produce urolithin C: (v) an enzyme described in the above section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins"; (vi) a protein encoded by a polynucleotide described in the above section "1-1-1(A). Polynucleotides"; (vii) a microorganism producing the enzyme described in (v) or the protein described in (vi); (viii) A treated product of the microorganism described in (vii) above.

[0088] (Process (I)) With regard to step (I), the contents described above in "1-3-1. Production method of urolithin C (first aspect)" are incorporated by reference.

[0089] (Step (II)) In step (II), one or more selected from the following (i) to (iv) are contacted with the urolithin M5 to produce urolithin D. (i) an enzyme described in the above section "1-1-1(B). Enzymes that dehydroxylate the hydroxyl group at position 10 of urolithins"; (ii) a protein encoded by a polynucleotide described in the above section "1-1-1(B). Polynucleotides"; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above.

[0090] In step (II), (i) the enzyme and (ii) the protein encoded by the polynucleotide each dehydroxylate the hydroxyl group at position 10 of urolithin M5 upon contact with the urolithin M5 having a hydroxyl group at position 10. In addition, in (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii), and (iv) a treated product of the microorganism described in (iii), the enzyme or protein contained in the microorganism or the treated product of the microorganism dehydroxylates the hydroxyl group at position 10 of urolithin M5 upon contact with the urolithin M5 having a hydroxyl group at position 10. The mode of this process is similar to the details described in the section "1-2-1(B). Method for dehydroxylating the hydroxyl group at position 10 of urolithins" above.

[0091] In step (III), one or more selected from the following (v) to (viii) are contacted with urolithin D to produce urolithin C. (v) an enzyme described in the above section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins"; (vi) a protein encoded by a polynucleotide described in the above section "1-1-1(A). Polynucleotides"; (vii) a microorganism producing the enzyme described in (v) or the protein described in (vi); (viii) A treated product of the microorganism described in (vii) above.

[0092] In step (III), (v) the enzyme and (vi) the protein encoded by the polynucleotide each come into contact with urolithin D having a hydroxyl group at position 4, thereby dehydroxylating the hydroxyl group at position 4 of the urolithin D. In addition, in (vii) a microorganism that produces the enzyme described in (v) or the protein described in (vi), and (viii) a treated product of the microorganism described in (vii), the enzyme or protein contained in the microorganism or the treated product of the microorganism comes into contact with urolithin D having a hydroxyl group at position 4, thereby dehydroxylating the hydroxyl group at position 4 of the urolithin D. The mode of this process is similar to the details described in the section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins."

[0093] Steps (I) to (III) may be carried out in the same system. The phrase "carried out in the same system" means that the series of steps from when urolithin M5 produced in step (I) is used as urolithin M5 in step (II) to produce urolithin D in step (II), and when urolithin D is used as urolithin D in step (III) to produce urolithin C in step (III) are carried out continuously in the same system. In other words, it does not include, for example, a step of separating and / or purifying urolithin M5 produced in step (I). When microorganisms are used in each step, the microorganisms in each step may be the same or different.

[0094] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing Urolithin C (First embodiment)."

[0095] <1-3-3. Method for producing urolithin A (first embodiment)> Another aspect of the present disclosure is a method for producing urolithin A (first aspect). This aspect is a method for producing urolithin A, which includes the following step (I): Step (I): A step of allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C produced in the above "1-3-1. Method for producing urolithin C (first aspect)".

[0096] In this aspect, the steps included in the above-mentioned "1-3-1. Production method of urolithin C (first aspect)" are described below. In this case, the above step (I) corresponds to the following step (IV).

[0097] That is, the present embodiment is a method for producing urolithin A, comprising the following steps (I) to (IV): Step (I): Allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin M6. (i) an enzyme described in the above section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins"; (ii) a protein encoded by a polynucleotide described in the above section "1-1-1(A). Polynucleotides"; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with the urolithin M6 to produce urolithin C. (v) an enzyme described in the above section "1-1-1(B). Enzymes that dehydroxylate the hydroxyl group at position 10 of urolithins"; (vi) a protein encoded by a polynucleotide described in the above section "1-1-1(B). Polynucleotides"; (vii) a microorganism producing the enzyme described in (v) or the protein described in (vi); (viii) A treated product of the microorganism described in (vii) above. Step (IV): Allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C.

[0098] (Step (I) to step (III)) With regard to steps (I) to (III), the contents described above in "1-3-1. Production method of urolithin C (first aspect)" are incorporated by reference.

[0099] (Step (IV)) In step (IV), a microorganism capable of producing urolithin A from urolithin C is allowed to produce urolithin A from the urolithin C. Examples of microorganisms capable of producing urolithin A from urolithin C include microorganisms belonging to the genus Clostridium.

[0100] Among microorganisms belonging to the genus Clostridium, microorganisms belonging to Clostridium bolteae, Clostridium asparagiforme, and Clostridium citroniae are preferred. Furthermore, among microorganisms belonging to Clostridium bolteae, the DSM 29485 strain, the DSM 15670 strain, and the JCM 12243 strain are more preferable, and among these, the DSM 15670 strain is even more preferable. Among microorganisms belonging to Clostridium asparagiforme, the DSM 15981 strain is preferred. Among microorganisms belonging to Clostridium citroniae, the DSM 19261 strain is preferred.

[0101] As a Clostridium sp. that belongs to the genus Clostridium but does not correspond to an existing species, Clostridium sp. DC 3656 (NITE ABP-02708) strain is preferred. The Clostridium sp. DC 3656 (NITE ABP-02708) strain was deposited in Japan on May 8, 2018 at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (NITE) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under the accession number NITE P-02708. Subsequently, a request was made to transfer the strain to an international deposit under the Budapest Treaty at the NITE Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) (receipt date: July 15, 2020), and the strain was assigned the receipt number NITE ABP-02708.

[0102] Clostridium sp. DC 3656 (NITE ABP-02708) is not limited to the same strain as the deposited strain, and may be a bacterium substantially equivalent to the deposited strain. The substantially equivalent bacterium is a microorganism belonging to the genus Clostridium, capable of producing the effect of the present embodiment, such as the ability to produce urolithin A from urolithin C, and further having a base sequence of its 16S rRNA gene that is preferably 98% or more, more preferably 99% or more, and more preferably 100% homologous to the base sequence of the 16S rRNA gene of the deposited strain, and preferably having the same bacteriological properties as the deposited strain. In addition, the bacterium may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, or the like, so long as the effect of the present disclosure is not impaired. This also applies to Clostridium bolteae strains DSM 29485, DSM 15670, and JCM 12243, Clostridium asparagiforme strain DSM 15981, and Clostridium citroniae strain DSM 19261.

[0103] Examples of methods for producing urolithin A from urolithin C include an embodiment in which the subject brought into contact with urolithins having a hydroxyl group at position 4 in the above section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins" is a microorganism that produces an enzyme described in the above section "1-1-1(A). Enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins", or a microorganism that produces a protein encoded by a polynucleotide described in the above section "1-1-1(A). Polynucleotide".

[0104] One or more steps selected from the group consisting of steps (I) to (IV) may be carried out in the same system. The phrase "carried out in the same system" means that the urolithin M5 produced in step (I) is used as is as urolithin M5 in step (II), urolithin M6 is produced in step (II), the urolithin M6 is used as is as urolithin M6 in step (III), and urolithin C produced in step (III) is used as is as urolithin C in step (IV), and a series of steps up to the production of urolithin A in step (IV) are carried out continuously in the same system. In other words, it means that the process does not include, for example, a step of separating and / or purifying urolithin M5 produced in step (I). When microorganisms are used in each step, the microorganisms in each step may be the same or different.

[0105] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing Urolithin C (First embodiment)."

[0106] <1-3-4. Method for producing urolithin A (second embodiment)> Another aspect of the present disclosure is a method for producing urolithin A (second aspect). This aspect is a method for producing urolithin A, which includes the following step (I): Step (I): A step of allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C produced in the above "1-3-2. Production method of urolithin C (second aspect)".

[0107] In this aspect, the steps included in the above "1-3-2. Production method of urolithin C (second aspect)" are described below. In this case, the above step (I) corresponds to the following step (IV).

[0108] That is, the present embodiment is a method for producing urolithin A, comprising the following steps (I) to (IV): Step (I): Allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin D. (i) an enzyme described in the above section "1-1-1(B). Enzymes that dehydroxylate the hydroxyl group at position 10 of urolithins"; (ii) a protein encoded by a polynucleotide described in the above section "1-1-1(B). Polynucleotides"; (iii) a microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with urolithin D to produce urolithin C: (v) an enzyme described in the above section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins"; (vi) a protein encoded by a polynucleotide described in the above section "1-1-1(A). Polynucleotides"; (vii) a microorganism producing the enzyme described in (v) or the protein described in (vi); (viii) A treated product of the microorganism described in (vii) above. Step (IV): Allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C.

[0109] (Step (I) to step (III)) With regard to steps (I) to (III), the contents described above in "1-3-1. Production method of urolithin C (first aspect)" are incorporated by reference.

[0110] (Step (IV)) In step (IV), a microorganism capable of producing urolithin A from urolithin C is allowed to produce urolithin A from the urolithin C. Details of step (IV) are referred to above in the section "1-3-3. Production method of urolithin A (first aspect)".

[0111] One or more steps selected from the group consisting of steps (I) to (IV) may be carried out in the same system. The phrase "carried out in the same system" means that the urolithin M5 produced in step (I) is used as is as urolithin M5 in step (II), urolithin D is produced in step (II), the urolithin D is used as is as urolithin D in step (III), and urolithin C produced in step (III) is used as is as urolithin C in step (IV), and a series of steps up to the production of urolithin A in step (III) are carried out continuously in the same system. In other words, it means that the process does not include, for example, a step of separating and / or purifying urolithin M5 produced in step (I). When microorganisms are used in each step, the microorganisms in each step may be the same or different.

[0112] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing Urolithin C (First embodiment)."

[0113] <2-1-1. Ellagic acid transporter> Another aspect of the present disclosure is an ellagic acid transporter. The ellagic acid transporter according to this embodiment is preferably derived from a microorganism belonging to the genus Gordonibacter. More preferably, the microorganism is one or more selected from the group consisting of microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and microorganisms belonging to Gordonibacter faecihominis. A preferred microorganism belonging to Gordonibacter urolithinfaciens is the Gordonibacter urolithinfaciens DSM 27213 strain. A preferred microorganism belonging to the genus Gordonibacter pamelaeae is the DSM 19378 strain. A preferred microorganism belonging to the genus Gordonibacter faecihominis is the JCM 16058 strain.

[0114] The amino acid sequence of the ellagic acid transporter derived from Gordonibacter urolithinfaciens DSM 27213 strain is the sequence shown in SEQ ID NO: 25. The nucleotide sequence of the gene encoding the ellagic acid transporter is the sequence shown in SEQ ID NO: 26. The amino acid sequence of the ellagic acid transporter derived from Gordonibacter pamelaeae DSM 19378 strain is the sequence shown in SEQ ID NO: 27. The nucleotide sequence of the gene encoding the ellagic acid transporter is the sequence shown in SEQ ID NO: 28. Therefore, the ellagic acid transporter according to this embodiment is preferably a protein (ellagic acid transporter) that includes the amino acid sequence represented by SEQ ID NO: 25 or the amino acid sequence represented by SEQ ID NO: 27.

[0115] Furthermore, as long as the ellagic acid transporter derived from the Gordonibacter urolithinfaciens DSM 27213 strain has ellagic acid transporter activity, it may be a protein consisting of amino acids in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added in the amino acid sequence shown in SEQ ID NO: 25. The term "one or more" means preferably 1 to 50, more preferably 1 to 49, even more preferably 1 to 48, still more preferably 1 to 30, particularly preferably 1 to 10, and even more particularly preferably 1 to 5, and the same applies when amino acids are added to the N-terminus and / or C-terminus.

[0116] The mode of substitution is the same as that described in the section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins."

[0117] Furthermore, the ellagic acid transporter derived from Gordonibacter urolithinfaciens DSM 27213 strain may be a protein that has a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more to the full length amino acid sequence represented by SEQ ID NO:25, as long as it has ellagic acid transporter activity.

[0118] Furthermore, the ellagic acid transporter derived from Gordonibacter urolithinfaciens DSM 27213 strain may be a protein encoded by a polynucleotide that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 26, so long as it has ellagic acid transporter activity. Examples of "stringent conditions" include conditions under which polynucleotides having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more hybridize with each other, and polynucleotides having a lower homology than that do not hybridize with each other.

[0119] The above amino acid substitutions, protein homology, stringent conditions, and the like also apply to the ellagic acid transporter derived from Gordonibacter pamelaeae strain DSM 19378. However, with respect to the ellagic acid transporter derived from the DSM 19378 strain, the above "one to more" preferably means 1 to 52, more preferably 1 to 51, further preferably 1 to 50, even further preferably 1 to 30, particularly preferably 1 to 10, and further particularly preferably 1 to 5.

[0120] The ellagic acid transporter activity of the ellagic acid transporter of this embodiment can be evaluated, for example, as described below in Experimental Example 18-1.

[0121] <2-1-2. Polynucleotides Containing Genes Encoding Ellagic Acid Transporters> Another embodiment of the present disclosure is a polynucleotide comprising the base sequence represented by SEQ ID NO: 26, or comprising the base sequence represented by SEQ ID NO: 28. Details of each base sequence are as described above.

[0122] The nucleotide sequence of the gene encoding the ellagic acid transporter derived from Gordonibacter urolithinfaciens DSM 27213 strain may be a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 26, so long as the protein encoded thereby has ellagic acid transporter activity. "Stringent conditions" include, for example, conditions under which polynucleotides having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more hybridize with each other, and polynucleotides having a lower homology than that do not hybridize with each other. This also applies to the nucleotide sequence of the gene encoding the ellagic acid transporter derived from Gordonibacter pamelaeae DSM 19378 strain.

[0123] <2-1-3. Genetic engineering aspects> Another aspect of the present disclosure is a method for producing a protein encoded by the polynucleotide, comprising the steps of: a recombinant vector comprising the "2-1-2. Polynucleotide comprising a gene encoding an ellagic acid transporter"; a transformant retaining the "2-1-2. Polynucleotide comprising a gene encoding an ellagic acid transporter" in an expressible state or retaining the vector in an expressible state; and culturing the transformant. The protein is the "ellagic acid transporter." The details are the same as those described in the above section "1-1-3(A). Genetic Engineering Aspects."

[0124] In addition, the transformant has enhanced uptake of ellagic acid from the outside of the transformant into the transformant. Therefore, a preferred embodiment is a method for enhancing uptake of ellagic acid from the outside of the transformant into the transformant, the method comprising the following step (I): Step (I): contacting the transformant with ellagic acid. Details of the transformant are the same as those described in the above section "1-1-3(A). Genetic engineering aspects." In addition, examples of "an aspect in which the transformant is contacted with ellagic acid" include an aspect in which a microorganism that produces the enzyme described in (i) above or a microorganism that produces the protein described in (ii) above is used in the above section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins."

[0125] <2-1-4. Method for producing urolithin M5 (first embodiment)> Another aspect of the present disclosure is a method for producing urolithin M5 (first aspect). This aspect is a method for producing urolithin M5, comprising the following step (I): Step (I): A step of contacting a transformant described in the above section "2-1-3. Genetic engineering aspects", whose host is a microorganism having the ability to produce urolithin M5 from ellagic acid, with ellagic acid to produce urolithin M5 from ellagic acid.

[0126] (Process (I)) In step (I), the transformant described above in "2-1-3. Genetic engineering aspects" is contacted with ellagic acid, wherein the host is a microorganism having the ability to produce urolithin M5 from ellagic acid, to produce urolithin M5 from ellagic acid.

[0127] The host microorganism capable of producing urolithin M5 from ellagic acid may be Escherichia coli, which is commonly used as a host for transformation, but examples include the microorganisms described in the above section "1-2-1(B). Method for dehydroxylating the hydroxyl group at position 10 of urolithins".

[0128] An example of the contact between the transformant and ellagic acid is the case where a microorganism that produces the enzyme described in (i) above or a microorganism that produces the protein described in (ii) above is used in the section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins".

[0129] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing urolithin C (first embodiment)". Furthermore, for example, urolithins may be produced according to the method for producing urolithins described herein using the produced urolithin M5. Furthermore, this embodiment may be practiced in the step of producing urolithin M5 from ellagic acid in the method for producing urolithins described herein.

[0130] <2-1-5. Method for producing urolithin C (third embodiment)> Another aspect of the present disclosure is a method for producing urolithin C (third aspect). This aspect is a method for producing urolithin C, comprising the following step (I): Step (I): A step of contacting the transformant described in the above section "2-1-3. Genetic engineering aspects", whose host is a microorganism having the ability to produce urolithin C from ellagic acid, with ellagic acid to produce urolithin C from ellagic acid.

[0131] (Process (I)) In step (I), the transformant described in the above section "2-1-3. Genetic engineering aspects", whose host is a microorganism having the ability to produce urolithin C from ellagic acid, is contacted with ellagic acid.

[0132] Examples of a microorganism capable of producing urolithin C from ellagic acid include Escherichia coli, which is commonly used as a host for transformation, but examples include the microorganisms described in the above section "1-3-1. Method for producing urolithin C (first aspect)".

[0133] An example of the contact between the transformant and ellagic acid is the case where a microorganism that produces the enzyme described in (i) above or a microorganism that produces the protein described in (ii) above is used in the section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins".

[0134] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing urolithin C (first embodiment)". Furthermore, for example, urolithins may be produced using the produced urolithin C according to the method for producing urolithins described herein. Furthermore, this embodiment may be practiced in the step of producing urolithin C from ellagic acid in the method for producing urolithins described herein.

[0135] <2-1-6. Method for producing urolithin A (third embodiment)> Another aspect of the present disclosure is a method for producing urolithin A (third aspect). This aspect is a method for producing urolithin A, comprising the following step (I): Step (I): A step of allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C produced in the above "2-1-5. Production method of urolithin C (third aspect)".

[0136] In this aspect, the steps included in the above "2-1-5. Production method of urolithin C (third aspect)" are described below. In this case, the above step (I) corresponds to the following step (II).

[0137] That is, the present embodiment is a method for producing urolithin A, comprising the following steps (I) and (II): Step (I): A step of contacting the transformant described in the above section "2-1-3. Genetic engineering aspects", whose host is a microorganism having the ability to produce urolithin C from ellagic acid, with ellagic acid to produce urolithin C from ellagic acid. Step (II): Allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C.

[0138] (Process (I)) With regard to step (I), the contents described above in "2-1-5. Production method of urolithin C (third aspect)" are incorporated by reference.

[0139] (Step (II)) In step (II), a microorganism capable of producing urolithin A from urolithin C is allowed to produce urolithin A from the urolithin C. Details of step (II) are referred to above in the section "1-3-3. Production method of urolithin A (first aspect)".

[0140] Step (I) and step (II) may be carried out in the same system. The phrase "carried out in the same system" means that urolithin C produced in step (I) is used as is as urolithin C in step (II), and the series of steps up to the production of urolithin A in step (II) are carried out continuously in the same system. In other words, it means that the process does not include, for example, a step of separating and / or purifying urolithin C produced in step (I). When microorganisms are used in each step, the microorganisms in each step may be the same or different.

[0141] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing Urolithin C (First embodiment)."

[0142] <2-2-1. Lactonase> Another aspect of the present disclosure is a lactonase. Lactonase has the activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, for example, the activity of catalyzing a reaction of producing urolithin M5 from ellagic acid.

[0143] The lactonase according to this embodiment is preferably derived from a microorganism belonging to the genus Gordonibacter. More preferably, the microorganism is one or more selected from the group consisting of microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and microorganisms belonging to Gordonibacter faecihominis. A preferred microorganism belonging to Gordonibacter urolithinfaciens is the Gordonibacter urolithinfaciens DSM 27213 strain. A preferred microorganism belonging to the genus Gordonibacter pamelaeae is the DSM 19378 strain. A preferred microorganism belonging to the genus Gordonibacter faecihominis is the JCM 16058 strain.

[0144] The amino acid sequence of the lactonase possessed by Gordonibacter urolithinfaciens DSM 27213 strain is the sequence shown in SEQ ID NO: 29. The nucleotide sequence of the gene encoding the lactonase is the sequence shown in SEQ ID NO: 30. The amino acid sequence of the lactonase derived from Gordonibacter pamelaeae DSM 19378 strain is the sequence shown in SEQ ID NO: 31. The nucleotide sequence of the gene encoding the lactonase is the sequence shown in SEQ ID NO: 32. Therefore, the lactonase according to this embodiment is preferably a protein (lactonase) comprising the amino acid sequence shown in SEQ ID NO:29 or comprising the amino acid sequence shown in SEQ ID NO:31.

[0145] Furthermore, so long as it has lactonase activity, the lactonase derived from Gordonibacter urolithinfaciens DSM 27213 strain may be a protein consisting of amino acids in which one or more amino acids have been substituted or deleted, or one or more amino acids have been inserted or added in the amino acid sequence shown in SEQ ID NO: 29. The term "one or more" means preferably 1 to 36, more preferably 1 to 35, even more preferably 1 to 20, still more preferably 1 to 10, and particularly preferably 1 to 5, and the same applies when amino acids are added to the N-terminus and / or C-terminus.

[0146] The mode of substitution is the same as that described in the section "1-1-1(A). Enzymes that dehydroxylate the hydroxyl group at position 4 of urolithins."

[0147] Furthermore, the lactonase derived from Gordonibacter urolithinfaciens DSM 27213 strain may be a protein having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more to the full length amino acid sequence represented by SEQ ID NO:29, as long as it has lactonase activity. Furthermore, lactonase derived from Gordonibacter urolithinfaciens DSM 27213 strain may be a protein encoded by a polynucleotide that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 30, so long as it has lactonase activity. "Stringent conditions" include, for example, conditions under which polynucleotides having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more hybridize with each other, and polynucleotides having a lower homology than that do not hybridize with each other.

[0148] The substitution of the amino acids, protein homology, stringent conditions, etc. are also applicable to the ellagic acid transporter derived from the strain Gordonibacter pamelaeae DSM 19378. However, with respect to the ellagic acid transporter derived from the strain Gordonibacter pamelaeae DSM 19378, the "1 to a plurality" preferably refers to 1 to 36, more preferably 1 to 35, still more preferably 1 to 20, even more preferably 1 to 10, and particularly preferably 1 to 5.

[0149] The lactonase activity by the lactonase according to the present disclosure can be evaluated, for example, as follows. Prepare a reaction solution containing 20 mM potassium phosphate buffer (pH 6.5), 1 mg / mL ellagic acid, and lactonase, and after reacting at 37°C for 3 hours, measure the generated urolithin M5 by HPLC under the following conditions.

[0150] <HPLC conditions> Column: Cosmosil 5C18-AR-II (inner diameter 4.6 mm × length 150 mm) Eluent: ultrapure water (Milli Q water) / acetonitrile / formic acid = 80 / 20 / 1 Flow rate: 1 mL / min Column temperature: 40°C Detection: UV (349 nm)

[0151] <2-2-2. Polynucleotide containing a gene encoding lactonase> Another aspect of the present disclosure is a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 30 or containing the nucleotide sequence represented by SEQ ID NO: 32. The details of each nucleotide sequence are as already described.

[0152] The nucleotide sequence of the gene encoding lactonase derived from Gordonibacter urolithinfaciens DSM 27213 strain may be a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 30, so long as the protein encoded thereby has lactonase activity. "Stringent conditions" include, for example, conditions under which polynucleotides having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more hybridize with each other, and polynucleotides having a lower homology than that do not hybridize with each other. This also applies to the nucleotide sequence of the gene encoding lactonase from Gordonibacter pamelaeae DSM 19378 strain.

[0153] <2-2-3. Genetic engineering aspects> Another aspect of the present disclosure is a method for producing a protein encoded by the polynucleotide, comprising the steps of: a recombinant vector comprising the "2-2-2. Polynucleotide comprising a gene encoding lactonase"; a transformant that retains the "2-2-2. Polynucleotide comprising a gene encoding lactonase" in an expressible state or that retains the vector in an expressible state; and culturing the transformant. The protein is the "lactonase". The details are the same as those described in the above section "1-1-3(A). Genetic Engineering Aspects."

[0154] The transformant also has an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid. Therefore, a preferred embodiment is a method for promoting a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, the method comprising the following step (I): Step (I): contacting the transformant with ellagic acid. Details of the transformant are the same as those described in the above section "1-1-3(A). Genetic engineering aspects." In addition, examples of "an aspect in which the transformant is contacted with ellagic acid" include an aspect in which a microorganism that produces the enzyme described in (i) above or a microorganism that produces the protein described in (ii) above is used in the above section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins."

[0155] In a transformant that expressibly retains the "2-2-2. Polynucleotide comprising a gene encoding lactonase" or that expressibly retains the vector, it is preferable that the "2-1-2. Polynucleotide comprising a gene encoding an ellagic acid transporter" is also expressed. In other words, it is preferable that the lactonase and the ellagic acid transporter are coexpressed in the transformant.

[0156] <2-2-4. Method for producing urolithin M5 (second embodiment)> Another aspect of the present disclosure is a method for producing urolithin M5 (second aspect). This aspect is a method for producing urolithin M5, comprising the following step (I): Step (I): A step of contacting the transformant described in the above section "2-2-3. Genetic engineering aspects" with ellagic acid to produce urolithin M5 from ellagic acid.

[0157] (Process (I)) In step (I), the transformant described in the above section "2-2-3. Genetic engineering aspects" is contacted with ellagic acid to produce urolithin M5 from ellagic acid.

[0158] The transformant in the section "2-2-3. Genetic engineering aspects" above is a microorganism that expresses lactonase and has the ability to produce urolithin M5 from ellagic acid.

[0159] An example of the contact between the transformant and ellagic acid is the case where a microorganism that produces the enzyme described in (i) above or a microorganism that produces the protein described in (ii) above is used in the section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins".

[0160] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing urolithin C (first embodiment)". Furthermore, for example, urolithins may be produced according to the method for producing urolithins described herein using the produced urolithin M5. Furthermore, this embodiment may be practiced in the step of producing urolithin M5 from ellagic acid in the method for producing urolithins described herein.

[0161] <2-2-5. Method for producing urolithin C (fourth aspect)> Another aspect of the present disclosure is a method for producing urolithin C (fourth aspect). This aspect is a method for producing urolithin C, comprising the following step (I): Step (I): A step of producing urolithin M5 from ellagic acid by contacting the transformant, which is described in the above section "2-2-3. Genetic engineering aspects" and whose host is a microorganism having the ability to produce urolithin C from urolithin M5, with ellagic acid, and producing urolithin C from the urolithin M5.

[0162] (Process (I)) In step (I), the transformant described in the section "2-2-3. Genetic engineering aspects" above, wherein the host is a microorganism having the ability to produce urolithin C from urolithin M5, is contacted with ellagic acid to produce urolithin M5 from ellagic acid, and urolithin C is produced from the urolithin M5.

[0163] The transformant in the section "2-2-3. Genetic engineering aspects" above is a microorganism that expresses lactonase and has the ability to produce urolithin M5 from ellagic acid. Furthermore, a microorganism capable of producing urolithin C from its host, urolithin M5, may be Escherichia coli, which is commonly used as a host for transformation, but examples of such microorganisms include those described in the above section "1-2-1(B). Method for dehydroxylating the hydroxyl group at position 10 of urolithins".

[0164] An example of the contact between the transformant and ellagic acid is the case where a microorganism that produces the enzyme described in (i) above or a microorganism that produces the protein described in (ii) above is used in the section "1-2-1(A). Method for dehydroxylating the hydroxyl group at position 4 of urolithins".

[0165] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing urolithin C (first embodiment)". Furthermore, for example, urolithins may be produced according to the method for producing urolithins described herein using the produced urolithin C. Furthermore, this embodiment may be practiced in the step of producing urolithin M5 from ellagic acid and the step of producing urolithin C from urolithin M5 in the method for producing urolithins described herein.

[0166] <2-2-6. Method for producing urolithin A (fourth aspect)> Another aspect of the present disclosure is a method for producing urolithin A (fourth aspect). This aspect is a method for producing urolithin A, comprising the following steps (I) and (II): Step (I): A step of producing urolithin M5 from ellagic acid by contacting the transformant, which is described in the above section "2-2-3. Genetic engineering aspects" and whose host is a microorganism having the ability to produce urolithin C from urolithin M5, with ellagic acid, and producing urolithin C from the urolithin M5. Step (II): Allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C.

[0167] (Process (I)) With regard to step (I), step (I) in the section "2-2-5. Production method of urolithin C (fourth aspect)" is referred to.

[0168] (Step (II)) In step (II), a microorganism capable of producing urolithin A from urolithin C is allowed to produce urolithin A from the urolithin C. Details of step (II) are referred to above in the section "1-3-3. Production method of urolithin A (first aspect)".

[0169] Step (I) and step (II) may be carried out in the same system. The phrase "carried out in the same system" means that urolithin C produced in step (I) is used as is as urolithin C in step (II), and the series of steps up to the production of urolithin A in step (II) are carried out continuously in the same system. In other words, it means that the process does not include, for example, a step of separating and / or purifying urolithin C produced in step (I). When microorganisms are used in each step, the microorganisms in each step may be the same or different.

[0170] This embodiment may include other steps, the details of which are similar to those described in the above section "1-3-1. Method for producing Urolithin C (First embodiment)." EXAMPLES

[0171] Examples are set forth below, but none of the examples are to be construed in a limiting sense.

[0172] [Experimental Example 1] Preparation of bacterial cells for proteome analysis Gordonibacter urolithinfaciens DSM 27213 strain was inoculated into 10 mL of ABB medium (ANAEROBE BASAL BROTH, Oxoid, Code CM0957) containing or not containing 0.1% ellagic acid (Sigma-Aldrich), and cultured at 37°C for 16 days under an anaerobic gas (N2 / H2 / CO2 = 80:10:10) environment. The culture medium was centrifuged to collect the bacterial cells, which were then used for proteome analysis.

[0173] [Experimental Example 2] Proteome analysis The bacterial cells prepared in Experimental Example 1 were lysed using a lysis solution (7 M urea, 2 M thiourea, 2% CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate), 10 mM DTT (dithiothreitol), 50 mM Tris-HCl buffer (pH 7.0), and proteome analysis was performed using the method described in AMB Express, 2:37 (2012). As a result, proteins whose expression levels were significantly increased when cultured in a medium containing ellagic acid compared to when cultured in a medium not containing ellagic acid, i.e., proteins whose expression was induced by ellagic acid, were found. Analysis was based on genome information, and CD2938, CD2940, CD2941, CD2942, and CD2943, as well as CD2949, CD2950, ​​and CD2952 were identified. GENETYX (Genetyx Corporation) was used for analysis of genetic information such as homology analysis.

[0174] [Experimental Example 3] Protein function prediction The amino acid sequences of CD2938, CD2940, CD2941, CD2942, and CD2943 were subjected to a homology search with known proteins to predict their functions. The results are as follows: CD2938 is predicted to be an anaerobic dehydrogenase having Se-Cys (selenocysteine) and Mo (molybdenum)-pterin binding domains. CD2940 is predicted to be a "hydrogenase component having an Fe-S (iron-sulfur) cluster." CD2941 was shown to be homologous to a "cation-diverse drug efflux pump." We speculate that CD2938, CD2940, and CD2941 may be subunits that compose the enzyme that dehydroxylates the hydroxyl group at position 4 of urolithins. We named these genes GuuroA1, GuuroA2, and GuuroA3, which encode GuUroA1, GuUroA2, and GuUroA3, respectively. CD2942 is presumed to be a "metal-dependent hydrolase," and the gene was named uroH. This gene is a lactonase gene. The lactonase has the activity of catalyzing the hydrolysis of at least one of the two ester bonds present in ellagic acid. CD 2943 is predicted to be a permease belonging to the major facilitator superfamily and was named uroT. It is speculated to function as a transporter that imports ellagic acid into the bacterial cell in the biosynthetic pathway of urolithins.

[0175] CD2949 and CD2950, ​​like CD2938 and CD2940, are predicted to be "anaerobic dehydrogenases having Se-Cys (selenocysteine) and Mo (molybdenum)-pterin binding domains" and "hydrogenase components having Fe-S (iron-sulfur) clusters," respectively. CD2952 is speculated to be related to "heme utilization or adsorption." Therefore, we speculate that CD2949, CD2950, ​​and CD2952 may be subunits that compose the enzyme that dehydroxylates the 10-hydroxyl group of urolithins. We named these genes GuuroB1, GuuroB2, and GuuroB3, which encode GuUroB1, GuUroB2, and GuUroB3, respectively.

[0176] The homologies between CD2938 and CD2949, between CD2940 and CD2950, ​​and between CD2941 and CD2951 were 42%, 50%, and 17%, respectively, suggesting that "CD2938 and CD2940," "CD2949 and CD2950," "CD2938, CD2940, and CD2941," and "CD2949, CD2950, ​​and CD2952" may function as complexes.

[0177] [Experimental Example 4] Purification of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins (Microbial Culture) For preculture, 20 mL of modified GAM medium (Nihon Pharmaceutical Co., Ltd.) containing 0.1 mg / mL ellagic acid was added to a 100 mL Erlenmeyer flask, and Gordonibacter urolithinfaciens DSM 27213 strain was inoculated into the medium. The medium was then cultured in an anaerobic chamber for 5 to 6 days at 37°C with shaking at 80 rpm. The preculture solution was inoculated into a 2 L Erlenmeyer flask containing 1.75 L of the same medium, and cultured in an anaerobic chamber for 7 to 9 days at 37°C with shaking at 80 rpm. The resulting culture solution was centrifuged (8,000 rpm, 15 minutes, 4° C.) to prepare bacterial cells as a precipitate fraction.

[0178] (Preparation of ultracentrifugation supernatant) The obtained bacteria were suspended in 50 mM potassium phosphate buffer (KPB, pH 6.5) and disrupted using a KUBOTA Insonator Model 201M (Kubota Corporation). The ultrasonic disruption solution was centrifuged at 8,500 rpm and 4°C for 30 minutes, and the supernatant was centrifuged at 20,000 g for 60 minutes to obtain a supernatant, which was used as a cell-free extract. The cell-free extract was ultracentrifuged at 100,000 g for 110 minutes to obtain the supernatant fraction.

[0179] (Enzyme purification (Mono Q 1st time)) The resulting supernatant from the ultracentrifugation was subjected to anion exchange chromatography using a Mono Q 5 / 50 column (GE Healthcare Japan, Ltd.). Eluent A: 50 mM KPB (pH 6.5) Eluent B: 50 mM KPB (pH 6.5) and 1 M NaCl Flow rate: 0.8 mL / min Gradient elution was performed from eluent A to B, and the enzyme active fraction was collected and concentrated by gel filtration to obtain the 1st Mono Q fraction.

[0180] (Enzyme purification (Superdex 200)) The obtained 1st Mono Q fraction was subjected to gel filtration chromatography using Superdex 200 Increase 10 / 300 GL (GE Healthcare Japan, Ltd.). Eluent: 50 mM KPB (pH 6.5) and 150 mM NaCl Flow rate: 0.25 mL / min The enzyme active fractions were collected and concentrated by ultrafiltration to give the Superdex fraction.

[0181] (Enzyme purification (Mono Q 2nd time)) The resulting Superdex fraction was purified again by anion exchange chromatography on Mono Q. Eluent A: 50 mM KPB (pH 6.5) Eluent B: 50 mM KPB (pH 6.5) and 1 M NaCl Flow rate: 0.8 mL / min Gradient elution was performed from eluent A to B, and the enzyme active fraction was collected and concentrated by gel filtration to obtain the 2nd Mono Q fraction.

[0182] The purification steps of the enzyme so far are summarized in Table 1.

[0183] [Experimental Example 5] Measurement of the activity of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins For each fraction obtained in Experimental Example 4, the activity of dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins was measured. Urolithin M5 was used as the urolithin having a hydroxyl group at the 4-position. When the hydroxyl group at the 4-position is dehydrated and oxidized, urolithin M6 is produced. 30 μL of a reaction solution for measuring enzyme activity containing 1 mg / mL urolithin M5, 10 mM NADPH (reduced nicotinamide adenine dinucleotide), 0.2 mM FAD (flavin adenine dinucleotide), 10 mM MV (methyl viologen), 50 mM KPB (potassium phosphate buffer, pH 6.5), and each fraction was placed in a 200 μL tube for PCR. The gas phase was set as the gas phase in an anaerobic chamber (COY vinyl anaerobic chamber), and the reaction was carried out at 37 °C for 90 minutes. 60 μL of N,N-dimethylacetamide containing 1% formic acid was added to the reaction-terminated solution. After mixing, the supernatant of the centrifugation was analyzed by HPLC.

[0184] HPLC was performed under the following conditions to quantify the produced urolithin M6 (detection wavelength: 348 nm). <HPLC Conditions> Column: COSMOSIL5C18-ARII (inner diameter 4.6 mm × length 150 mm, manufactured by Nacalai Tesque) Eluent: ultrapure water (Milli Q water) / acetonitrile (CH3CN) / formic acid = 80 / 20 / 1 Flow rate: 1.0 mL / min Temperature: 40 °C Detection: PDA (photodiode array) One unit of enzyme activity was defined as the amount of enzyme that catalyzes the production of 1 μmol of urolithin M6 in 90 minutes. The results are shown in Table 1.

[0185]

Table 1

[0186] 〔Experimental Example 6〕Native-PAGE of the enzyme that dehydrates and oxidizes the hydroxyl group at the 4-position of urolithins and the enzyme that dehydrates and oxidizes the hydroxyl group at the 10-position of urolithins The purified enzyme fraction was subjected to non-denaturing polyacrylamide electrophoresis (Native-PAGE) using a 5-20% gradient gel (PAGEL, NPG-520L, ATTO). The resulting bands were semi-dry blotted onto a PVDF membrane (Immobilon-P Transfer membrane, Millipore) using a Polyase Prot AE6677G (Atto). Each band was excised and its sequence was analyzed using an amino acid sequencer.

[0187] The results are shown in Figure 1. In Figure 1, lane 1 is the molecular weight marker lane, lane 2 is the ultracentrifugation supernatant fraction lane, lane 3 is the Mono Q 1st fraction lane, lane 4 is the Superdex fraction lane, and lane 5 is the Mono Q 2nd fraction lane. Band I is predicted to be formate-tetrahydrofolate ligase based on its amino acid sequence. Two types of amino acid sequences were obtained from band II, which are presumed to be GuUroB1 (protein corresponding to gene GuuroB1) and GuUroB2 (protein corresponding to gene GuuroB2). Two types of amino acid sequences were also obtained for band III, which are presumed to be GuUroA1 (a protein corresponding to the GuuroA1 gene) and GuUroA2 (a protein corresponding to the GuuroA2 gene).

[0188] [Experimental Example 7] SDS-PAGE of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins and the enzyme that dehydroxylates the 10-hydroxyl group of urolithins The purified enzyme fraction was subjected to sodium dodecyl sulfate-electrophoresis (SDS-PAGE) using a 5-20% gradient gel (e-PAGEL, E-R520L, manufactured by ATTO) to evaluate its molecular weight. Protein Molecular Weight Marker (Broad) manufactured by Takara Bio was used as a molecular weight marker. The obtained bands were semi-dry blotted onto a PVDF membrane (Immobilon-P Transfer membrane, Millipore) using a Polizeprot AE6677G (Atto), and each band was excised and its sequence was analyzed using an amino acid sequencer.

[0189] The results are shown in Figure 2. In Figure 2, lane 1 is the molecular weight marker lane, lane 2 is the ultracentrifugation supernatant fraction lane, lane 3 is the Mono Q 1st fraction lane, lane 4 is the Superdex fraction lane, and lane 5 is the Mono Q 2nd fraction lane. Analysis of the amino acid sequence of band I and band II obtained from the 2nd Mono Q fraction in lane 5 revealed two different amino acid sequences. They were identified as GuUroA1 (protein corresponding to the GuuroA1 gene) and GuUroB1 (protein corresponding to the GuuroB1 gene), respectively. The molecular masses of the bands were estimated to be 90.0 kDa and 87.0 kDa. Band III is predicted to be formate-tetrahydrofolate ligase based on its amino acid sequence.

[0190] [Experimental Example 8] Effect of cofactors on the activity of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins Cofactors were added at the concentrations shown in Table 2 to the reaction solution obtained by removing NADPH, FAD, and MV from the reaction solution for measuring enzyme activity described in Experimental Example 5, and the amount of urolithin M6 produced after the reaction was quantified. The 1st Mono Q fraction was used as the enzyme. The amount of urolithin M6 produced under the conditions shown in No. 12 in Table 2 (10 mM MV, 10 mM NADPH, 0.2 mM FAD) was set to 100%, and the amount of urolithin M6 produced under each condition was expressed as relative activity.

[0191] The results are summarized in Table 2. When NADH, NADPH, FMN, or MV was added alone, activity was confirmed compared to when none of them was added. It was confirmed that in the presence of MV, the activity was synergistically increased when NADH or NADPH was also present. In the presence of MV and NADPH, and also in the presence of FAD or FMN, a very large activity was observed. The optimal conditions were a combination of 10 mM MV, 10 mM NADPH, and 0.2 mM FAD. In the following experimental examples, these conditions were used as standard activity measurement conditions.

[0192] [Table 2]

[0193] [Experimental Example 9] Temperature dependence of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins The activity was measured under the standard activity measurement conditions described in Experimental Example 8, with the reaction temperature being changed. The results are shown in Figure 3. The optimum temperature was 42°C, and the activity at that temperature was taken as 100%. In the range of 37 to 50°C, the activity was 50% or more.

[0194] [Experimental Example 10] pH dependence of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins Under the standard activity measurement conditions described in Experimental Example 8, activity measurements were performed while changing the pH by using the following buffer solutions. Citrate buffer: 50 mM citric acid-sodium citrate buffer Acetate buffer: 50 mM acetic acid-sodium acetate buffer Potassium phosphate buffer: 50 mM K2HPO4-KH2PO4 buffer Tris-HCl buffer: 50 mM Tris-HCl buffer Carbonate buffer: 50 mM Na2CO3-NaHCO3 buffer Sodium phosphate buffer: 50 mM Na3PO4-Na2HPO4 buffer The results are shown in Figure 4. The optimum pH was 6.0, and the activity was 80% or more of that under the optimum conditions in the pH range of 6.0 to 7.0.

[0195] [Experimental Example 11] Temperature stability of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins The enzyme was kept at 4 to 60° C. for 12 hours, and then activity was measured under the standard activity measurement conditions described in Experimental Example 8. The results are shown in Figure 5. The optimum temperature was 10°C, and assuming the residual activity at 10°C to be 100%, the residual activity was 80% or more at 4 to 20°C.

[0196] [Experimental Example 12] pH stability of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins 90 μL of each of the following 100 mM buffer solutions was added to 10 μL of the 1st Mono Q fraction as the enzyme, and the mixture was concentrated 10-fold by ultrafiltration and then kept at 4° C. for 12 hours. Then, 60 μL of 50 mM KPB (pH 6.5) was added to return the pH to normal, and the activity was measured under the standard activity measurement conditions described in Experimental Example 8. Citrate buffer: 50 mM citric acid-sodium citrate buffer Acetate buffer: 50 mM acetic acid-sodium acetate buffer Potassium phosphate buffer: 50 mM K2HPO4-KH2PO4 buffer Tris-HCl buffer: 50 mM Tris-HCl buffer Carbonate buffer: 50 mM Na2CO3-NaHCO3 buffer Sodium phosphate buffer: 50 mM Na3PO4-Na2HPO4 buffer The results are shown in Figure 6. The most stable pH was 6.0 (potassium phosphate buffer). Therefore, assuming the activity at pH 6.0 to be 100%, the enzyme showed a residual activity of 75% or more in the pH range of 5.0 to 9.0. The enzyme also showed a residual activity of 75% or more in the pH range of 10.0 to 11.0 (carbonate buffer).

[0197] [Experimental Example 13] Construction of a plasmid expressing ellagic acid transporter Genomic DNA was prepared from Gordonibacter urolithinfaciens DSM 27213. Using this as a template, the ellagic acid transporter gene (uroT) was amplified by PCR using the following primer set, and inserted into the BamHI and EcoRI sites of the expression vector pRSFDuet-1 to construct pRSFDuet-1_uroT. 5'-AAAGGATCCATGAGCAATCCGAATGCCGCCGTAGCGGCAAGC-3' (SEQ ID NO: 33) 5'-GTTGAATTCTTAGTGCTGTGCGGCTGCCTCGTCTGCGGGTTT-3' (SEQ ID NO: 34)

[0198] [Experimental Example 14] Construction of a plasmid expressing lactonase Genomic DNA was prepared from Gordonibacter urolithinfaciens DSM 27213 strain according to the standard method. The lactonase gene (uroH) was amplified by PCR using the following primer set with this as a template, and inserted into the BamHI and EcoRI sites of the expression vector pET-21b(+) to construct pET21b_UroH. 5'-GCCGGATCCCATGGCAGACAACAAGGTCATCGACATCAACATG-3' (SEQ ID NO: 35) 5'-TATGAATTCCTACAGGTTGAACAGCTTCGCCGCGTTGCCGCC-3' (SEQ ID NO: 36)

[0199] [Experimental Example 15] Expression of ellagic acid transporter in Escherichia coli E. coli Rosetta2 (DE3) strain was transformed with pRSFDuet-1_uroT by the Ca method. The resulting transformant was cultured in 5 mL of LB medium containing 34 μg / mL chloramphenicol and / or 39 μg / mL kanamycin at 37° C. for 4 hours with shaking. IPTG was added to the culture solution to a concentration of 1 mM, and induction culture was further carried out at 30° C. for 4 hours. The resulting culture medium was centrifuged and washed with 0.85% NaCl to prepare wet cells.

[0200] [Experimental Example 16] Expression of lactonase and ellagic acid transporter in Escherichia coli E. coli Rosetta2 (DE3) strain was transformed with pET21b_UroH and pRSFDuet-1_uroT by the Ca method. The resulting transformant was cultured in 5 mL of LB medium containing 34 μg / mL chloramphenicol and / or 39 μg / mL kanamycin at 37° C. for 4 hours with shaking. IPTG was added to the culture solution to a concentration of 1 mM, and induction culture was further carried out at 30° C. for 4 hours. The resulting culture medium was centrifuged and washed with 0.85% NaCl to prepare wet cells.

[0201] [Experimental Example 17] Expression of lactonase in Escherichia coli E. coli Rosetta2 (DE3) strain was transformed with pET21b_UroH by the Ca method. The resulting transformant was cultured in 5 mL of LB medium containing 34 μg / mL chloramphenicol and / or 39 μg / mL kanamycin at 37° C. for 4 hours with shaking. IPTG was added to the culture solution to a concentration of 1 mM, and induction culture was further carried out at 30° C. for 4 hours. The resulting culture medium was centrifuged and washed with 0.85% NaCl to prepare wet cells.

[0202] [Experimental Example 18-1] Production of urolithin M5 from ellagic acid by transformants The bacterial cells contained in 3 mL of each culture medium obtained in Experimental Examples 15, 16, and 17 were reacted in 100 mM KPB (pH 6.5) buffer containing 5 mg of ellagic acid at 37° C. for 4 hours, and the amount of urolithin M5 produced was measured. The amount of urolithin M5 was quantified in the same manner as in Experimental Example 5.

[0203] The results are shown in Table 3. The multiplication factors in Table 3 are relative values ​​to the amount of urolithin M5 produced when only the uroH gene was introduced (Experimental Example 17). When only the uroT gene was introduced (Experimental Example 15), urolithin M5 was not produced at all. When only the uroH gene was introduced (Experimental Example 17), 0.960 mM urolithin M5 was produced. When the uroH gene and the uroT gene were introduced (Experimental Example 16), 1.22 mM urolithin M5 was produced, which was a 1.3-fold increase compared to when only the uroH gene was introduced (Experimental Example 17).

[0204] [Table 3]

[0205] [Experimental Example 18-2] Production of urolithin M5 from ellagic acid by the deposited strain ABB medium (Oxoid) was added with ellagic acid at a final concentration of 1 g / L, then heat sterilized and the gas phase was replaced with N2:CO2:H2 (80% / 10% / 10%) gas to be used as the base medium. Gordonibacter faecihominis JCM 16058 strain was inoculated into the base medium and cultured anaerobically at 37°C for 14 days.

[0206] After cultivation, urolithins in the culture medium were quantified by the following method. Urolithins were extracted with an equal volume of ethyl acetate per 5 mL of culture solution, and the resulting ethyl acetate phase was concentrated under reduced pressure and dried. The dried product thus obtained was redissolved in 0.5 mL of methanol, and quantitative analysis of urolithins was performed by HPLC.

[0207] HPLC was performed under the conditions described below. HPLC analysis conditions: Column: Inertsil ODS-3 (250 x 4.6 mm) (GL Science) Eluent: water / acetonitrile / acetic acid = 74 / 25 / 1 Flow rate: 1.0mL / min Column temperature: 40℃ Detection: 305 nm

[0208] As a standard, urolithins manufactured by DALTON PHARMA were dissolved in DMSO and used. As a result, 450 μM of urolithin M5 was obtained.

[0209] 〔Experimental Example 19-1〕Production of urolithin A from urolithin C To ABB medium (manufactured by Oxoid), urolithin C was added as a precursor of urolithin A so that the final concentration was 1.0 g / L, followed by heat sterilization, and the one with the gas phase replaced with N2: CO2: H2 (80% / 10% / 10%) gas was used as the basic medium. Clostridium bolteae strain JCM 12243, DSM 15670 strain, or DSM 29485 strain was inoculated into the basic medium and anaerobically cultured at 37°C. After the cultivation was completed, urolithins were extracted from 5 mL of the culture solution with an equal volume of ethyl acetate, and the obtained ethyl acetate phase was concentrated under reduced pressure and dried. The dried product thus obtained was redissolved in 0.5 mL of methanol, and quantitative analysis of urolithins was performed by HPLC.

[0210] The HPLC conditions are as follows. <HPLC conditions> Column: Inertsil ODS-3 (inner diameter 4.6 mm × length 250 mm, 5 μm) (manufactured by GL Science) Eluent A: 1% formic acid B: Acetonitrile containing 1% formic acid Flow rate: 1 mL / min Column temperature: 40°C Detection: UV (305 nm)

[0211] Also, urolithins manufactured by DALTON PHARMA were used as a standard and dissolved in DMSO for use. As a result, by culturing for 2 weeks, 89%, 100%, and 89% of the added urolithin C was converted to urolithin A, respectively.

[0212] 〔Experimental Example 19-2〕Production of urolithin A from urolithin C The same procedure was followed as in Experimental Example 19-1, except that Clostridium asparagiforme DSM 15981 strain was used and cultured for 5 days. As a result, 95% of the added urolithin C was converted to urolithin A.

[0213] [Experimental Example 19-3] Production of urolithin A from urolithin C The same procedure was followed as in Experimental Example 19-1, except that Clostridium citronellae DSM 19261 strain was used and cultured for 5 days. As a result, 82% of the added urolithin C was converted to urolithin A.

[0214] [Experimental Example 19-4] Production of urolithin A from urolithin C Clostridium volteae JCM 12243 strain and Gordonibacter pamelaeae DSM 19378 strain were inoculated into ABB medium (Oxoid) containing 0.1% ellagic acid (SIGMA) and cultured in the same manner as in Experimental Example 19-1. After two weeks of culture, 67% of the added ellagic acid was converted to urolithin A.

[0215] [Experimental Example 19-5] Production of urolithin A from urolithin C Cultivation was performed in the same manner as in Experimental Example 19-4, except that Clostridium volteae JCM 12243 strain and Gordonibacter urolithifaciens DSM 27213 strain were used. As a result, after 2 weeks of cultivation, 62% of the added ellagic acid was converted to urolithin A.

[0216] [Experimental Example 19-6] Production of urolithin A from urolithin C Cultivation was performed in the same manner as in Experimental Example 19-4, except that Clostridium asparagiforme strain DSM 15981 and Gordonibacter urolithifaciens strain DSM 27213 were used. As a result, after 5 days of cultivation, 60% of the added ellagic acid was converted to urolithin A.

[0217] [Experimental Example 19-7] Production of urolithin A from urolithin C Except for using Clostridium citroniae DSM 19261 strain and Gordonibacter urolithinfaciens DSM 27213 strain, the culture was carried out in the same manner as in Experimental Example 19-4. As a result, 60% of the added ellagic acid was converted to urolithin A after 5 days of culture.

[0218] 〔Experimental Example 19-8〕Production of urolithin A from urolithin C After adding urolithin C as a substrate to ABB medium (manufactured by Oxoid), it was heat-sterilized, and the one with the gas phase replaced with N2: CO2: H2 (80% / 10% / 10%) gas was used as the medium. Clostridium sp. DC 3656 (NITE ABP-02708) strain was inoculated into the medium containing urolithin C at a final concentration of 1.0 g / L and cultured anaerobically at 37 °C for 5 days. After the culture was completed, the culture broth was analyzed by HPLC under the following conditions.

[0219] <HPLC conditions> Column: Inertsil ODS-3 (inner diameter 4.6 mm × length 250 mm) (manufactured by GL Science) Eluent: water / acetonitrile / acetic acid = 74 / 25 / 1 Flow rate: 1.0 mL / min Column temperature: 40 °C Detection: UV (305 nm)

[0220] As a result, urolithin A was produced from urolithin C with a molar yield of 89%.

[0221] 〔Experimental Example 19-9〕Production of urolithin C from ellagic acid by the deposited strain After adding ellagic acid at a final concentration of 1 g / L to ABB medium (manufactured by Oxoid), it was heat-sterilized, and the one with the gas phase replaced with N2:CO2:H2 (80% / 10% / 10%) gas was used as the basic medium. Eggerthella sp. DC3563 (NITE BP-02376) strain was inoculated into the basic medium and cultured anaerobically at 37 °C for 2 weeks. The quantification of urolithins in the culture broth after culture was carried out by the following method. Urolithins were extracted with an equal volume of ethyl acetate per 5 mL of culture solution, and the resulting ethyl acetate phase was concentrated under reduced pressure and dried. The dried product thus obtained was redissolved in 0.5 mL of methanol, and quantitative analysis of urolithins was performed by HPLC.

[0222] HPLC was performed under the conditions described below. HPLC analysis conditions: Column: Inertsil ODS-3 (250 x 4.6 mm) (GL Science) Eluent: water / acetonitrile / acetic acid = 74 / 25 / 1 Flow rate: 1.0mL / min Column temperature: 40℃ Detection: 305 nm

[0223] As a standard sample, urolithins manufactured by Dalton Pharma were dissolved in DMSO and used. As a result, urolithin C was obtained in a molar yield of 90.3% based on the added ellagic acid.

[0224] [Experimental Example 20] Measurement of lactonase activity Using genomic DNA from Gordonibacter urolithinfaciens DSM 27213 strain as a template, the lactonase gene was cloned by PCR using the primer set below and inserted into pET-28a(+) to construct an expression plasmid pET-28a_GuUroH-Histag(N) capable of expressing lactonase as a His-tagged protein at the N-terminus. 5'-GCCGGATCCATGGCAGACAACAAGGTCATCGACATCAACATG-3' (SEQ ID NO: 37) 5'-TATGAATTCCTACAGGTTGAACAGCTTCGCCGCGTTGCCGCC-3' (SEQ ID NO: 38)

[0225] E. coli Rosetta2 (DE3) strain was transformed with pET-28a_GuUroH-Histag(N) by the Ca method. The obtained transformant was cultured with shaking at 37 °C for 2.5 hours in 5 mL of LB medium containing 34 μg / mL chloramphenicol and 30 μg / mL kanamycin. IPTG was added to the culture solution to a final concentration of 0.1 mM, and induction was carried out at 30 °C for 7 hours. The obtained cells were disrupted to prepare a cell-free extract. Using the obtained cell-free extract, the reaction was carried out at 37 °C for 7 hours, and urolithin M5 produced from ellagic acid as a raw material (substrate) was quantified by HPLC. The conditions are as follows.

[0226] <HPLC conditions> Column: Cosmosil 5C18-AR-II (inner diameter 4.6 mm × length 150 mm) Eluent A: 1% formic acid B: Acetonitrile containing 1% formic acid Flow rate: 1 mL / min Column temperature: 40 °C Detection: UV (urolithin M5, 349 nm; urolithin M6, 348 nm; urolithin C, 337 nm)

[0227] As a result, the specific activity of the cell-free extract was 15.7 mU / mg-protein. Note that 1 U refers to the activity that catalyzes the production of 1 μmol of urolithin M5 per minute under the above conditions.

[0228] 〔Experimental Example 20-1〕Construction of a vector expressing an enzyme that dehydrates and oxidizes the 4-position hydroxyl group of urolithins, and a transformant The Rhodococcus genus expression vector pTipQC1 (Hokkaido System Science Co., Ltd.) was digested with the restriction enzymes EcoRI and BamHI, and a linearized plasmid was obtained by gel extraction. Using the primer sets described in Table 4, each gene fragment was amplified by PCR using the genomic DNA of Gordonibacter urolithinfaciens DSM 27213 strain as a template, and each inserted DNA fragment was obtained by gel extraction. The linearized plasmid and each insert DNA fragment were ligated using NEBuilder HiFi DNA Assembly Mastermix (New England Biolabs).

[0229] [Table 4]

[0230] The base sequences of the primers in the table are as follows: uroA1_f: 5'-catcaccatatgggaattATGGCCGATTCAGCCCAGGCCCCCGTGCAGGAG-3' (SEQ ID NO: 39) uroA1_r: 5'-agagatctaagcttgCTAGTCGGCCTTGCGGATCTTGCACATCATGGC-3' (SEQ ID NO: 40) uroA2_r: 5'-agagatctaagcttgCTACGCCTCGTTGATGGCGACGATCATCTGCTT-3' (SEQ ID NO: 41) uroA3_r: 5'-agagatctaagcttgTTACCGGTAGATCGGTGTAACTTCGCGCTCCGC-3' (SEQ ID NO: 42)

[0231] Details of the constructed plasmids in the table are as follows: pTIpQC1_uroA123 is a plasmid in which GuuroA1, GuuroA2, and GuuroA3 are inserted into the EcoR1 and BamHI sites of pTipQC1. pTIpQC1_uroA12 is a plasmid in which GuuroA1 and GuuroA2 are inserted into the EcoR1 and BamHI sites of pTipQC1. pTIpQC1_uroA1 is a plasmid in which GuuroA1 is inserted between the EcoR1 and BamHI sites of pTipQC1.

[0232] The vector was cloned using Escherichia coli DH5α as a host, with ampicillin used as a selection marker. The constructed plasmid was introduced into Rhodococcus erythropolis L88 (Hokkaido System Science Co., Ltd.) by electroporation. Chloramphenicol was used as a selection marker. The formed colonies were cultured (28°C) in LB medium (20 μg / mL chloramphenicol), and glycerol stocks (30% glycerol) were prepared from the culture medium and stored (-80°C) for use in heterologous expression and enzyme activity measurements.

[0233] [Experimental Example 20-2] Expression of an enzyme that dehydroxylates the 4-hydroxyl group of urolithins As a preculture, 5 mL of LB medium containing 20 μg / mL chloramphenicol was added to a 10 mL test tube, the transformant was inoculated, and cultured at 28° C. with shaking at 300 rpm. 1 mL of the preculture solution was inoculated into a 50 mL Erlenmeyer flask containing 23 mL of LB medium containing 20 μg / mL chloramphenicol, and cultured at 28°C and 200 rpm for 7 hours with shaking. After that, 0.1 μg / mL thiostrepton was added to induce the enzyme for 16 hours. The cells were collected by centrifugation (8,000 rpm, 10 minutes, 4° C.) and washed twice with physiological saline to obtain resting cells, which were then used in the reaction for measuring enzyme activity.

[0234] [Experimental Example 20-3] Measurement of the activity of the enzyme that dehydroxylates the 4-hydroxyl group of urolithins The reaction for measuring activity was carried out under the following conditions. A reaction solution containing 0.5 mg / mL urolithin M5 or urolithin M6, 10 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH), 0.2 mM flavin adenine dinucleotide sodium (FAD), 10 mM methyl viologen (MV), and 50 mM potassium phosphate buffer (pH 6.5) was reacted in an Anaeropack at 37°C for 3 hours with shaking at 200 spm. 100 μL of the reaction solution was added to 200 μL of N,N-dimethylacetamide (DMA) containing 1% formic acid, mixed, and then centrifuged. The supernatant was analyzed by HPLC to quantify the product.

[0235] The results are shown in Table 5. Urolithin M6 was not detectable in the host strain L88 (R. erythropolis L88), but production of urolithin M6 was confirmed in the transformed strain containing GuuroA1, GuuroA2, and GuuroA3 (R. erythropolis_uroA123) and the transformed strain containing GuuroA1 and GuuroA2 (R. erythropolis_uroA12). Furthermore, production of urolithin M6 was not confirmed in the transformed strain containing only GuuroA1 (R. erythropolis_uroA1). These results confirmed that at least GuuroA1 and GuuroA2 are required for the synthesis of urolithin M6 from urolithin M5. Regarding the function of GuuroA3, it is possible that the function is complemented by host-derived proteins in the host L88 strain.

[0236] [Table 5]

[0237] [Experimental Example 21-1] Construction of a vector expressing an enzyme that dehydroxylates the 10-hydroxyl group of urolithins and a transformant The Rhodococcus expression vector pTipQC1 (Hokkaido System Science Co., Ltd.) was digested with restriction enzymes EcoRI and BamHI, and a linearized plasmid was obtained by gel extraction. Using the primer sets shown in Table 6, each gene fragment was amplified by PCR using the genomic DNA of Gordonibacter urolithinfaciens DSM 27213 strain as a template, and each inserted DNA fragment was obtained by gel extraction. The linearized plasmid and each insert DNA fragment were ligated using NEBuilder HiFi DNA Assembly Mastermix (New England Biolabs).

[0238] [Table 6]

[0239] The primer sequences in the table are as follows: uroB1_f: 5'-catcaccatatgggaattATGGTAGAGAACGCTGTCGCGGAGAAGGACC-3' (SEQ ID NO: 43) uroB1_r: 5'-agagatctaagcttgTTACAGTTCCTCCTCCTTGCAGCGGTACACCTT-3' (SEQ ID NO: 44) uroB2_r: 5'-agagatctaagcttgCTACGCGCAGGGCGGCACCCACAGGTACTGCTG-3' (SEQ ID NO: 45) uroB3_r: 5'-agagatctaagcttgCTAGCCCTTCTTCGCTGGCACGGGGTCCCGCTC-3' (SEQ ID NO: 46)

[0240] Details of the constructed plasmids in the table are as follows: pTIpQC1_uroB123 is a plasmid in which GuuroB1, GuuroB2, and GuuroB3 are inserted into the EcoR1 and BamHI sites of pTipQC1. pTIpQC1_uroB12 is a plasmid in which GuuroB1 and GuuroB2 are inserted into the EcoR1 and BamHI sites of pTipQC1. pTIpQC1_uroB1 is a plasmid in which GuuroB1 is inserted between the EcoR1 and BamHI sites of pTipQC1.

[0241] The vector was cloned using Escherichia coli DH5α as a host, with ampicillin used as a selection marker. The constructed plasmid was introduced into Rhodococcus erythropolis L88 (Hokkaido System Science Co., Ltd.) by electroporation. Chloramphenicol was used as a selection marker. The formed colonies were cultured (28°C) in LB medium (20 μg / mL chloramphenicol), and glycerol stocks (30% glycerol) were prepared from the culture medium and stored (-80°C) for use in heterologous expression and enzyme activity measurements.

[0242] [Experimental Example 21-2] Expression of an enzyme that dehydroxylates the 10-hydroxyl group of urolithins As a preculture, 5 mL of LB medium containing 20 μg / mL chloramphenicol was added to a 10 mL test tube, the transformant was inoculated, and cultured with shaking at 28° C. and 300 rpm for 2 to 3 days. The preculture solution was inoculated at 1% into a 50 mL Erlenmeyer flask containing 25 mL of LB medium containing 20 μg / mL chloramphenicol, and cultured at 28°C and 200 rpm for 7 hours with shaking. After that, 0.2 μg / mL thiostrepton was added and the enzyme was induced for 48 hours. The cells were collected by centrifugation (8,000 rpm, 10 minutes, 4° C.) and washed twice with physiological saline to obtain resting cells, which were then used in the reaction for measuring enzyme activity.

[0243] [Experimental Example 21-3] Measurement of the dehydroxylation activity of bacteria that dehydroxylates the 10-hydroxyl group of urolithins The reaction for measuring activity was carried out under the following conditions. A reaction solution containing 0.5 mg / mL urolithin M6, 10 mM methyl viologen (MV), 2 mg / mL sodium hyposulfite, 2 mg / mL sodium bicarbonate, and 50 mM potassium phosphate buffer (pH 6.5) was reacted in an Anaeropack at 37°C for 12 hours with shaking at 200 spm. 100 μL of the reaction solution was added to 200 μL of N,N-dimethylacetamide (DMA) containing 1% formic acid, mixed, and then centrifuged. The supernatant was analyzed by HPLC to quantify the product.

[0244] The results are shown in Figure 7. Urolithin C was not detectable in the host strain L88 (R. erythropolis L88), but production of urolithin M6 was confirmed in the transformed strain containing GuuroB1, GuuroB2, and GuuroB3 (R. erythropolis_uroB123, uroB123 in Figure 7) and the transformed strain containing GuuroB1 and GuuroB2 (R. erythropolis_uroB12, uroB12 in Figure 7). Furthermore, production of urolithin C was not confirmed in the transformed strain containing only GuuroB1 (R. erythropolis_uroA1, uroB1 in Figure 7). These results confirmed that at least GuuroB1 and GuuroB2 are required for the production of urolithin C from urolithin M6. Regarding the function of GuuroB3, it is possible that the function is complemented by host-derived proteins in the host L88 strain.

[0245] [Experimental Example 22-1] Production of an enzyme that dehydroxylates the 10-hydroxyl group of urolithins Gordonibacter urolithinfaciens DSM 27213 strain was inoculated into 20 mL of modified GAM medium (Nissui Pharmaceutical) containing 0.1 mg / mL ellagic acid and cultured with shaking at 37°C for 5-6 days. 10 mL of the obtained preculture solution was inoculated into 1.75 L of the same medium and cultured with shaking at 37°C for 7-9 days. The cells were collected by centrifugation and washed twice with physiological saline to obtain washed cells.

[0246] [Experimental Example 22-2] Partial purification of the enzyme that dehydroxylates the 10-hydroxyl group of urolithins The washed cells were suspended in a buffer solution and disrupted by ultrasonication for 40 minutes. The cell lysate was centrifuged (8,500 rpm, 30 minutes, 4°C), and the supernatant obtained by further high-speed centrifugation (20,000g, 60 minutes, 4°C) was used as the cell-free extract. The supernatant obtained by further ultracentrifugation (100,000g, 110 minutes, 4°C) of the cell-free extract was used as the ultracentrifugation supernatant fraction. The subsequent analysis of the enzymatic properties was performed using this ultracentrifugation supernatant fraction.

[0247] 〔Experimental Example 23-1〕Method for Measuring the Activity of an Enzyme that Dehydrates and Oxidizes the Hydroxyl Group at the 10th Position of Urolithins The activity of dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins was measured for the ultracentrifugation supernatant fraction obtained in Experimental Example 22-2. Urolithin M6 was used as the urolithin having a hydroxyl group at the 10th position. When the hydroxyl group at the 10th position is dehydrated and oxidized, urolithin C is produced. 20 μL of a reaction solution for measuring enzyme activity containing 0.5 mg / mL urolithin M6, 10 mM NADPH (reduced nicotinamide adenine dinucleotide), 0.2 mM FAD (flavin adenine dinucleotide), 10 mM MV (methyl viologen), 50 mM KPB (potassium phosphate buffer, pH 6.5), and the fraction obtained in Experimental Example 22-1 was placed in a 200 μL tube for PCR. The gas phase was set as the gas phase in an anaerobic chamber (COY vinyl anaerobic chamber), and the reaction was carried out at 37°C for 120 minutes. 40 μL of N,N-dimethylacetamide containing 1% formic acid was added to the reaction-terminated solution. After mixing, the supernatant of the centrifugation was analyzed by HPLC.

[0248] HPLC was performed under the following conditions to quantify the produced urolithin C (detection wavelength: 348 nm). <HPLC Conditions> Column: COSMOSIL5C18-ARII (inner diameter 4.6 mm × length 150 mm, manufactured by Nacalai Tesque) Eluent: Ultrapure water (Milli Q water) / acetonitrile (CH3CN) / formic acid = 80 / 20 / 1 Flow rate: 1.0 mL / min Temperature: 40°C Detection: PDA (photodiode array) (Urolithin C is measured by UV absorption at 337 nm)

[0249] The results of this experiment correspond to the results of No. 9 in Table 7 of Experimental Example 23-2.

[0250] [Experimental Example 23-2] Effect of cofactors on the activity of the enzyme that dehydroxylates the 10-hydroxyl group of urolithins The enzyme solution obtained in Experimental Example 22-2 was used to prepare a reaction solution obtained by removing NADPH, FAD, and MV from the reaction solution for measuring enzyme activity described in Experimental Example 23-1, to which cofactors were added at the concentrations shown in Table 7, and the amount of urolithin C produced after the reaction was quantified. The amount of urolithin C produced under the conditions shown in No. 9 of Table 7 (10 mM MV, 10 mM NADPH, 0.2 mM FAD) was set to 100%, and the amount of urolithin C produced under each condition was expressed as relative activity. In the following experimental examples, the conditions shown in No. 9 of Table 7 were used as the standard activity measurement conditions.

[0251] [Table 7]

[0252] [Experimental Example 24] Temperature dependence of the enzyme that dehydroxylates the 10-hydroxyl group of urolithins Activity measurements were performed under the standard activity measurement conditions described in Experimental Example 23-2, with the reaction temperature being changed. The results are shown in Figure 8. The optimum temperature was 42°C.

[0253] [Experiment 25] pH Dependence of the Enzyme That Dehydroxylates the 10-Hydroxy Group of Urolithins Under the standard activity measurement conditions described in Experimental Example 23-2, activity measurements were performed while changing the pH by using the following buffer solutions. Citrate buffer: 50 mM citric acid-sodium citrate buffer Acetate buffer: 50 mM acetic acid-sodium acetate buffer Potassium phosphate buffer: 50 mM K2HPO4-KH2PO4 buffer Tris-HCl buffer: 50 mM Tris-HCl buffer Carbonate buffer: 50 mM Na2CO3-NaHCO3 buffer Sodium phosphate buffer: 50 mM Na3PO4-Na2HPO4 buffer The results are shown in Figure 9. The optimum pH was 5.5.

[0254] Example 26: Production of urolithin C from urolithin M5 by the deposited strain Urolithin M5 was added to ABB medium (Oxoid) to a final concentration of 3.3 mM, followed by heat sterilization and replacement of the gas phase with N2:CO2:H2 (80% / 10% / 10%) gas to prepare a basic medium. Gordonibacter urolithinfaciens DSM 27213 strain was inoculated into the basic medium and cultured anaerobically at 37°C. After the culture was completed, an equal volume of DMSO was added per 1 mL of culture solution to dissolve the urolithins, and quantitative analysis of the urolithins was performed by HPLC.

[0255] HPLC was performed under the conditions described below. HPLC conditions: Column: Inertsil ODS-3 (φ4.6 mm × 250 mm, 5 μm) (GL Science) Eluent A: 1% formic acid B: acetonitrile containing 1% formic acid Flow rate: 1mL / min Column temperature: 40℃ Detection: UV (305 nm)

[0256] As a result, after 14 days of culture, 0.0707 mM urolithin C was produced.

Claims

1. The following (1) and (2) are derived from microorganisms belonging to the genus Gordonibacter: An enzyme having the properties of 2). (1) The hydroxyl group at the 4-position of urolithins, which are compounds represented by the following general formula (1), and which have a hydroxyl group at the 4-position, is dehydroxylated. (2) In the presence of methyl viologen (MV), it is activated with one or more selected from the group consisting of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN). 【Chemistry 1】 (In the formula, R1 to R6 each independently represent a hydroxyl group, a hydrogen atom, or a methoxy group, and at least one of R1 to R6 is a hydroxyl group.)

2. The enzyme according to claim 1, having the following properties (3) and (4). (3) The optimum pH is 5.5 or higher and 7.5 or lower. (4) The results of SDS-PAGE include a band showing a molecular weight of 81,000 or more and 99,000 or less.

3. The enzyme according to claim 1 or 2, having the following property (5): (5) The optimum temperature is 37°C or higher and 50°C or lower.

4. The microorganism belonging to the genus Gordonibacter is Gordonibacter u Microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and The enzyme according to any one of claims 1 to 3, which is one or more selected from the group consisting of microorganisms belonging to Gordonibacter faecihominis.

5. The amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2 are included, or The amino acid sequence represented by SEQ ID NO: 13 and the amino acid sequence represented by SEQ ID NO: 14 are included. The enzyme according to any one of claims 1 to 4.

6. The amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence represented by SEQ ID NO: 2, and the amino acid sequence represented by SEQ ID NO: 3, or The amino acid sequence represented by SEQ ID NO: 13, the amino acid sequence represented by SEQ ID NO: 14, and the amino acid sequence represented by SEQ ID NO: 15 are included. The enzyme according to any one of claims 1 to 5.

7. The base sequence represented by SEQ ID NO: 7 and the base sequence represented by SEQ ID NO: 8, or It consists of the base sequence represented by SEQ ID NO: 19 and the base sequence represented by SEQ ID NO: 20, Polynucleotide.

8. A base sequence represented by SEQ ID NO: 7, a base sequence represented by SEQ ID NO: 8, and a base sequence represented by SEQ ID NO: 9, or A base sequence represented by SEQ ID NO: 19, a base sequence represented by SEQ ID NO: 20, and a base sequence represented by SEQ ID NO:

21. Polynucleotide.

9. A recombinant vector comprising the polynucleotide of claim 7 or 8.

10. A transformant comprising the polynucleotide according to claim 7 or 8 in an expressible state, or the vector according to claim 9 in an expressible state.

11. The method according to claim 10, wherein the host is a microorganism belonging to the genus Rhodococcus. Transformant.

12. A method for producing a protein encoded by the polynucleotide according to claim 7 or 8, comprising the step of culturing the transformant according to claim 10 or 11.

13. A method for dehydroxylating a hydroxyl group at the 4-position of urolithins, which are compounds represented by the following general formula (1) and have a hydroxyl group at the 4-position, comprising the following step (I): Step (I): A step of contacting one or more selected from the following (i) to (iv) with a urolithin, which is a compound represented by general formula (1) below and has a hydroxyl group at the 4-position, to dehydroxylate the hydroxyl group at the 4-position. (i) an enzyme according to any one of claims 1 to 6; (ii) a protein encoded by the polynucleotide of claim 7 or 8; (iii) a microorganism producing the enzyme according to (i) above or the protein according to (ii) above; (iv) A treated product of the microorganism described in (iii) above. 【Chemistry 2】 (In the formula, R1 to R6 each independently represent a hydroxyl group, a hydrogen atom, or a methoxy group, and at least one of R1 to R6 is a hydroxyl group.)

14. the urolithin having a hydroxyl group at position 4 is urolithin M5, urolithin D, or urolithin E, The products produced by dehydroxylation of the hydroxyl group at the 4-position of the urolithins are urolithin M6, urolithin C, and urolithin M7, respectively. The method of claim 13.

15. The following (1) and (2) are derived from microorganisms belonging to the genus Gordonibacter: An enzyme having the properties of 2). (1) In urolithins, which are compounds represented by the following general formula (1), which have a hydroxyl group at the 10-position, the hydroxyl group at the 10-position is dehydroxylated. (2) In the presence of methyl viologen (MV), it is activated with one or more selected from the group consisting of reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN). 【Chemistry 3】 (In the formula, R1 to R6 each independently represent a hydroxyl group, a hydrogen atom, or a methoxy group, and at least one of R1 to R6 is a hydroxyl group.)

16. The enzyme according to claim 15, having the following properties (3) and (4). (3) The optimum pH is 5.0 or higher and 7.0 or lower. (4) The optimum temperature is 37°C or higher and 42°C or lower.

17. The microorganism belonging to the genus Gordonibacter is Gordonibacter u Microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and Selected from the group consisting of microorganisms belonging to Gordonibacter faecihominis 17. The enzyme according to claim 15 or 16, wherein the enzyme is one or more selected from the group consisting of

18. The amino acid sequence represented by SEQ ID NO: 4 and the amino acid sequence represented by SEQ ID NO: 5 are included, or The amino acid sequence represented by SEQ ID NO: 16 and the amino acid sequence represented by SEQ ID NO: 17 are included. The enzyme according to any one of claims 15 to 17.

19. The amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, and the amino acid sequence represented by SEQ ID NO: 6, or The amino acid sequence represented by SEQ ID NO: 16, the amino acid sequence represented by SEQ ID NO: 17, and the amino acid sequence represented by SEQ ID NO: 18 are included. The enzyme according to any one of claims 15 to 18.

20. It consists of the base sequence represented by SEQ ID NO: 10 and the base sequence represented by SEQ ID NO: 11, or It consists of the base sequence represented by SEQ ID NO: 22 and the base sequence represented by SEQ ID NO: 23, Polynucleotide.

21. A base sequence represented by SEQ ID NO: 10, a base sequence represented by SEQ ID NO: 11, and a base sequence represented by SEQ ID NO: 12, or A base sequence represented by SEQ ID NO: 22, a base sequence represented by SEQ ID NO: 23, and a base sequence represented by SEQ ID NO:

24. Polynucleotide.

22. A recombinant vector comprising the polynucleotide of claim 20 or 21.

23. A transformant carrying the polynucleotide according to claim 20 or 21 in an expressible manner, or carrying the vector according to claim 22 in an expressible manner.

24. The method according to claim 23, wherein the host is a microorganism belonging to the genus Rhodococcus. Transformant.

25. A method for producing a protein encoded by the polynucleotide of claim 20 or 21, comprising the step of culturing the transformant of claim 23 or 24.

26. The method of claim 1, further comprising culturing a microorganism belonging to the genus Gordonibacter.

20. A method for producing the enzyme according to any one of 15 to 19.

27. The microorganism belonging to the genus Gordonibacter is Gordonibacter u Microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and 27. The method of claim 26, wherein the microorganism is one or more selected from the group consisting of microorganisms belonging to Gordonibacter faecihominis.

28. A method for dehydroxylating a hydroxyl group at the 10-position of urolithins, which are compounds represented by the following general formula (1) and have a hydroxyl group at the 10-position, comprising the following step (I): Step (I): A step of contacting one or more selected from the following (i) to (iv) with a urolithin, which is a compound represented by general formula (1) below and has a hydroxyl group at the 10-position, to dehydroxylate the hydroxyl group at the 10-position: (i) an enzyme according to any one of claims 15 to 19; (ii) a protein encoded by the polynucleotide of claim 20 or 21; (iii) a microorganism producing the enzyme according to (i) above or the protein according to (ii) above; (iv) A treated product of the microorganism described in (iii) above. 【Chemistry 4】 (In the formula, R1 to R6 each independently represent a hydroxyl group, a hydrogen atom, or a methoxy group, and at least one of R1 to R6 is a hydroxyl group.)

29. the urolithin having a hydroxyl group at position 10 is urolithin M5, urolithin M6, or urolithin M7, The products produced by dehydroxylation of the hydroxyl group at position 10 of the urolithins are urolithin D, urolithin C, and urolithin A, respectively.

30. The method of claim 28.

30. A method for producing urolithin C, comprising the following steps (I) to (III): Step (I): A step of allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin M6: (i) an enzyme according to any one of claims 1 to 6; (ii) a protein encoded by the polynucleotide of claim 7 or 8; (iii) a microorganism producing the enzyme according to (i) above or the protein according to (ii) above; (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with the urolithin M6 to produce urolithin C: (v) an enzyme according to any one of claims 15 to 19; (vi) a protein encoded by the polynucleotide of claim 20 or 21; (vii) a microorganism producing the enzyme according to (v) or the protein according to (vi); (viii) A treated product of the microorganism described in (vii).

31. A method for producing urolithin C, comprising the following steps (I) to (III): Step (I): A step of allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin D. (i) an enzyme according to any one of claims 15 to 19; (ii) a protein encoded by the polynucleotide of claim 20 or 21; (iii) a microorganism producing the enzyme according to (i) above or the protein according to (ii) above; (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with urolithin D to produce urolithin C: (v) an enzyme according to any one of claims 1 to 6; (vi) a protein encoded by the polynucleotide of claim 7 or 8; (vii) a microorganism producing the enzyme according to (v) or the protein according to (vi); (viii) A treated product of the microorganism described in (vii).

32. A method for producing urolithin A, comprising the following steps (I) to (IV): Step (I): A step of allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin M6. (i) an enzyme according to any one of claims 1 to 6; (ii) a protein encoded by the polynucleotide of claim 7 or 8; (iii) a microorganism producing the enzyme according to (i) above or the protein according to (ii) above; (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with the urolithin M6 to produce urolithin C: (v) an enzyme according to any one of claims 15 to 19; (vi) a protein encoded by the polynucleotide of claim 20 or 21; (vii) a microorganism producing the enzyme according to (v) or the protein according to (vi); (viii) A treated product of the microorganism described in (vii). Step (IV): Allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C.

33. A method for producing urolithin A, comprising the following steps (I) to (IV): Step (I): A step of allowing a microorganism having the ability to produce urolithin M5 from ellagic acid to produce urolithin M5 from ellagic acid. Step (II): A step of contacting one or more selected from the following (i) to (iv) with the urolithin M5 to produce urolithin D. (i) an enzyme according to any one of claims 15 to 19; (ii) a protein encoded by the polynucleotide of claim 20 or 21; (iii) a microorganism producing the enzyme according to (i) above or the protein according to (ii) above; (iv) A treated product of the microorganism described in (iii) above. Step (III): A step of contacting one or more selected from the following (v) to (viii) with urolithin D to produce urolithin C: (v) an enzyme according to any one of claims 1 to 6; (vi) a protein encoded by the polynucleotide of claim 7 or 8; (vii) a microorganism producing the enzyme according to (v) or the protein according to (vi); (viii) A treated product of the microorganism described in (vii). Step (IV): Allowing a microorganism capable of producing urolithin A from urolithin C to produce urolithin A from the urolithin C.

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