Enzyme capable of dehydroxylating hydroxyl group in urolithin

Abstract

To provide, at least, an enzyme that dehydroxylates hydroxyl groups at predetermined positions of urolithins having hydroxyl groups at the predetermined positions.SOLUTION: The present invention provides an enzyme having the following properties (1) and (2): (1) dehydroxylating a hydroxyl group at the 4-position of urolithins; and (2) in the presence of methyl viologen (MV), being activated by 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).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to an enzyme that dehydrates and oxidizes the hydroxyl group of urolithins.

Background Art

[0002] Urolithins such as urolithin A and urolithin C are known as intestinal metabolites of ellagic acid derived from ellagitannins contained in berries such as pomegranate, raspberry, blackberry, cloudberry, strawberry, and walnut.

[0003] As a method for synthesizing these urolithins, a method has been reported in which 2-bromo-5-methoxybenzoic acid is used as a starting material, demethylated to 2-bromo-5-hydroxybenzoic acid, and reacted with resorcinol to obtain urolithin A (Non-Patent Document 1). However, such a chemical synthesis method is not suitable for using urolithins as raw materials for functional foods (including beverages and supplements).

[0004] On the other hand, it is known that ellagitannins and ellagic acid are metabolized by the intestinal microbiota in the body and converted into urolithins after ingestion. In recent years, microorganisms belonging to Gordonibacter urolithinfaciens and microorganisms belonging to Gordonibacter pamelaeae, which are intestinal bacteria that produce urolithin C, a type of urolithin, from ellagic acid, have been found. Using these intestinal bacteria, a method for producing urolithin C by fermentation of ellagic acid has been reported (Patent Document 1, Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Non-Patent Documents

[0006]

Non - Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present disclosure is to provide an enzyme that dehydrates and oxidizes a hydroxyl group at a specific position of urolithins having a hydroxyl group at a predetermined position, at least.

Means for Solving the Problems

[0008] The present inventors found, by proteome analysis of a specific strain of Gordonibacter urolithinfaciens, that there are proteins with significantly higher expression levels when cultured in a medium containing ellagic acid than when cultured in a medium not containing ellagic acid. Furthermore, as a result of purifying an enzyme that catalyzes the reaction of dehydrating and oxidizing the hydroxyl group at the 4 - position of urolithins having a hydroxyl group at the 4 - position from a predetermined strain and performing proteome analysis, it was found that the obtained purified enzyme coincides with a part of the protein whose expression level was significantly increased when cultured in a medium containing ellagic acid. By introducing the gene encoding the enzyme into a heterologous microorganism, the expression of the enzyme was successful, and the function of the gene could be specified. In addition, around the genome of the gene encoding the enzyme, there are genes encoding enzymes that have homology with the gene and catalyze the reaction of dehydrating and oxidizing the hydroxyl group at the 10 - position of urolithins having a hydroxyl group at the 10 - position, as well as genes encoding ellagic acid lactonase that hydrolyzes the lactone of ellagic acid to produce urolithin M5 It was found that there are genes encoding urolithin transporters. The present disclosure includes the following aspects.

[0009] 〔1〕An enzyme having the following properties (1) and (2). (1) Dehydrate and oxidize the hydroxyl group at the 4-position of urolithins. (2) In the presence of methyl viologen (MV), it is activated by 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 more and 7.5 or less. (4) In the result of SDS-PAGE, a band showing a molecular weight of 81,000 or more and 99,000 or less is included. [3] The enzyme according to [1] or [2], having the following property (5). (5) The optimum temperature is 37°C or more and 50°C or less. [4] The enzyme according to any one of [1] to [3], derived from a microorganism belonging to the genus Gordonibacter. [5] 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, the enzyme according to [4]. [6] The enzyme according to any one of [1] to [5], comprising the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2, or comprising the amino acid sequence represented by SEQ ID NO: 13 and the amino acid sequence represented by SEQ ID NO: 14. [7] The enzyme according to any one of [1] to [5], comprising 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 Comprising the amino acid sequence represented by SEQ ID NO: 13, comprising the amino acid sequence represented by SEQ ID NO: 14, and comprising the amino acid sequence represented by SEQ ID NO: 15 The enzyme according to any one of [1] to [6]. [8] Comprising the nucleotide sequence represented by SEQ ID NO: 7 and the nucleotide sequence represented by SEQ ID NO: 8, or Comprising the nucleotide sequence represented by SEQ ID NO: 19 and the nucleotide sequence represented by SEQ ID NO: 20 Polynucleotide [9] Comprising the nucleotide sequence represented by SEQ ID NO: 7, the nucleotide sequence represented by SEQ ID NO: 8, and the nucleotide sequence represented by SEQ ID NO: 9, or Comprising the nucleotide sequence represented by SEQ ID NO: 19, the nucleotide sequence represented by SEQ ID NO: 20, and the nucleotide sequence represented by SEQ ID NO: 21 Polynucleotide

[10] A recombinant vector comprising the polynucleotide according to [8] or [9].

[0010]

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

[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 dehydrating and oxidizing the 4-position hydroxyl group of urolithins, comprising the following step (I): Step (I): Contacting one or more selected from the following (i) to (iv) with urolithins having a hydroxyl group at the 4-position to dehydrate and oxidize the 4-position hydroxyl group. (i) The enzyme according to any one of [1] to [7]; (ii) The protein encoded by the polynucleotide according to [8] or [9]; (iii) A microorganism that produces the enzyme according to (i) or the protein according to (ii); (iv) The processed product of the microorganism described in (iii) above.

[15] The urolithins are urolithin M5, urolithin D, or urolithin E, and the products formed by the dehydration oxidation of the hydroxyl group at the 4-position 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) Dehydration oxidizes the hydroxyl group at the 10-position of urolithins. (2) Is activated by 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) in the presence of methyl viologen (MV).

[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] An enzyme according to

[16] or

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

[19] 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. The enzyme according to

[18] .

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

[16] to

[19] .

[0011] 〔21〕Comprising 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, Comprising 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, The enzyme according to any one of

[16] to

[20] . 〔22〕Comprising the nucleotide sequence represented by SEQ ID NO: 10 and the nucleotide sequence represented by SEQ ID NO: 11, or, A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 22 and the nucleotide sequence represented by SEQ ID NO: 23. 〔23〕Comprising the nucleotide sequence represented by SEQ ID NO: 10, the nucleotide sequence represented by SEQ ID NO: 11, and the nucleotide sequence represented by SEQ ID NO: 12, or, Comprising the nucleotide sequence represented by SEQ ID NO: 22, the nucleotide sequence represented by SEQ ID NO: 23, and the nucleotide sequence represented by SEQ ID NO: 24, The polynucleotide according to

[22] . 〔24〕A recombinant vector comprising the polynucleotide according to

[22] or

[23] . 〔25〕A transformant that can expressibly hold the polynucleotide according to

[22] or

[23] , or that can expressibly hold the vector according to

[24] . 〔26〕The transformant according to

[25] , wherein the host is a microorganism belonging to the genus Rhodococcus thereof. A method for producing a protein encoded by the polynucleotide according to

[22] or

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

[27] ,

[25] , or

[26] . 〔28〕Comprising the step of culturing a microorganism belonging to the genus Gordonibacter, A method for producing the enzyme according to any one of

[16] to

[21] .

[29] The microorganism belonging to the genus Gordonibacter is Gordonibacter Microorganisms belonging to the species Gordonibacter urolithinfaciens, Gordonibacter pamelaeae, and Gordonibacter The method according to

[28] , wherein the bacterium is one or more selected from the group consisting of 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 described in [8] or [9]; (iii) A microorganism that produces the enzyme described in (i) above or the protein described in (ii) above; (iv) A processed 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 urolicin M6 to produce urolicin C. (v) An enzyme described in any one of

[16] to

[21] ; (vi) A protein encoded by the polynucleotide described in

[22] or

[23] ; (vii) A microorganism that produces the enzyme described in (v) above or the protein described in (vi) above; (viii) A processed product of the microorganism described in (vii) above.

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

[16] to

[21] ; (ii) A protein encoded by the polynucleotide described in

[22] or

[23] ; (iii) A microorganism that produces the enzyme described in (i) above or the protein described in (ii) above; (iv) A processed 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 urolicin D to produce urolicin C. (v) An enzyme described in any one of [1] to [7]; (vi) A protein encoded by the polynucleotide described in [8] or [9]; (vii) A microorganism that produces the enzyme described in (v) above or the protein described in (vi) above; (viii) The processed product of the microorganism described in (vii).

[34] A method for producing urolithin A, comprising the following step (I): Step (I): A step of causing a microorganism having the ability to produce 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 comprising the amino acid sequence represented by SEQ ID NO: 27.

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

[37] A recombinant vector comprising the polynucleotide described in

[36] .

[38] A transformant that retains the polynucleotide described in

[36] in an expressible manner or retains the vector described in

[37] in an expressible manner.

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

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

[38] .

[40] A method for promoting the uptake of ellagic acid from the extracellular to the intracellular 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 a transformant described in

[38] , wherein the 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 a transformant described in claim 38, wherein the 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): Process (I): A step of contacting a transformant described in

[38] , wherein the host is a microorganism having the ability to produce urolithin C from ellagic acid, with ellagic acid to produce urolithin C from ellagic acid. Process (II): A step of causing a microorganism having the ability to produce urolithin A from urolithin C to produce urolithin A from the urolithin C. A protein comprising the amino acid sequence represented by SEQ ID NO: 29 or comprising the amino acid sequence represented by SEQ ID NO: 31. A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 30 or comprising the nucleotide sequence represented by SEQ ID NO: 32. A recombinant vector comprising the polynucleotide described in

[45] . A transformant that retains the polynucleotide described in

[45] in an expressible manner or retains the vector described in

[46] in an expressible manner. A method for producing a protein encoded by the polynucleotide described in

[45] , comprising a step of culturing the transformant described in

[47] . A method for producing urolithin M5, comprising the following process (I): Process (I): A step of contacting the transformant described in

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

[0014] A method for producing urolithin A, comprising the following processes (I) and (II): Process (I): A step of contacting a transformant described in

[47] , wherein the host is a microorganism having the ability to produce urolithin C from urolithin M5, with ellagic acid to produce urolithin M5 from ellagic acid and then produce urolithin C from the urolithin M5. Process (II): A step of causing a microorganism having the ability to produce urolithin A from urolithin C to produce urolithin A from the urolithin C.

Advantages of the Invention

[0015] The present disclosure can achieve at least the effect of providing an enzyme that dehydrates and oxidizes the hydroxyl group at a specific position of urolithins having a hydroxyl group at a predetermined position.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Modes for Carrying Out the Invention

[0017] Hereinafter, the present disclosure will be described in detail. Each configuration and their combinations, etc. in each aspect are examples, and within the scope not departing from the gist of the present invention, addition, omission, substitution, and other changes of the configuration can be made as appropriate. The present disclosure is not limited by the aspects, but is limited only by the scope of the claims. Also, each aspect disclosed in this specification can be combined with any other features disclosed in this specification.

[0018] In the present disclosure, the deposit numbers of strains starting with the phrase "DSM" are numbers assigned to microorganisms stored in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). It is the number. Also, the deposit numbers of strains starting with the phrase "JCM" are numbers assigned to microorganisms stored in the RIKEN BioResource Center. It is the number assigned to the microorganisms stored in the center.

[0019] Urolithins are represented by the following general formula (1).

Chemical formula

[0020] <1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins> One aspect of the present disclosure is an enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins. Specifically, it is an enzyme that catalyzes the reaction of dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins having a hydroxyl group at the 4-position. 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) Dehydrates and oxidizes the hydroxyl group at the 4-position of urolithins. (2) In the presence of methyl viologen (MV), it is activated by 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 the 4-position of these is dehydrated and oxidized by the enzyme according to this embodiment, urolithin M6, urolithin C, and urolithin M7 are produced, respectively.

[0023] The enzyme according to this embodiment is activated by a cofactor. Examples of the cofactor include methyl viologen (MV), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN). The cofactor in this embodiment preferably uses MV and NADH, more preferably uses MV and NADPH, even more preferably uses MV, NADPH, and FMN, and even more preferably uses MV, NADPH, and FAD, because it increases the activity of the enzyme according to this embodiment to dehydrate and oxidize the hydroxyl group at the 4-position of urolithins.

[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. On the other hand, it 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, a band showing a molecular weight of preferably 81,000 or more, more preferably 85,000 or more, still more preferably 87,000 or more, and on the other hand, preferably 99,000 or less, more preferably 95,000 or less, still more preferably 93,000 or less is included.

[0026] The enzyme according to this embodiment preferably has the property of the following (5). (5) The optimum temperature is preferably 37°C or more, more preferably 38°C or more, still more preferably 40°C or more, and on the other hand, preferably 50°C or less, more preferably 46°C or less, still more preferably 44°C or less.

[0027] The enzyme according to this embodiment is preferably derived from a microorganism belonging to the genus Gordonibacter. It is more preferably 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. Among the microorganisms belonging to Gordonibacter urolithinfaciens, the preferred one is the Gordonibacter urolithinfaciens DSM 27213 strain. Among the microorganisms belonging to Gordonibacter pamelaeae, the preferred one is the DSM 19378 strain. Among the microorganisms belonging to Gordonibacter faecihominis, the preferred one is the JCM 16058 strain. It is the DSM 19378 strain. It is the JCM 16058 strain.

[0028] The strain Gordonibacter urolithinfaciens DSM 27213 is not limited to the same strain, and may be bacteria substantially equivalent to the deposited strain. Substantially equivalent bacteria are microorganisms belonging to Gordonibacter urolithinfaciens, capable of exhibiting the effects according to this aspect, such as expressing the enzyme according to this aspect. Furthermore, the nucleotide sequence of its 16S rRNA gene has preferably 98% or more, more preferably 99% or more, and even more preferably 100% homology with the nucleotide sequence of the 16S rRNA gene of the deposited strain, and is preferably a microorganism having the same mycological properties as the deposited strain. Also, as long as the effects of this aspect are not impaired, it may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, etc. This also applies to the strain Gordonibacter pamelaeae DSM 19378 and the strain Gordonibacter faecihominis JCM 16058. This also applies.

[0029] Microorganisms belonging to the genus Gordonibacter are cultured in common media used for culturing anaerobic bacteria, such as Anaerobe Basal Broth (CM0957 manufactured by ThermoFisher Scientific), Wilkins-Chalgren Anaerobe Broth (CM0643 manufactured by ThermoFisher Scientific), 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. On the other hand, 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 a carbon source. Examples of water-soluble organic substances include, for example, sorbose, fructose, glucose, dextrin, soluble starch , sugars such as these; alcohols such as methanol; organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, succinic acid; amino acids such as arginine, methionine, phenylalanine, valine, glutamic acid, and the like. The concentration of the organic substance added to the medium as a carbon source can be appropriately adjusted for efficient growth. Usually, it can be selected in the range of 0.1 to 10 wt / vol%.

[0031] In addition to the above carbon source, a nitrogen source may be added to the medium. As the nitrogen source, various nitrogen compounds that can be used in normal culture and fermentation can be used. Preferred inorganic nitrogen sources include ammonium salts and nitrates. More preferred inorganic nitrogen sources include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, ammonium citrate, potassium nitrate, and sodium nitrate. On the other hand, preferred organic nitrogen sources include amino acids, yeast extract, peptones (peptones derived from milk, soybeans, soybean peptides, etc.), meat extract (for example, Lab-Lemco powder, bonito extract, tuna extract, bonito extract, broth, shellfish extract, etc.), liver extract, digested serum powder, and the like. More preferred organic nitrogen sources include arginine, cysteine, citrulline, lysine, tryptophan, yeast extract, peptones, and the like.

[0032] Furthermore, in addition to carbon sources and nitrogen sources, microbial growth factors such as extracts, vitamins, metal salts, and inorganic compounds can also be added to the medium. Examples of extracts include hemin, heme iron, digested serum powder, liver extract, and blood digest. Examples of vitamins include biotin, folic acid, pyridoxal, thiamine, riboflavin, nicotinic acid, nicotinamide, pantothenic acid, vitamin B12, thioctic acid, p-aminobenzoic acid, and vitamin Ks. Examples of 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, ammonium ferrous 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 solution by adding growth assisting factors derived from animals and plants such as these inorganic compounds and vitamins are known. The medium can be liquid, semi-solid, or solid. A preferred form of the medium is a liquid medium.

[0033] 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, which is 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 as to have a concentration in the medium of 0.01 g / L or more and 20 g / L or less.

[0034] Produced by a microorganism belonging to the genus Gordonibacter according to this embodiment To recover the enzyme, after the production of the enzyme according to this embodiment, the culture is recovered, and the microorganism is disrupted in a buffer solution containing a reducing agent such as cysteine, 2-mercaptoethanol or dithiothreitol, and a protease inhibitor such as phenylmethanesulfonyl fluoride (PMFS), pepstatin A, or ethylenediaminetetraacetic acid to obtain a cell-free extract. The cell-free extract can be purified by appropriately combining fractionation based on protein solubility and various chromatographies. All of these can be carried out according to conventional methods.

[0035] One subunit, two subunits, or three subunits that are presumed to constitute the enzyme according to this embodiment and are possessed by Gordonibacter urolithinfaciens DSM 27213 strain are preferably selected from the combination of GuUroA1, GuUroA2, and GuUroA3. The amino acid sequences of GuUroA1, GuUroA2, and GuUroA3 are the sequences represented by SEQ ID NOs: 1 to 3, respectively. The nucleotide sequences of the genes encoding GuUroA1, GuUroA2, and GuUroA3 are the sequences represented by SEQ ID NOs: 7 to 9, respectively. Also, as described above, the enzyme according to this embodiment preferably has the property of (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] One subunit, two subunits, or three subunits that are presumed to constitute the enzyme according to this embodiment and are possessed by Gordonibacter pamelaeae DSM 19378 strain are preferably selected from the combination of GpUroA1, GpUroA2, and GpUroA3. The amino acid sequences of GpUroA1, GpUroA2, and GpUroA3 are the sequences represented by SEQ ID NOs: 13 to 15, respectively. is an array represented as: The nucleotide sequences of the genes encoding GpUroA1, GpUroA2, and GpUroA3 are SEQ ID NO: The sequence is represented by numbers 19 to 21.

[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] In addition, when GuUroA1 acts as a subunit by itself or forms a unit together with other subunits, GuUroA2 and / or GuUroA3, it catalyzes the formation of a water molecule at the 4-position of urolithins. As long as it has the activity of dehydroxylating an acid group, 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: 1. "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, and the same applies when amino acids are added to the N-terminus and / or C-terminus. This also applies to GpUroA1.

[0039] Conservative substitution is preferable. Conservative substitution is, for example, 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, and between Lys when the substitution site is a basic amino acid. , between Arg and His, in the case of acidic amino acids, between Asp and Glu, and in the case of amino acids having a hydroxyl group, between Ser and Thr, it means that they can be substituted for each other. Specifically, as conservative substitutions, 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 Leu, Met, Val or Phe, substitution of Leu with Ile, Met, Val or Phe, substitution of Lys with Asn, Glu, Gln, His or Arg, substitution of Met with Ile, Leu, Val or Phe, substitution of Phe with Trp, Tyr, Met, Ile or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe or Trp, and substitution of Val with Met, Ile or Leu can be mentioned. This also applies to GpUroA1. It applies.

[0040] Also, as long as GuUroA1 has the activity of dehydrating and oxidizing the 4-position hydroxyl group of urolithins when it alone forms a subunit or forms a unit together with other subunits GuUroA2 and / or GuUroA3, it may be a protein having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more with respect to the entire length of the amino acid sequence represented by SEQ ID NO: 1. This also applies to GpUroA1. It applies.

[0041] In addition, as long as GuUroA1 has the activity of dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins when it alone constitutes a unit or forms a unit together with GuUroA2 and / or GuUroA3 which are other subunits, it may be a protein encoded by a polynucleotide that hybridizes with the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions. "Stringent conditions" include, for example, conditions under which polynucleotides having 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more homology hybridize with each other, and polynucleotides having lower homology do not hybridize with each other. This also applies to GpUroA1. This also applies to GuUroA1. This also applies to GpUroA1.

[0042] The amino acid substitution, protein homology, stringent conditions, etc. also apply to GuUroA2. However, for GuUroA2, the "1 to a plurality of" preferably means 1 to 12, more preferably 1 to 11, still more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. This also applies to GpUroA2. This also applies to GpUroA2. This also applies to GpUroA2. This also applies to GpUroA2. In addition, the amino acid substitution, protein homology, stringent conditions, etc. also apply to GuUroA3. However, for GuUroA3, the "1 to a plurality of" preferably means 1 to 7, more preferably 1 to 6, still more preferably 1 to 5, even more preferably 1 to 3, and particularly preferably 1 to 2. This also applies to GpUroA3. This also applies to GpUroA3.

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

[0044] <1-1-2(A). Polynucleotide> Other aspects of the present disclosure are a polynucleotide comprising one or more selected from the group consisting of the nucleotide sequence represented by SEQ ID NO: 7, the nucleotide sequence represented by SEQ ID NO: 8, and the nucleotide sequence represented by SEQ ID NO: 9, or a polynucleotide comprising one or more selected from the group consisting of the nucleotide sequence represented by SEQ ID NO: 19, the nucleotide sequence represented by SEQ ID NO: 20, and the nucleotide sequence represented by SEQ ID NO: 21. Details of each nucleotide sequence are as described in the column of "1-1-1(A). Dehydrating oxidase of the 4-position hydroxyl group of urolithins" which has been previously described.

[0045] The nucleotide sequence of the gene encoding GuUroA1 may be a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 7 as long as GuUroA1 itself alone or when constituting a unit together with GuUroA2 and / or GuUroA3 which are other subunits has the activity of dehydrating and oxidizing the 4-position hydroxyl group of urolithins. "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, still more preferably 97% or more, and particularly preferably 99% or more hybridize with each other, and polynucleotides having a lower homology do not hybridize with each other. This also applies to the nucleotide sequence of the gene encoding GuUroA2 and the nucleotide sequence of the gene encoding GuUroA3. Also, regarding 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 it also applies.

[0046] <1-1-3(A). Genetic engineering aspects> Other aspects of the present disclosure include a recombinant vector containing the polynucleotide; a transformant that retains the polynucleotide in an expressible manner or retains the vector in an expressible manner; and a method for producing a protein encoded by the polynucleotide, which includes the step of culturing the transformant. The protein is the "enzyme that dehydrates and oxidizes the hydroxyl group at the 4-position of urolithins". By inserting the polynucleotide into a known expression vector, an expression vector that expresses the enzyme can be obtained. Then, by using the expression vector to transform a microorganism or the like, a transformant can be obtained, and by culturing the transformant or the like to produce the enzyme, the enzyme can be obtained.

[0047] Examples of the host-vector system include, for lactic acid bacteria, Lactococcus lactis subsp. Cremoris - vector pNZ8148 (MoBiTech), Lactococcus lactis - pGKV11 (Appl. Environ. Microbiol., 50, 540 - 542 (1985)), Lactococcus lactis / Streptococcus thermophilus / Streptococcus faecalis, etc. - pBE194 (Japanese Patent Laid-Open No. 6-253861), Lactococcus lactis subsp. lactis / Lactobacillus delbrueckii, etc. - pSYE1 (Japanese Patent Laid-Open No. 5-176776) , examples of Bifidobacterium include Bifidobacterium adolescentis - pKKT427 (Nucleic Acids Research, 2009, Vol. 37, No. 1 e3 doi:10.1093 / nar / gkn884), a wide range of Bifidobacterium 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)), ACE vector (Anaerobe, 41, 104 - 112 (2016)), etc. Although an anaerobic environment is required for the expression of the enzyme, an anaerobic host - vector system is not necessarily required for heterologous gene expression. For example, the Rhodococcus erythropolis - pTip, pNit, pCpi Vector series (Hokkaido System Science, Biotechnol. Bioeng., 86, 136 - 148 (2004)) can be preferably used. Specific examples of the host include the L88 strain, and a specific example of the vector is pTipQC1. In addition to microorganisms, various host - vector systems have been developed in 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 can also be used.

[0048] <1 - 2 - 1(A). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 4 - Position of Urolithins> Another aspect of the present disclosure is a method for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins. Specifically, it is a method for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins having a hydroxyl group at the 4-position. This aspect is a method for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins, including the following step (I): Step (I): contacting one or more selected from the following (i) to (iv) with urolithins having a hydroxyl group at the 4-position to dehydrate and oxidize the hydroxyl group at the 4-position. (i) The enzyme described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins"; (ii) The protein encoded by the polynucleotide described in the column of "1-1-2(A). Polynucleotide"; (iii) A microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) A treated product of the microorganism described in (iii).

[0049] (Urolithins) Details of the urolithins in this aspect are as described in the previously presented column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins".

[0050] (Step (I)) In step (I), (i) the enzyme and (ii) the protein encoded by the polynucleotide each contact urolithins having a hydroxyl group at the 4-position to dehydrate and oxidize the hydroxyl group at the 4-position present in the urolithins. Also, in (iii) the microorganism that produces the enzyme described in (i) or the protein described in (ii) and (iv) the treated product of the microorganism described in (iii), the enzyme or the protein contained in the microorganism or the treated product of the microorganism contacts urolithins having a hydroxyl group at the 4-position to dehydrate and oxidize the hydroxyl group at the 4-position present in the urolithins.

[0051] ​(iii) A microorganism that produces the enzyme described in (i) above or the protein described in (ii) above; (iv) As the microorganism in the processed product of the microorganism described in (iii) above, it may be a microorganism obtained by genetic engineering techniques. Examples of such microorganisms include microorganisms belonging to the genus Gordonibacter. Preferred specific examples of microorganisms belonging to the genus Gordonibacter are the same as those described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins" mentioned above.

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

[0053] When contacting one or more selected from the above (i) to (iv) with urolithins having a hydroxyl group at the 4-position, it 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 and other organic solvents; or in a two-phase mixing system of the organic solvent and an aqueous medium such as ethanol or acetone. It is also possible to carry out by immobilizing one or more selected from the above (i) to (iv), and it is also possible to carry out using a membrane reactor or the like. In addition, when one or more selected from the above (i) to (iv) is a microorganism that produces the enzyme described in (i) above or the protein described in (ii) above, it may be carried out in a solution usually 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, still more preferably 33°C or higher, while on the other hand, it is preferably 55°C or lower, more preferably 50°C or lower, still more preferably 42°C or lower. The pH in step (I) is preferably 4.0 or higher, more preferably 5.0 or higher, still more preferably 5.5 or higher, while on the other hand, it is preferably 9.0 or lower, more preferably 8.0 or lower, still more preferably 7.0 or lower. The concentration of urolithins having a hydroxyl group at the 4-position, which is the raw material (substrate) in step (I), in the reaction solution is 0.001 g / L or higher, preferably 0.01 g / L or higher, more preferably 0.1 g / L or higher, while on the other hand, it is 100 g / L or lower, preferably 20 g / L or lower, more preferably 10 g / L or lower.

[0055] This embodiment may include a step of quantifying the obtained product (quantification step). The quantification method can follow conventional methods. For example, ethyl acetate added with an acid such as formic acid as needed is added to the culture solution, stirred vigorously, then centrifuged, and the ethyl acetate layer is taken out. If necessary, the same operation is performed several times, and these 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. It can be exemplified that this is filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insolubles, and then quantified using high-performance liquid chromatography.

[0056] Moreover, this embodiment may include a step of recovering the obtained product. The recovery step includes a purification step, a concentration step, etc. As the purification treatment in the purification step, sterilization of microorganisms by heat or the like; sterilization by microfiltration (MF), ultrafiltration (UF), etc.; removal of solids and macromolecular substances; extraction with an organic solvent or an ionic liquid; adsorption and decolorization using a hydrophobic adsorbent, an ion exchange resin, an activated carbon column, etc. can be performed. Also, as the concentration treatment in the concentration step, concentration by an evaporator, a reverse osmosis membrane, etc. can be mentioned. Furthermore, the solution containing the obtained product can be powdered by freeze-drying, spray-drying, or the like. In powdering, excipients such as lactose, dextrin, and corn starch can also be added.

[0057] <1-1-1(B). Enzyme that dehydrates and oxidizes the hydroxyl group at the 10th position of urolithins> Another aspect of the present disclosure is an enzyme that dehydrates and oxidizes the hydroxyl group at the 10th position of urolithins. Specifically, it is an enzyme that catalyzes the reaction of dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins having a hydroxyl group at the 10th position. The enzyme is presumed to be composed of one subunit, two subunits, or three subunits.

[0058] Regarding the enzyme according to this aspect, the content described in the column of "1-1-1(A). Enzyme that dehydrates and oxidizes the hydroxyl group at the 4th position of urolithins" is incorporated by reference, but since there are also differences, the differences will be described.

[0059] The urolithins in this aspect are preferably urolithin M5, urolithin M6, or urolithin M7. When the hydroxyl groups at the 10th positions of these are dehydrated and oxidized by the enzyme according to this aspect, urolithin D, urolithin C, and urolithin A are produced, respectively.

[0060] The enzyme according to this aspect is activated by a cofactor. Examples of the cofactor include methyl viologen (MV), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin adenine mononucleotide (FMN). Since the cofactor in this aspect increases the activity of the enzyme according to this aspect in dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins, it is preferable to use MV and NADH, more preferably to use MV, NADPH, and FMN, even more preferably to use MV and NADPH, even more preferably to use MV, NADH, and FAD, extremely preferably to use MV, NADH, and FMN, and even more extremely preferably to use MV, NADPH, and FAD.

[0061] The enzyme according to this aspect 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, 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, and on the other hand, preferably 50 °C or lower, more preferably 42 °C or lower.

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

[0063] One subunit, two subunits, or three subunits that are presumed to constitute the enzyme according to this aspect, which are possessed by Gordonibacter urolithinfaciens DSM 27213 strain, are preferably selected from the combination of GuUroB1, GuUroB2, and GuUroB3. The amino acid sequences of GuUroB1, GuUroB2, and GuUroB3 are the sequences represented by SEQ ID NOs: 4 to 6, respectively. respectively. The nucleotide sequences of the genes encoding GuUroB1, GuUroB2, and GuUroB3 are the sequences represented by SEQ ID NOs: 10 to 12, respectively. respectively. Also, as described above, the enzyme according to this aspect preferably has the property (4) described in the column of "1-1-1(A). Enzyme that dehydrates and oxidizes the 4-position hydroxyl group of urolithins". The molecular weight of 78,000 or more and 96,000 or less is presumed to be the molecular weight of the GuUroB1 subunit. respectively. ​

[0064] One subunit, two subunits, or three subunits that are presumed to constitute the enzyme according to this embodiment and are possessed by the strain Gordonibacter pamelaeae DSM 19378 are preferably selected from the combination of GpUroB1, GpUroB2, and GpUroB3 . The amino acid sequences of GpUroB1, GpUroB2, and GpUroB3 are represented by SEQ ID NOs: 16 to 18, respectively The sequences are as follows The nucleotide sequences of the genes encoding GpUroB1, GpUroB2, and GpUroB3 are represented by SEQ ID NOs: 22 to 24, respectively The sequences are as follows

[0065] Therefore, the enzyme according to this embodiment preferably contains 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 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, and is an enzyme In addition, as long as GuUroB1 has the activity of dehydrating and oxidizing the 10-position hydroxyl group of urolithins when it alone forms a unit as one subunit or forms a unit together with other subunits GuUroB2 and / or GuUroB3, in the amino acid sequence represented by SEQ ID NO: 4, it may be a protein consisting of one or more amino acids substituted or deleted, or one or more amino acids inserted or added. One or more preferably refers to 1 to 80, more preferably 1 to 79, still more preferably 1 to 50, even more preferably 1 to 30, and particularly preferably 1 to 10. The same applies when amino acids are added to the N-terminal side and / or C-terminal side. This also applies to GpUroB1

[0066] ​ In addition, regarding amino acid substitution, protein homology, stringent conditions, etc. are the same as the enzymes described in the column of "1-1-1(A). Enzyme that dehydrates and oxidizes the hydroxyl group at the 4th position of urolithins". This also applies to GpUroB1.

[0067] In addition, the above amino acid substitution, protein homology, stringent conditions, etc. also apply to GuUroB2. However, regarding GuUroB2, the "1 to a plurality" preferably means 1 to 12, more preferably 1 to 11, still more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. This also applies to GpUroB2. This also applies to it. In addition, the above amino acid substitution, protein homology, stringent conditions, etc. also apply to GuUroB3. However, regarding GuUroB3, the "1 to a plurality" preferably means 1 to 20, more preferably 1 to 19, still 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 dehydrate and oxidize the hydroxyl group at the 10th position of urolithins can be evaluated, for example, in the same manner as the method described in Experimental Example 5 below, by quantifying urolithin C produced as a product from urolithin M6.

[0069] <1-1-2(B). Polynucleotide> Another aspect of the present disclosure is a polynucleotide comprising one or more selected from the group consisting of the base sequence represented by SEQ ID NO: 10, the base sequence represented by SEQ ID NO: 11, and the base sequence represented by SEQ ID NO: 12, or 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. The details of each nucleotide sequence are as described in the column of the previously disclosed "1-1-1(B). Enzyme that dehydrates and oxidizes the 10-position hydroxyl group of urolithins".

[0070] The nucleotide sequence of the gene encoding GuUroB1 may be a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 10 as long as GuUroB1 itself or when it forms a unit together with GuUroB2 and / or GuUroB3 which are other subunits has the activity of dehydrating and oxidizing the 10-position hydroxyl group of urolithins. "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, still more preferably 97% or more, particularly preferably 99% or more hybridize with each other, and polynucleotides having a lower homology do not hybridize with each other. This also applies to the nucleotide sequence of the gene encoding GuUroB2 and the nucleotide sequence of the gene encoding GuUroB3. Also, regarding 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 also applies.

[0071] <1-1-3(B). Genetic engineering aspects> Other aspects in the present disclosure include a recombinant vector containing the polynucleotide; a transformant that retains the polynucleotide in an expressible manner or retains the vector in an expressible manner; a method for producing a protein encoded by the polynucleotide, which includes the step of culturing the transformant. The protein is the "enzyme that dehydrates and oxidizes the 10-position hydroxyl group of urolithins". By inserting the polynucleotide into a known expression vector, an expression vector that expresses the enzyme can be obtained. Then, by transforming a microorganism or the like with the expression vector, a transformant can be obtained, and by culturing the transformant and the like to produce the enzyme the enzyme can be obtained. For other aspects, the description in the column of "1-1-3(A). Genetic Engineering Aspects" is incorporated by reference.

[0072] <1-2-1(B). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 10th Position of Urolithins> Another aspect of the present disclosure is a method for dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins. Specifically, it is a method for dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins having a hydroxyl group at the 10th position. This aspect is a method for dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins, including the following step (I): Step (I): contacting one or more selected from the following (i) to (iv) with urolithins having a hydroxyl group at the 10th position to dehydrate and oxidize the hydroxyl group at the 10th position. (i) The enzyme described in the column of "1-1-1(B). Enzyme for Dehydrating and Oxidizing the Hydroxyl Group at the 10th Position of Urolithins"; (ii) The protein encoded by the polynucleotide described in the column of "1-1-2(B). Polynucleotide"; (iii) The microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) The treated product of the microorganism described in (iii).

[0073] (Urolithins) Details of the urolithins in this aspect incorporate the content described in the previously presented column of "1-1-1(B). Enzyme for Dehydrating and Oxidizing the Hydroxyl Group at the 10th Position of Urolithins".

[0074] (Step (I)) In step (I), (i) the enzyme and (ii) the protein encoded by the polynucleotide each dehydrate and oxidize the hydroxyl group at the 10-position of urolithins by contacting the urolithins having a hydroxyl group at the 10-position. Further, (iii) the enzyme described in (i) above or the microorganism producing the protein described in (ii) above, and (iv) the treated product of the microorganism described in (iii) above, the enzyme or the protein contained in the microorganism or the treated product of the microorganism dehydrates and oxidizes the hydroxyl group at the 10-position of the urolithins by contacting the urolithins having a hydroxyl group at the 10-position.

[0075] (iii) The microorganism that produces the enzyme described in (i) above or the protein described in (ii) above; (iv) The microorganism in the treated product of the microorganism described in (iii) above may be a microorganism obtained by genetic engineering techniques. Examples of the microorganism include microorganisms belonging to the genus Gordonibacter and microorganisms belonging to the genus Eggerthella. Preferred specific examples of the microorganisms belonging to the genus Gordonibacter are the same as the microorganisms described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins" described above. 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 under existing species. The Eggerthella sp. DC3563 (NITE BP-02376) strain was deposited internationally based on the Budapest Treaty on November 11, 2016, with the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (address: Room 122, 2-5-8 Kazusa Kamashita, Kisarazu City, Chiba Prefecture 292-0818, Japan). It has been deposited internationally in accordance with the 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. A bacterium substantially equivalent is a microorganism belonging to the genus Eggerthella, having a hydroxyl group at the 10th position, and capable of exerting the effects according to this aspect, such as dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins. Further, the nucleotide sequence of its 16S rRNA gene preferably has a homology of 98% or more, more preferably 99% or more, and even more preferably 100% with respect to the nucleotide sequence of the 16S rRNA gene of the deposited strain, and preferably has the same mycological properties as the deposited strain. Also, as long as the effects of this aspect are not impaired, it may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, etc. For other matters, the description in the section "1-2-1(A). Method for dehydrating and oxidizing the hydroxyl group at the 4th position of urolithins" is incorporated by reference. For other matters, the description in the section "1-2-1(A). Method for dehydrating and oxidizing the hydroxyl group at the 4th position of urolithins" is incorporated by reference.

[0076] For other matters, the description in the section "1-2-1(A). Method for dehydrating and oxidizing the hydroxyl group at the 4th position of urolithins" is incorporated by reference.

[0077] <1-3-1. Method for producing urolithin C (first aspect)> Another aspect of the present disclosure is a method for producing urolithin C (first aspect). This aspect is a method for producing urolithin C, including the following steps (I) to (III): Step (I): A step of causing 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 urolithin M5 with one or more selected from the following (i) to (iv) to produce urolithin M6. (i) The enzyme described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins"; (ii) The protein encoded by the polynucleotide described in the column of "1-1-1(A). Polynucleotide"; (iii) A microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) A processed product of the microorganism described in (iii). Step (III): A step of contacting urolithin M6 with one or more selected from the following (v) to (viii) to produce urolithin C. (v) The enzyme described in the column of "1-1-1(B). Enzyme for dehydrating and oxidizing the hydroxyl group at the 10-position of urolithins"; (vi) The protein encoded by the polynucleotide described in the column of "1-1-1(B). Polynucleotide"; (vii) A microorganism that produces the enzyme described in (v) or the protein described in (vi); (viii) A processed product of the microorganism described in (vii).

[0078] (Step (I)) In Step (I), a microorganism having the ability to produce urolithin M5 from ellagic acid is caused to produce urolithin M5 from ellagic acid.

[0079] Examples of the microorganism having the ability to produce urolithin M5 from ellagic acid include the microorganisms described in Step (I) of <1-2-1(B). Method for dehydrating and oxidizing the hydroxyl group at the 10-position of urolithins>.

[0080] As a method for producing urolithin M5 from ellagic acid, in the column of "1-2-1(B). Method for dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins", the object contacting urolithins having a hydroxyl group at the 10th position is a microorganism producing the enzyme described in the column of "1-1-1(B). Enzyme for dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins", or a microbe producing the protein encoded by the polynucleotide described in the column of "1-1-1(B). Polynucleotide". Examples of the embodiment include the case where the microbe is as described above.

[0081] (Step (II)) In step (II), one or more selected from the following (i) to (iv) are brought into contact with the urolithin M5 to produce urolithin M6. (i) The enzyme described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4th position of urolithins"; (ii) The protein encoded by the polynucleotide described in the column of "1-1-1(A). Polynucleotide"; (iii) A microorganism producing the enzyme described in (i) or the protein described in (ii); (iv) A processed product of the microorganism described in (iii).

[0082] In step (II), (i) the enzyme and (ii) the protein encoded by the polynucleotide dehydrate and oxidize the hydroxyl group at the 4th position present in the urolithin M5 by contacting the urolithin M5 having a hydroxyl group at the 4th position. Further, in (iii) the microorganism producing the enzyme described in (i) or the protein described in (ii), and (iv) the processed product of the microorganism described in (iii), the enzyme or the protein contained in the microorganism or the processed product of the microorganism dehydrates and oxidizes the hydroxyl group at the 4th position present in the urolithin M5 by contacting the urolithin M5 having a hydroxyl group at the 4th position. The embodiment is the same as the details described in the column of the previously described "1-2-1(A). Method for dehydrating and oxidizing the hydroxyl group at the 4th position of urolithins".

[0083] (Step (III)) In step (III), one or more selected from the following (v) to (viii) are brought into contact with the urolithin M6 to produce urolithin C. (v) The enzyme described in the column of "1-1-1(B). Enzyme for dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins"; (vi) The protein encoded by the polynucleotide described in the column of "1-1-1(B). Polynucleotide"; (vii) A microorganism that produces the enzyme described in (v) or the protein described in (vi); (viii) A processed product of the microorganism described in (vii).

[0084] In step (III), (v) the enzyme and (vi) the protein encoded by the polynucleotide each contact urolithin M6 having a hydroxyl group at the 10th position to dehydrate and oxidize the hydroxyl group at the 10th position present in the urolithin M6. Further, in (vii) the microorganism that produces the enzyme described in (v) or the protein described in (vi), and (viii) the processed product of the microorganism described in (vii), the enzyme or the protein contained in the microorganism or the processed product of the microorganism contacts urolithin M6 having a hydroxyl group at the 10th position to dehydrate and oxidize the hydroxyl group at the 10th position present in the urolithin M6. As an embodiment thereof, it is the same as the details described in the column of the previously described "1-2-1(B). Method for dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins".

[0085] One or more selected from the group consisting of step (I) to step (III) may be carried out in the same system. The "being carried out in the same system" means that urolithin M5 produced in step (I) is directly used as urolithin M5 in step (II), urolithin M6 is produced in step (II), and the urolithin M6 is directly used as urolithin M6 in step (III), and a series of processes until urolithin C is produced in step (III) are continuously carried out in the same system. It means that it is carried out. That is, for example, it means not including a step of separating and / or purifying urolithin M5 produced in step (I). In addition, when using microorganisms 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 purification (purification step), and a step of concentration (concentration step). The details thereof are the same as the details described in the column of "1-2-1(A). Method for dehydrating and oxidizing the 4-position hydroxyl group of urolithins" already mentioned. Furthermore, it may include a step of powdering the solution containing urolithin C by freeze-drying, spray-drying, etc. In powdering, 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 embodiment). This embodiment is a method for producing urolithin C including the following steps (I) to (III): Step (I): A step of causing 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 described in the column of "1-1-1(B). Enzyme for dehydrating and oxidizing the 10-position hydroxyl group of urolithins"; (ii) The protein encoded by the polynucleotide described in the column of "1-1-1(B). Polynucleotide"; (iii) A microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) A processed product of the microorganism described in (iii). Step (III): A step of contacting one or more selected from the following (v) to (viii) with the urolithin D to produce urolithin C. (v) The enzyme described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins"; (vi) The protein encoded by the polynucleotide described in the column of "1-1-1(A). Polynucleotide"; (vii) A microorganism that produces the enzyme described in (v) or the protein described in (vi); (viii) A processed product of the microorganism described in (vii).

[0088] (Step (I)) Regarding Step (I), the content described in "1-3-1. Method for producing urolithin C (first aspect)" is incorporated by reference.

[0089] (Step (II)) In Step (II), one or more selected from the following (i) to (iv) are brought into contact with the urolithin M5 to produce urolithin D. (i) The enzyme described in the column of "1-1-1(B). Enzyme for dehydrating and oxidizing the hydroxyl group at the 10-position of urolithins"; (ii) The protein encoded by the polynucleotide described in the column of "1-1-1(B). Polynucleotide"; (iii) A microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) A processed product of the microorganism described in (iii).

[0090] In Step (II), (i) the enzyme and (ii) the protein encoded by the polynucleotide each contact urolithin M5 having a hydroxyl group at the 10-position to dehydrate and oxidize the hydroxyl group at the 10-position present in the urolithin M5. Further, in (iii) the microorganism that produces the enzyme described in (i) or the protein described in (ii), and (iv) the processed product of the microorganism described in (iii), the enzyme or the protein contained in the microorganism or the processed product of the microorganism contacts urolithin M5 having a hydroxyl group at the 10-position to dehydrate and oxidize the hydroxyl group at the 10-position present in the urolithin M5. The mode is the same as the details described in the previously presented section "1-2-1(B). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 10th Position of Urolithins".

[0091] In step (III), one or more selected from the following (v) to (viii) are brought into contact with the urolithin D to produce urolithin C. (v) The enzyme described in the section "1-1-1(A). Enzyme for Dehydrating and Oxidizing the Hydroxyl Group at the 4th Position of Urolithins"; (vi) The protein encoded by the polynucleotide described in the section "1-1-1(A). Polynucleotide"; (vii) A microorganism that produces the enzyme described in (v) or the protein described in (vi); (viii) A processed product of the microorganism described in (vii).

[0092] In step (III), (v) the enzyme and (vi) the protein encoded by the polynucleotide each contact urolithin D having a hydroxyl group at the 4th position, thereby dehydrating and oxidizing the hydroxyl group at the 4th position present in the urolithin D. Further, in (vii) the microorganism that produces the enzyme described in (v) or the protein described in (vi), and (viii) the processed product of the microorganism described in (vii), the enzyme or the protein contained in the microorganism or the processed product of the microorganism contacts urolithin D having a hydroxyl group at the 4th position, thereby dehydrating and oxidizing the hydroxyl group at the 4th position present in the urolithin D. The mode is the same as the details described in the previously presented section "1-2-1(A). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 4th Position of Urolithins".

[0093] Steps (I) to (III) may be carried out in the same system. The term "performed in the same system" means that the urolithin M5 generated in step (I) is directly used as the urolithin M5 in step (II), urolithin D is generated in step (II), the urolithin D is directly used as the urolithin D in step (III), and a series of processes until urolithin C is generated in step (III) are continuously performed in the same system. That is, for example, it means not including steps such as separating and / or purifying the urolithin M5 generated in step (I). Incidentally, when microorganisms are used in each step, the microorganisms in each step may be the same or different.

[0094] This aspect may include other steps. The details are the same as those described in the column of the previously mentioned "1-3-1. Method for producing urolithin C (first aspect)".

[0095] <1-3-3. Method for producing urolithin A (first aspect)> Another aspect of the present disclosure is a method for producing urolithin A (first aspect). This aspect is a method for producing urolithin A including the following step (I). Step (I): A step of causing a microorganism having the ability to generate urolithin A from urolithin C to generate urolithin A from the urolithin C produced by the "1-3-1. Method for producing urolithin C (first aspect)".

[0096] This aspect includes the steps included in the "1-3-1. Method for producing urolithin C (first aspect)" When described, it is as follows. At this time, the above step (I) corresponds to the following step (IV).

[0097] That is, this aspect is a method for producing urolithin A including the following steps (I) to (IV): Step (I): A step of causing a microorganism having the ability to generate urolithin M5 from ellagic acid to generate urolithin M5 from ellagic acid. Step (II): A step of bringing one or more selected from the following (i) to (iv) into contact with the urolithin M5 to generate urolithin M6. (i) The enzyme described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins"; (ii) The protein encoded by the polynucleotide described in the column of "1-1-1(A). Polynucleotide"; (iii) A microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) A processed product of the microorganism described in (iii). Step (III): A step of contacting urolithin M6 with one or more selected from the following (v) to (viii) to produce urolithin C. (v) The enzyme described in the column of "1-1-1(B). Enzyme for dehydrating and oxidizing the hydroxyl group at the 10-position of urolithins"; (vi) The protein encoded by the polynucleotide described in the column of "1-1-1(B). Polynucleotide"; (vii) A microorganism that produces the enzyme described in (v) or the protein described in (vi); (viii) A processed product of the microorganism described in (vii). Step (IV): A step of causing a microorganism having the ability to produce urolithin A from urolithin C to produce urolithin A from the urolithin C.

[0098] (Steps (I) to (III)) For Steps (I) to (III), the content described in "1-3-1. Method for producing urolithin C (first aspect)" is incorporated by reference.

[0099] (Step (IV)) In Step (IV), a microorganism having the ability to produce urolithin A from urolithin C is caused to produce urolithin A from the urolithin C. Examples of the microorganism having the ability to produce urolithin A from urolithin C include microorganisms belonging to the genus Clostridium.

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

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

[0102] Clostridium sp. DC 3656 (NITE ABP-02708) is not limited to the same strain, and may be bacteria substantially equivalent to the deposited strain. Substantially equivalent bacteria are microorganisms belonging to the genus Clostridium, which can produce urolithin A from urolithin C, etc., and can exhibit the effects according to this aspect. Further, the base sequence of its 16S rRNA gene preferably has a homology of 98% or more, more preferably 99% or more, still more preferably 100% with respect to the base sequence of the 16S rRNA gene of the deposited strain, and preferably has the same mycological properties as the deposited strain. Also, as long as the effects of the present disclosure are not impaired, it may be a strain bred from the deposited strain or a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, etc. This also applies to Clostridium bolteae DSM 29485 strain, DSM 15670 strain, JCM 12243 strain, Clostridium asparagiforme DSM 15981 strain, and Clostridium citroniae DSM19261 strain. This also applies to Clostridium bolteae DSM 29485 strain, DSM 15670 strain, JCM 12243 strain, Clostridium asparagiforme DSM 15981 strain, and Clostridium citroniae DSM19261 strain.

[0103] As a method for producing urolithin A from urolithin C, in the column of "1-2-1(A). Method for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins", when the object contacting urolithins having a hydroxyl group at the 4-position is a microorganism producing the enzyme described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins" or a microorganism producing the protein encoded by the polynucleotide described in the column of "1-1-1(A). Polynucleotide", the embodiments can be exemplified.

[0104] One or more selected from the group consisting of steps (I) to (IV) may be carried out in the same system. The "carried out in the same system" means that urolithin M5 produced in step (I) is directly used as urolithin M5 in step (II), urolithin M6 is produced in step (II), urolithin M6 is directly used as urolithin M6 in step (III), urolithin C produced in step (III) is directly used as urolithin C in step (IV), and a series of processes until urolithin A is produced in step (IV) are continuously carried out in the same system. That is, for example, it means not including 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 are the same as those described in the column of the previously mentioned "1-3-1. Method for producing urolithin C (the first embodiment)".

[0106] <1-3-4. Method for producing urolithin A (the second embodiment)> Another embodiment of the present disclosure is a method for producing urolithin A (the second embodiment). This embodiment is a method for producing urolithin A including the following step (I). Step (I): A step of producing urolithin A from urolithin C produced in the "1-3-2. Method for producing urolithin C (the second embodiment)" by a microorganism having the ability to produce urolithin A from urolithin C.

[0107] This aspect is described as follows when listing the steps included in the above "1-3-2. Method for Producing Urolithin C (Second Aspect)". At this time, the above step (I) corresponds to the following step (IV).

[0108] That is, this aspect is a method for producing urolithin A, including the following steps (I) to (IV): Step (I): A step of causing 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 urolithin M5 with one or more selected from the following (i) to (iv) to produce urolithin D. (i) The enzyme described in the column of "1-1-1(B). Enzyme for dehydrating and oxidizing the hydroxyl group at the 10th position of urolithins"; (ii) The protein encoded by the polynucleotide described in the column of "1-1-1(B). Polynucleotide"; (iii) A microorganism that produces the enzyme described in (i) or the protein described in (ii); (iv) A processed product of the microorganism described in (iii). Step (III): A step of contacting urolithin D with one or more selected from the following (v) to (viii) to produce urolithin C. (v) The enzyme described in the column of "1-1-1(A). Enzyme for dehydrating and oxidizing the hydroxyl group at the 4th position of urolithins"; (vi) The protein encoded by the polynucleotide described in the column of "1-1-1(A). Polynucleotide"; (vii) A microorganism that produces the enzyme described in (v) or the protein described in (vi); (viii) A processed product of the microorganism described in (vii). Step (IV): A step of causing a microorganism having the ability to produce urolithin A from urolithin C to produce urolithin A from the urolithin C.

[0109] (Steps (I) to (III)) For step (I) to step (III), the content described in the above "1-3-1. Method for producing urolithin C (first aspect)" is incorporated by reference.

[0110] (Step (IV)) In step (IV), a microorganism having the ability to produce urolithin A from urolithin C is caused to produce urolithin A from the urolithin C. For the details of this step (IV), the content described in the column of the previously mentioned "1-3-3. Method for producing urolithin A (first aspect)" is incorporated by reference.

[0111] One or more selected from the group consisting of step (I) to step (IV) may be carried out in the same system. The "carried out in the same system" means that urolithin M5 produced in step (I) is directly used as urolithin M5 in step (II), urolithin D is produced in step (II), the urolithin D is directly used as urolithin D in step (III), urolithin C produced in step (III) is directly used as urolithin C in step (IV), and a series of processes until urolithin A is produced in step (III) are continuously carried out in the same system. That is, for example, it means not including a step of separating and / or purifying urolithin M5 produced in step (I). In addition, when microorganisms are used in each step the microorganisms in each step may be the same or different.

[0112] This aspect may include other steps. The details are the same as those described in the column of the previously mentioned "1-3-1. Method for producing urolithin C (first aspect)".

[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 aspect is preferably derived from a microorganism belonging to the genus Gordonibacter. More preferably, it 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. Among the microorganisms belonging to Gordonibacter urolithinfaciens, the preferred one is the Gordonibacter urolithinfaciens DSM 27213 strain. Among the microorganisms belonging to Gordonibacter pamelaeae, the preferred one is the DSM 19378 strain. Among the microorganisms belonging to Gordonibacter faecihominis, the preferred one is the JCM 16058 strain.

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

[0115] In addition, 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 are substituted or deleted, or one or more amino acids are inserted or added, in the amino acid sequence represented by SEQ ID NO: 25. The "one or more" is preferably 1 to 50, more preferably 1 to 49, still more preferably 1 to 48, even more preferably 1 to 30, particularly preferably 1 to 10, and still particularly preferably 1 to 5. The same applies when amino acids are added to the N-terminal side and / or C-terminal side.

[0116] The mode of substitution is the same as the mode described in the column of "1-1-1(A). Enzyme that dehydrates and oxidizes the 4-position hydroxyl group of urolithins" described above.

[0117] In addition, 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 having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, with respect to the full length of the amino acid sequence represented by SEQ ID NO: 25.

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

[0119] The amino acid substitution, protein homology, stringent conditions, etc. are also applicable to the ellagic acid transporter derived from Gordonibacter pamelaeae DSM 19378 strain. However, regarding the ellagic acid transporter derived from the DSM 19378 strain, the "1 to a plurality of" preferably means 1 to 52, more preferably 1 to 51, still more preferably 1 to 50, even more preferably 1 to 30, particularly preferably 1 to 10, and even particularly preferably 1 to 5.

[0120] The ellagic acid transporter activity by the ellagic acid transporter according to this aspect can be evaluated, for example, as in Experimental Example 18-1 described later.

[0121] <2-1-2. Polynucleotide containing a gene encoding an ellagic acid transporter> Another aspect of the present disclosure is a polynucleotide containing the nucleotide sequence represented by SEQ ID NO: 26 or containing the nucleotide sequence represented by SEQ ID NO: 28. The details of each nucleotide sequence are as already described.

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

[0123] <2-1-3. Genetic engineering aspects> Another aspect of the present disclosure is a recombinant vector containing the above-mentioned "polynucleotide containing a gene encoding an ellagic acid transporter"; a transformant that can express the above-mentioned "polynucleotide containing a gene encoding an ellagic acid transporter" or that can express the above-mentioned vector; and a method for producing a protein encoded by the polynucleotide, which includes a step of culturing the transformant. The protein is the above-mentioned "ellagic acid transporter". Details are the same as those described in the "1-1-3(A). Genetic engineering aspects" section mentioned above.

[0124] In addition, the transformant is one in which the uptake of ellagic acid from the extracellular to the intracellular is promoted. Therefore, a method for promoting the uptake of ellagic acid from the extracellular to the intracellular of the transformant, which includes the following step (I), is also a preferred aspect. Step (I): A step of bringing the transformant into contact with ellagic acid. The details of the transformant are the same as those described in the previously mentioned "1-1-3(A). Genetic Engineering Aspects" section. Also, as the "mode of contacting the transformant with ellagic acid", the mode in the previously mentioned "1-2-1(A). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 4-Position of Urolithins" section when using the microorganism that produces the enzyme described in (i) above or the microorganism that produces the protein described in (ii) above can be exemplified.

[0125] <2-1-4. Method for Producing Urolithin M5 (First Aspect)> Another aspect of the present disclosure is a method for producing urolithin M5 (first aspect). This aspect is a method for producing urolithin M5, including the following step (I): Step (I): Contacting a transformant described in the "2-1-3. Genetic Engineering Aspects" section, wherein the 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] (Step (I)) In step (I), a transformant described in the "2-1-3. Genetic Engineering Aspects" section, wherein the host is a microorganism having the ability to produce urolithin M5 from ellagic acid, is contacted with ellagic acid to produce urolithin M5 from ellagic acid.

[0127] As the microorganism having the ability to produce urolithin M5 from ellagic acid, which is the host, Escherichia coli commonly used as a host for transformation may be used, but the microorganisms described in the previously mentioned "1-2-1(B). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 10-Position of Urolithins" section can be exemplified.

[0128] As the mode of contacting the transformant with ellagic acid, the mode in the previously mentioned "1-2-1(A). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 4-Position of Urolithins" section when using the microorganism that produces the enzyme described in (i) above or the microorganism that produces the protein described in (ii) above can be exemplified.

[0129] This aspect may include other steps. The details are the same as those described in the previously mentioned section "1-3-1. Method for Producing Urolithin C (First Aspect)". Also, for example, urolithins may be produced according to the method for producing urolithins described in this specification using the generated urolithin M5. Further, this aspect may be implemented in the step of generating urolithin M5 from ellagic acid in the method for producing urolithins described in this specification.

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

[0131] (Step (I)) In step (I), a transformant described in the section "2-1-3. Genetic Engineering Aspect", wherein the host of the transformant is a microorganism having the ability to produce urolithin C from ellagic acid, is contacted with ellagic acid.

[0132] As the microorganism having the ability to produce urolithin C from ellagic acid, Escherichia coli, which is widely used as a host for transformation, may be used, but the microorganisms described in the previously mentioned section "1-3-1. Method for Producing Urolithin C (First Aspect)" can be exemplified.

[0133] As an aspect of the contact between the transformant and ellagic acid, the aspect in the case of using the microorganism that produces the enzyme described in (i) or the microorganism that produces the protein described in (ii) in the previously mentioned section "1-2-1(A). Method for Dehydroxyoxidizing the Hydroxyl Group at the 4-Position of Urolithins" can be exemplified.

[0134] This aspect may include other steps. The details are the same as those described in the previously mentioned "1-3-1. Method for Producing Urolithin C (First Aspect)" section. Also, for example, urolithins may be produced according to the method for producing urolithins described in this specification using the generated urolithin C. Further, this aspect may be implemented in the step of generating urolithin C from ellagic acid in the method for producing urolithins described in this specification.

[0135] <2-1-6. Method for Producing Urolithin A (Third Aspect)> Another aspect of the present disclosure is a method for producing urolithin A (third aspect). This aspect is a method for producing urolithin A, including the following step (I): Step (I): A step of causing a microorganism having the ability to generate urolithin A from urolithin C to generate urolithin A from the urolithin C produced by the method described in the above "2-1-5. Method for Producing Urolithin C (Third Aspect)".

[0136] When this aspect describes the steps included in the above "2-1-5. Method for Producing Urolithin C (Third Aspect)", the above step (I) corresponds to the following step (II).

[0137] That is, this aspect is a method for producing urolithin A, including the following steps (I) and (II): Step (I): A step of contacting a transformant described in the "2-1-3. Genetic Engineering Aspect" section, wherein the host of the transformant is a microorganism having the ability to generate urolithin C from ellagic acid, with ellagic acid to generate urolithin C from ellagic acid. Step (II): A step of causing a microorganism having the ability to generate urolithin A from urolithin C to generate urolithin A from the urolithin C.

[0138] (Step (I)) Regarding step (I), the content described in the above "2-1-5. Method for Producing Urolithin C (Third Aspect)" is incorporated by reference.

[0139] (Step (II)) In Step (II), a microorganism having the ability to produce urolithin A from urolithin C is caused to produce urolithin A from the urolithin C. For details of this Step (II), the content described in the column of the previously mentioned "1-3-3. Method for producing urolithin A (first aspect)" is incorporated by reference.

[0140] Step (I) and Step (II) may be carried out in the same system. The "being carried out in the same system" means that the urolithin C produced in Step (I) is directly used as the urolithin C in Step (II), and a series of processes until urolithin A is produced in the Step (II) are continuously carried out in the same system. That is, for example, it means not including a process of separating and / or purifying the urolithin C produced in Step (I). In addition, when using microorganisms in each step, the microorganisms in each step may be the same or different.

[0141] This aspect may include other steps. The details are the same as the details described in the column of the previously mentioned "1-3-1. Method for producing urolithin C (first aspect)".

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

[0143] The lactonase according to this aspect is preferably derived from a microorganism belonging to the genus Gordonibacter and belonging to the genus. More preferably, it 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. Among the microorganisms belonging to Gordonibacter urolithinfaciens, the preferred one is the Gordonibacter urolithinfaciens DSM 27213 strain. Among the microorganisms belonging to Gordonibacter pamelaeae, the preferred one is the DSM 19378 strain. Among the microorganisms belonging to Gordonibacter faecihominis, the preferred one is the JCM 16058 strain.

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

[0145] In addition, as long as the lactonase derived from the Gordonibacter urolithinfaciens DSM 27213 strain has lactonase activity, it may be a protein consisting of amino acids in which 1 to a plurality of amino acids are substituted or deleted, or 1 to a plurality of amino acids are inserted or added in the amino acid sequence represented by SEQ ID NO: 2 9. The "1 to a plurality of" 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. The same applies when amino acids are added to the N-terminal side and / or C-terminal side.

[0146] The mode of substitution is the same as the mode described in the column of "1-1-1(A). Enzyme that dehydrates and oxidizes the 4-position hydroxyl group of urolithins" described above.

[0147] In addition, as long as the lactonase derived from the Gordonibacter urolithinfaciens DSM 27213 strain has lactonase activity, it may be a protein having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more with respect to the entire length of the amino acid sequence represented by SEQ ID NO: 2 9. In addition, as long as the lactonase derived from the Gordonibacter urolithinfaciens DSM 27213 strain has lactonase activity, it may be a protein encoded by a polynucleotide that hybridizes with the nucleotide sequence represented by SEQ ID NO: 3 0 under stringent conditions. "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, still more preferably 9 7% or more, and particularly preferably 99% or more hybridize with each other, and polynucleotides having a lower homology 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 Gordonibacter pamelaeae DSM 19378 strain. However, with respect to the ellagic acid transporter derived from the Gordonibacter pamelaeae DSM 19378 strain, the "1 to a plurality" preferably means 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 lact onase, and after reacting at 37°C for 3 hours, measure the produced urolithin M5 by HPLC under the following cond itions.

[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 the lactonase derived from the 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 as long as the protein it encodes has lactonase activity. "Stringent conditions" include, for example, conditions under which polynucleotides having 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more homology hybridize with each other, and polynucleotides having lower homology do not hybridize with each other. This also applies to the nucleotide sequence of the gene encoding the lactonase derived from the Gordonibacter pamelaeae DSM 19378 strain.

[0153] <2-2-3. Genetic engineering aspects> Another aspect of the present disclosure is a recombinant vector containing the "polynucleotide containing a gene encoding lactonase" described above; a transformant that can express the "polynucleotide containing a gene encoding lactonase" or that can express the vector; and a method for producing the protein encoded by the polynucleotide, which includes the step of culturing the transformant. The protein is the "lactonase" described above. Details are the same as those described in the details in the previously mentioned "1-1-3(A). Genetic engineering aspects" section.

[0154] In addition, the transformant has the activity of catalyzing a reaction that hydrolyzes at least one of the two ester bonds present in ellagic acid. Therefore, a method for promoting a reaction that hydrolyzes at least one of the two ester bonds present in ellagic acid, which includes the following step (I), is also a preferred aspect. Step (I): A step of bringing the transformant into contact with ellagic acid. The details of the transformant are the same as those described in the previously described "1-1-3(A). Genetic engineering aspects" section. Further, as the "mode of contacting the transformant with ellagic acid", the mode in the case of using the microorganism that produces the enzyme described in the above (i) or the microorganism that produces the protein described in the above (ii) in the previously described "1-2-1(A). Method for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins" section can be exemplified.

[0155] In the transformant that retains the "2-2-2. Polynucleotide containing a gene encoding lactonase" in an expressible manner or retains the vector in an expressible manner, it is preferable that the expression of the "2-1-2. Polynucleotide containing a gene encoding an ellagic acid transporter" is also enabled. That is, in the transformant, it is preferable that lactonase and the ellagic acid transporter are co-expressed.

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

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

[0158] Since the transformant in the "2-2-3. Genetic engineering aspects" section expresses lactonase, it is a microorganism having the ability to generate urolithin M5 from ellagic acid.

[0159] As an aspect of the contact between the transformant and ellagic acid, the aspects in the column of the previously described "1-2-1(A). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 4th Position of Urolithins" can be exemplified when using the microorganism that produces the enzyme described in the above (i) or the microorganism that produces the protein described in the above (ii).

[0160] This aspect may include other steps. The details are the same as those described in the column of the previously described "1-3-1. Method for Producing Urolithin C (First Aspect)". Also, for example, urolithins may be produced according to the method for producing urolithins described in this specification using the generated urolithin M5. Further, this aspect may be implemented in the step of generating urolithin M5 from ellagic acid in the method for producing urolithins described in this specification.

[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 including the following step (I): Step (I): A transformant described in the column of the above "2-2-3. Genetic Engineering Aspect", wherein the host is a microorganism having the ability to produce urolithin C from urolithin M5, is brought into contact with ellagic acid to produce urolithin M5 from ellagic acid and produce urolithin C from the urolithin M5.

[0162] (Step (I)) In step (I), a transformant described in the column of the above "2-2-3. Genetic Engineering Aspect", wherein the host is a microorganism having the ability to produce urolithin C from urolithin M5, is brought into contact with ellagic acid to produce urolithin M5 from ellagic acid and cause the production of urolithin C from the urolithin M5.

[0163] The transformant in the column of the above "2-2-3. Genetic Engineering Aspect" is a microorganism having the ability to produce urolithin M5 from ellagic acid because it expresses lactonase. In addition, as the microorganism having the ability to produce urolithin C from its host, urolithin M5, Escherichia coli, which is widely used as a host for transformation, may be used, but the microorganisms described in the column of the previously disclosed "1-2-1(B). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 10th Position of Urolithins" can be exemplified.

[0164] As the mode of contact between the transformant and ellagic acid, the mode in the case of using the microorganism producing the enzyme described in the above (i) or the microorganism producing the protein described in the above (ii) in the column of the previously disclosed "1-2-1(A). Method for Dehydrating and Oxidizing the Hydroxyl Group at the 4th Position of Urolithins" can be exemplified.

[0165] This mode may include other steps. The details are the same as those described in the column of the previously disclosed "1-3-1. Method for Producing Urolithin C (First Mode)". Further, for example, using the produced urolithin C, urolithins may be produced according to the method for producing urolithins described in this specification. Further, this mode may be implemented in the step of producing urolithin M5 from ellagic acid and the step of producing urolithin C from the urolithin M5 in the method for producing urolithins described in this specification.

[0166] <2-2-6. Method for Producing Urolithin A (Fourth Mode)> Another mode of the present disclosure is a method for producing urolithin A (fourth mode). This mode is a method for producing urolithin A including the following steps (I) and (II): Step (I): A step of generating urolithin M5 from ellagic acid by contacting a transformant described in the column of the above "2-2-3. Genetic Engineering Mode", wherein the host is a microorganism having the ability to produce urolithin C from urolithin M5, and generating urolithin C from the urolithin M5. Step (II): A step of causing a microorganism having the ability to generate urolithin A from urolithin C to generate urolithin A from the urolithin C.

[0167] (Step (I)) For step (I), refer to step (I) in the section "2-2-5. Method for producing urolithin C (the fourth aspect)".

[0168] (Step (II)) In step (II), a microorganism having the ability to produce urolithin A from urolithin C is allowed to produce urolithin A from the urolithin C. For the details of this step (II), refer to the content described in the previously mentioned section "1-3-3. Method for producing urolithin A (the first aspect)".

[0169] Steps (I) and (II) may be carried out in the same system. The phrase "carried out in the same system" means that the urolithin C produced in step (I) is directly used as the urolithin C in step (II), and the series of processes until urolithin A is produced in step (II) are continuously carried out in the same system. That is, for example, it means not including steps such as separating and / or purifying the urolithin C produced in step (I). When using microorganisms in each step, the microorganisms in each step may be the same or different.

[0170] This aspect may include other steps. The details are the same as those described in the previously mentioned section "1-3-1. Method for producing urolithin C (the first aspect)".

Examples

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

[0172] 〔Experimental Example 1〕Preparation of Bacterial Cells for Proteome Analysis The Gordonibacter urolithinfaciens DSM 27213 strain was inoculated into a 10 mL ABB medium (ANAEROBE BASAL BROTH, Oxoid, Code CM0957) containing 0.1% ellagic acid (Sigma-Aldrich) or a medium without it, and cultured at 37°C for 16 days under an anaerobic gas (N2 / H2 / CO2 = 80:10:10) environment. The culture broth was centrifuged to recover the cells, which were then used for proteome analysis.

[0173] 〔Experimental Example 2〕Proteome analysis The 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 according to the method described in AMB Express, 2:37 (2012). As a result, compared with the case of culturing in a medium without ellagic acid, when culturing in a medium containing ellagic acid, proteins with a significantly increased expression level, that is, proteins induced by ellagic acid, were found. Analyzing based on genomic information, CD2938, CD2940, CD2941, CD2942, CD2943, and CD2949, CD2950, and CD2952 were identified. For the analysis of gene information such as homology analysis, GENETYX (Genetics Co., Ltd.) was used.

[0174] 〔Experimental Example 3〕Function prediction of proteins The amino acid sequences of CD2938, CD2940, CD2941, CD2942, and CD2943 were predicted for their functions by homology search with known proteins. The results are as follows. CD2938 is presumed to be "anaerobic dehydrogenase having a Se-Cys (selenocysteine) and Mo (molybdenum)-pterin binding region". CD2940 is presumed to be "a hydrogenase component having an Fe-S (iron-sulfur) cluster" CD2941 showed homology with "cationic and various drug efflux pumps". CD2938, CD2940, and CD2941 are presumably subunits that constitute an enzyme for dehydrating and oxidizing the hydroxyl group at the 4-position of urolithins. These genes were named GuuroA1, GuuroA2, and GuuroA3, respectively, and are genes encoding GuUroA1, GuUroA2, and GuUroA3, respectively. CD2942 is presumably a "metal-dependent hydrolase", and its gene was named uroH. This gene is a lactonase gene, and the lactonase has an activity to catalyze a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid. CD2943 is presumably a "permease belonging to the major facilitator superfamily" and was named uroT. In the biosynthetic pathway of urolithins, it is presumably functioning as a transporter for taking ellagic acid into the bacterial cell.

[0175] Similar to CD2938 and CD2940, CD2949 and CD2950 are presumably an "anaerobic dehydrogenase having a Se-Cys (selenocysteine) and Mo (molybdenum)-pterin binding region" and a "hydrogenase component having an Fe-S (iron-sulfur) cluster", respectively. CD2952 is presumably related to "hem utilization or adsorption". Therefore, it is presumed that CD2949, CD2950, and CD2952 may be subunits that constitute an enzyme for dehydrating and oxidizing the hydroxyl group at the 10-position of urolithins. These genes were named Guur oB1, GuuroB2, and GuuroB3, respectively, and are genes encoding GuUroB1, GuUroB2, and GuUroB3, respectively.

[0176] Since the homologies between CD2938 and CD2949, between CD2940 and CD2950, and between CD2941 and CD2951 were 42%, 50%, and 17%, respectively, "CD2938 and CD2940", "CD2949 and CD2950" ,"CD2938, CD2940, and CD2941", and "CD2949, CD2950, and CD2952" were each suggested to function as a complex.

[0177] [Experimental Example 4] Purification of the Enzyme that Dehydrates and Oxidizes the Hydroxyl Group at the 4-Position of Urolithins (Cultivation of Microorganisms) As a preculture, 20 mL of a modified GAM medium (manufactured by Nippon Pharmaceutical Co., Ltd.) containing 0.1 mg / mL of ellagic acid was added to a 100 mL Erlenmeyer flask, inoculated with Gordonibacter urolithinfaciens DSM 27213 strain, and then incubated with shaking at 37 °C and 80 rpm for 5 to 6 days in an anaerobic chamber. The preculture solution was inoculated into a 2 L Erlenmeyer flask containing 1.75 L of the same medium, and incubated with shaking at 37°C and 80 rpm for 7 to 9 days in an anaerobic chamber. The obtained culture solution was centrifuged (8,000 rpm, 15 minutes, 4°C) to prepare cells as a precipitate fraction.

[0178] (Preparation of Ultracentrifugation Supernatant) The obtained cells were suspended in 50 mM potassium phosphate buffer (KPB, pH 6.5), and the cells were disrupted using a KUBOTA Insonator Model 201M (manufactured by 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 obtained cell-free extract was ultracentrifuged at 100,000 g for 110 minutes, and the supernatant fraction was obtained.

[0179] (Purification of Enzyme (First Mono Q)) Anion exchange chromatography was performed on the obtained supernatant of ultracentrifugation using a Mono Q 5 / 50 column (manufactured by GE Healthcare Japan Co., 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 from eluate A to B was performed, the active fraction of the enzyme was recovered, concentrated by gel filtration, and designated as the 1st Mono Q fraction.

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

[0181] (Purification of enzyme (2nd Mono Q)) The obtained Superdex fraction was purified again by anion exchange chromatography using 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 from eluate A to B was performed, the active fraction of the enzyme was recovered, concentrated by gel filtration, and designated as the 2nd Mono Q fraction.

[0182] The purification steps of the enzyme up to this point were summarized in Table 1.

[0183] [Experimental Example 5] Measurement of the activity of an enzyme that dehydrates and oxidizes the 4-position 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 4-position hydroxyl group of urolithins and the enzyme that dehydrates and oxidizes the 10-position hydroxyl group of urolithins Using a 5-20% gradient gel (PAGEL, NPG-520L, manufactured by ATTO), the enzyme fraction purified so far was subjected to non-denaturing polyacrylamide electrophoresis (Native-PAGE), and the resulting bands were semi-dry blotted onto a PVDF membrane (Immobilon-P Transfer membrane, manufactured by Milipore) using a Polarize Plot AE6677G (manufactured by Atto), each band was cut out, and the sequence was analyzed with an amino acid sequencer.

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

[0188] [Experimental Example 7] SDS-PAGE of the enzyme that dehydrates and oxidizes the 4-position hydroxyl group of urolithins and the enzyme that dehydrates and oxidizes the 10-position 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 Polize Blot AE6677G (Atto), and each band was cut out. The 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. Amino acid sequence analysis of bands I and II obtained in the 2nd Mono Q fraction in lane 5 As a result, two types of amino acid sequences were obtained. The bands were identified as GuUroB1 (protein corresponding to the gene GuuroB1) and GuUroB2 (protein corresponding to the gene GuuroB1). The molecular masses of the bands were estimated to be 90.0 kDa and 87.0 kDa, respectively. 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 The reaction mixture for measuring the enzyme activity described in Experimental Example 5 was prepared by removing NADPH, FAD, and MV, and the reaction mixture was prepared as shown in Table 2. The cofactor was added at the concentration shown in Table 2, and the amount of urolithin M6 produced after the reaction was quantified. The 1st Mono Q fraction was used as the enzyme. The conditions shown in No. 12 of Table 2 (10 mM MV, 10 mM NADPH, 0. The amount of urolicin M6 produced with 2 mM FAD was taken as 100%, and the production amount of urolicin M6 under each condition was expressed as relative activity. The results are summarized in Table 2.

[0191] The results are summarized in Table 2. When NADH, NADPH, FMN, or MV was added alone, activity was confirmed as compared with the case where none was added. In the presence of MV, when NADH or NADPH was further present, it was confirmed that the activity increased synergistically. This was confirmed. In the presence of MV and NADPH, when FAD or FMN was further present, very high activity was confirmed. This was confirmed. The optimal conditions were a combination of 10 mM MV, 10 mM NADPH, and 0.2 mM FAD. In subsequent experimental examples, these conditions were used as the standard activity measurement conditions.

[0192]

Table 2

[0193] 〔Experimental Example 9〕Temperature dependence of the enzyme that dehydrates and oxidizes the 4-position hydroxyl group of urolicins Under the standard activity measurement conditions described in Experimental Example 8, the activity was measured while changing the reaction temperature. The results are shown in Figure 3. The optimal temperature was 42°C, and the activity at that time was taken as 100%. In the range of 37 - 50°C, more than 50% activity was shown.

[0194] 〔Experimental Example 10〕pH dependence of the enzyme that dehydrates and oxidizes the 4-position hydroxyl group of urolicins Under the standard activity measurement conditions described in Experimental Example 8, the activity was measured 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-hydrochloric acid buffer Carbonate buffer: 50 mM Na2CO3-NaHCO3 buffer Sodium phosphate buffer: 50 mM Na3PO4-Na2HPO4 buffer The results are shown in Figure 4. The optimal pH was 6.0, and it showed more than 80% of the activity under the optimal conditions in the range of pH 6.0 to 7.0.

[0195] [Experimental Example 11] Temperature stability of the enzyme that dehydrates and oxidizes the hydroxyl group at the 4-position of urolithins The enzyme was held at 4 to 60 °C for 12 hours, and then under the standard activity measurement conditions described in Experimental Example 8 the activity was measured. The results are shown in Figure 5. The optimal temperature was 10 °C. Assuming the residual activity at 10 °C is 100%, it showed more than 80% of the residual activity at 4 to 20 °C.

[0196] [Experimental Example 12] pH stability of the enzyme that dehydrates and oxidizes the hydroxyl group at the 4-position of urolithins To 10 μL of the 1st Mono Q fraction as the enzyme, 90 μL of each of the following 100 mM buffers was added, concentrated 10-fold by ultrafiltration, and then held at 4 °C for 12 hours. Then, after adding 60 μL of 50 mM KPB (pH 6.5) to return the pH, 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-hydrochloric acid buffer Carbonate buffer: 50 mM Na2CO3-NaHCO3 buffer Sodium phosphate buffer: 50 mM Na3PO4-Na2HPO4 buffer The results are shown in Fig. 6. The most stable pH was 6.0 (potassium phosphate buffer). Therefore, when the activity at pH 6.0 was taken as 100%, residual activities of 75% or more were shown in the range of pH 5.0 to 9.0. Moreover, in the range of pH 10.0 to pH 11.0 (carbonate buffer), residual activities of 75% or more were shown.

[0197] 〔Experimental Example 13〕Construction of plasmid expressing ellagic acid transporter Genomic DNA was prepared from Gordonibacter urolithinfaciens DSM 27213 strain. Using this as a template, PCR was performed using the following primer set to amplify the ellagic acid transporter gene (uroT), which was 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'-AAAGGATCCATGAGCAATCCGAATGCCGCCGTAGCGGCAAGC-3' (SEQ ID NO: 33) 5'-GTTGAATTCTTAGTGCTGTGCGGCTGCCTCGTCTGCGGGTTT-3' (SEQ ID NO: 34)

[0198] 〔Experimental Example 14〕Construction of plasmid expressing lactonase Genomic DNA was prepared from Gordonibacter urolithinfaciens DSM 27213 strain according to a conventional method. Using this as a template, the lactonase gene (uroH) was amplified by PCR using the following primer set, 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]

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

[0200]

[0199] Example 16: Expression of lactonase and ellagic acid transporter in Escherichia coli Escherichia coli Rosetta2 (DE3) strain was transformed with pET21b_UroH and pRSFDuet-1_uroT by the Ca method. The obtained transformant was cultured with shaking at 37 °C for 4 hours in 5 mL of LB medium containing 34 μg / mL chloramphenicol and / or 39 μg / mL kanamycin. IPTG was added to the culture solution to a concentration of 1 mM and further cultured with induction at 30 °C for 4 hours. The obtained culture solution was centrifuged, washed with 0.85% NaCl, and wet bacterial cells were prepared.

[0201]

[0199] Example 17: Expression of lactonase in Escherichia coli Escherichia coli Rosetta2 (DE3) strain was transformed with pET21b_UroH by the Ca method. The obtained transformant was cultured with shaking at 37 °C for 4 hours in 5 mL of LB medium containing 34 μg / mL chloramphenicol and / or 39 μg / mL kanamycin. IPTG was added to the culture solution to a concentration of 1 mM and further cultured with induction at 30 °C for 4 hours. The obtained culture solution was centrifuged, washed with 0.85% NaCl, and wet bacterial cells were prepared.

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

[0203] The results are shown in Table 3. The magnification in Table 3 is the relative value with respect 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), no urolithin M5 was produced at all. When only the uroH gene was introduced (Experimental Example 17), 0.960 mM of urolithin M5 was produced. When the uroH gene and the uroT gene were introduced (Experimental Example 16), 1.22 mM of urolithin M5 was produced, and the amount of urolithin M5 produced increased 1.3-fold compared to the case where 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 After adding ellagic acid at a final concentration of 1 g / L to ABB medium (manufactured by Oxoid), heat sterilization was performed, and the one with the gas phase replaced with N2:CO2:H2 (80% / 10% / 10%) gas was used as the basal medium. The Gordonibacter faecihominis JCM 16058 strain was inoculated into the basal medium and anaerobically cultured at 37°C for 14 days.

[0206] The quantification of urolithins in the culture solution after culturing was performed 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 Urolithin C was added to ABB medium (Oxoid) as a precursor of urolithin A to a final concentration of 1.0 g / L, which was then sterilized by heating and the gas phase was replaced with N2:CO2:H2 (80% / 10% / 10%) gas. The basal medium was prepared by substituting Clostridium voltease JCM 12243. strain, DSM 15670 strain, or DSM 29485 strain was inoculated and cultured anaerobically at 37°C. After the culture was completed, 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 the urolithins were quantitatively analyzed by HPLC.

[0210] The HPLC conditions are as follows. <HPLC条件> 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] In addition, urolithins manufactured by DALTON PHARMA were used as standards and dissolved in DMSO for use. As a result, after 2 weeks of culture, 89%, 100%, and 89% of the added urolithin C was converted to urolithin A in each case.

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

[0213] 〔Experimental Example 19-3〕Production of urolithin A from urolithin C The same procedure as in Experimental Example 19-1 was performed except that Clostridium citroniae DSM 19261 was 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 bolteae JCM 12243 strain and Gordonia pamelaeae DSM 19378 strain were inoculated into ABB medium (manufactured by Oxoid) containing 0.1% ellagic acid (manufactured by SIGMA) and cultured in the same manner as in Experimental Example 19-1. As a result, after 2 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 Clostridium bolteae JCM 12243 strain and Gordonibacter urolithinfaciens DSM 27213 strain were cultured in the same manner as in Experimental Example 19-4 except that they were used. As a result of culturing for 2 weeks, 62% of the added ellagic acid was converted to urolithin A.

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

[0217] [Experimental Example 19-7] Production of urolithin A from urolithin C Clostridium citroniae DSM 19261 strain and Gordonibacter urolithifaci ence DSM 27213 strain were cultured in the same manner as in Experimental Example 19-4 except that they were used. As a result of culturing for 5 days, 60% of the added ellagic acid was converted to urolithin A.

[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 culturing 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 in 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 sterilized by heating, and the one with the gas phase replaced with N2:CO2:H2 (80% / 10% / 10%) gas was used as the basal medium. The basal medium was inoculated with the strain Eggerthella sp. DC3563 (NITE BP-02376) and cultured anaerobically at 37 °C for 2 weeks. The quantification of urolithins in the culture broth after culture was performed by the following method. Urolithins were extracted from 5 mL of the culture broth 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.

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

[0223] As a standard, 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% with respect to the added ellagic acid.

[0224] 〔Experimental Example 20〕Measurement of Lactonase Activity Using genomic DNA derived from Gordonibacter urolithinfaciens DSM 27213 strain as a template, the lactonase gene was cloned by PCR using the following primer set and inserted into pET-28a(+), thereby constructing an expression plasmid pET-28a_GuUroH-Histag(N) capable of expressing a protein with an His-tag added to the N-terminus of lactonase. 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 generated from the raw material (substrate) ellagic acid 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. Here, 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 the linearized plasmid was obtained by gel extraction. was obtained. Using the primer sets shown 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 inserted DNA fragment were ligated using NEBuillder HiFi DNA Assembly Mastermix (New England Biolabs).

[0229]

Table 4

[0230] The nucleotide 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 and. pTIpQC1_uroA1 is a plasmid in which GuuroA1 is inserted into the EcoR1 and BamHI sites of pTipQC1 and.

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

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

[0234] [Experimental Example 20-3] Measurement of the activity of the enzyme that dehydrates and oxidizes the hydroxyl group at the 4-position of urolithins The reaction for activity measurement 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 with shaking at 37 °C for 3 hours and 200 spm in an anaerobic pack. 100 μL of the reaction solution was added to 200 μL of N,N-dimethylacetamide (DMA) containing 1% formic acid, and after mixing, the supernatant obtained by centrifugation was analyzed by HPLC to quantify the product.

[0235] ​​​​The results are shown in Table 5. Urolithin M6 could not be detected in the host L88 strain (R. erythropolis L88), but its production was confirmed in the transformant strains containing GuuroA1, GuuroA2, and GuuroA3 (R. erythropolis_uroA123), as well as in the transformant strains containing GuuroA1 and GuuroA2 (R. erythropolis_uroA12). In addition, the production of urolithin M6 could not be confirmed in the transformant strain containing only GuuroA1 (R. erythropolis_uroA1). From this, it was confirmed that at least GuuroA1 and GuuroA2 are required for the production of urolithin M6 from urolithin M5. Regarding the function of GuuroA3, there is a possibility that host-derived proteins complement its function in the host L88 strain.

[0236] [Table 5]

[0237] [Experimental Example 21-1] Construction of a vector expressing an enzyme that dehydrates and oxidizes the 10-position hydroxyl group of urolithins, and transformant strains The Rhodococcus -derived expression vector pTipQC1 (Hokkaido System Science Co., Ltd.) was digested with the restriction enzymes EcoRI and BamHI, and the 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 the Gordonibacter urolithinfaciens DSM 27213 strain as a template, and each inserted DNA fragment was obtained by gel extraction. The linearized plasmid and each inserted DNA fragment were ligated using NEBuillder 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] The 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 and. pTIpQC1_uroB1 is a plasmid in which GuuroB1 is inserted into the EcoR1 and BamHI sites of pTipQC1 is.

[0241] The vector was cloned using Escherichia coli DH5α strain as the host. Selection Ampicillin was used as the marker. The prepared 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 (at 28 °C) in an LB medium (20 μg / mL chloramphenicol), and glycerol stocks (30% glycerol) were prepared from the culture broth and stored (-80 °C), and were used for heterologous expression and measurement of enzyme activity. and measurement of enzyme activity.

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

[0243] [Experimental Example 21-3] Measurement of the dehydration oxidation activity of cells that dehydrate and oxidize the 10-position hydroxyl group of urolithins The reaction for activity measurement 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 metabisulfite, 2 mg / mL sodium hydrogen carbonate, and 50 mM potassium phosphate buffer (pH 6.5) was reacted with shaking at 37 °C for 12 hours and 200 spm in an anaerobic pack. 100 μL of the reaction solution was added to 200 μL of N,N-dimethylacetamide (DMA) containing 1% formic acid , After mixing, the centrifuged supernatant was analyzed by HPLC to quantify the product.

[0244] The results are shown in Fig. 7. Urolithin C could not be detected in the host L88 strain (R. erythropolis L88), but production of urolithin M6 was confirmed in the transformants containing GuuroB1, GuuroB2, and GuuroB3 (R. erythropolis_uroB123, uroB123 in Fig. 7), as well as in the transformants containing GuuroB1 and GuuroB2 (R. erythropolis_uroB12, uroB12 in Fig. 7). Also, production of urolithin C could not be confirmed in the transformant containing only GuuroB1 (R. erythropolis_uroA1, uroB1 in Fig. 7). From this, it was confirmed that at least GuuroB1 and GuuroB2 are required for the production of urolithin C from urolithin M6. Regarding the function of GuuroB3, there is a possibility that host-derived proteins in the host L88 strain complement its function.

[0245] 〔Experimental Example 22-1〕Production of an enzyme that dehydrates and oxidizes the 10-position hydroxyl group of urolithins The Gordonibacter urolithinfaciens DSM 27213 strain was inoculated into 20 mL of a modified GAM medium (Nissui Pharmaceutical) containing 0.1 mg / mL ellagic acid and cultured with shaking at 37 ℃ for 5 to 6 days. 10 mL of the obtained preculture was inoculated into 1.75 L of the same medium and cultured with shaking at 37℃ for 7 to 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 an enzyme that dehydrates and oxidizes the 10-position hydroxyl group of urolithins The washed cells were suspended in a buffer and sonicated for 40 minutes. The cell lysate was centrifuged (8,500 rpm, 30 minutes, 4℃), and then further centrifuged at high speed (20,000g, 60 The supernatant obtained by centrifugation (4°C) was used as the cell-free extract. The supernatant obtained by further ultracentrifugation (100,000 g, 110 minutes, 4°C) of the cell-free extract was used as the ultracentrifugation supernatant fraction. The following 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 10-position hydroxyl group of urolithins The activity of dehydrating and oxidizing the 10-position hydroxyl group of urolithins was measured for the ultracentrifugation supernatant fraction obtained in Experimental Example 22-2. Urolithin M6, which has a hydroxyl group at the 10-position, was used as the urolithin. When the 10-position hydroxyl group is dehydrated and oxidized, urolithin C is produced. 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 enzyme contained in the fraction obtained in Experimental Example 22-1 20 μL of the reaction solution for activity measurement 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 as follows.

[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 was measured by UV absorption at 337 nm)

[0249] The results of this experimental example 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 dehydrates and oxidizes the hydroxyl group at the 10th position of urolithins Using the enzyme solution obtained in Experimental Example 22-2, cofactors at the concentrations described in Table 7 were added to the reaction solution from which NADPH, FAD, and MV were removed in the reaction solution for measuring enzyme activity described in Experimental Example 23-1, and the urolithin C produced after the reaction was quantified. Taking 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) as 100%, the production amount of urolithin C under each condition was expressed as relative activity. In the subsequent 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 dehydrates and oxidizes the hydroxyl group at the 10th position of urolithins Under the standard activity measurement conditions described in Experimental Example 23-2, the activity was measured by changing the reaction temperature. The results are shown in Figure 8. The optimum temperature was 42 °C.

[0253] 〔Experiment 25〕pH dependence of the enzyme that dehydrates and oxidizes the hydroxyl group at the 10th position of urolithins Under the standard activity measurement conditions described in Experimental Example 23-2, the activity was measured by changing the pH 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-hydrochloric acid buffer Carbonate buffer: 50 mM Na2CO3 - NaHCO3 buffer Sodium phosphate buffer: 50 mM Na3PO4 - Na2HPO4 buffer The results are shown in Figure 9. The optimal pH was 5.5.

[0254] [Example 26] Production of urolithin C from urolithin M5 by a deposited strain To ABB medium (manufactured by Oxoid), urolithin M5 was added so that the final concentration was 3.3 mM, and then the mixture was sterilized by heating. The resulting medium with the gas phase replaced with N2:CO2:H2 (80% / 10% / 10%) gas was used as the basal medium. The basal medium was inoculated with Gordonibacter urolithinfaciens DSM 27213 strain and cultured anaerobically at 37°C. After completion of the culture, an equal volume of DMSO was added to 1 mL of the culture solution to dissolve urolithins, and quantitative analysis of 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) (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)

[0256] As a result, 0.0707 mM of urolithin C was produced by culturing for 14 days.

Claims

1. A protein comprising the amino acid sequence represented by SEQ ID NO:25, or the amino acid sequence represented by SEQ ID NO:

27.

2. A polynucleotide comprising the base sequence represented by SEQ ID NO:26 or the base sequence represented by SEQ ID NO:

28.

3. A recombinant vector comprising the polynucleotide of claim 2.

4. A transformant comprising the polynucleotide according to claim 2 in an expressible state, or the vector according to claim 3 in an expressible state.

5. A method for producing a protein encoded by the polynucleotide of claim 2, comprising the step of culturing the transformant of claim 4.

6. A method for promoting the uptake of ellagic acid from outside the cell into the cell of the transformant described in claim 4, comprising the following step (I): Step (I): A step of contacting the transformant described in claim 4 with ellagic acid.

7. A method for producing urolithin M5, comprising the following step (I): Step (I): A step of contacting the transformant described in claim 4, 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.

8. A method for producing urolithin C, comprising the following step (I): Step (I): A step of contacting the transformant described in claim 4, 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.

9. A method for producing urolithin A, comprising the following steps (I) and (II): Step (I): A step of contacting the transformant described in claim 4, 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.

10. A protein comprising the amino acid sequence represented by SEQ ID NO:29, or the amino acid sequence represented by SEQ ID NO:

31.

11. A polynucleotide comprising the base sequence represented by SEQ ID NO:30 or the base sequence represented by SEQ ID NO:

32.

12. A recombinant vector comprising the polynucleotide of claim 11.

13. A transformant comprising the polynucleotide according to claim 11 in an expressible state, or the vector according to claim 12 in an expressible state.

14. A method for producing a protein encoded by the polynucleotide of claim 11, comprising the step of culturing the transformant of claim 13.

15. A method for producing urolithin M5, comprising the following step (I): Step (I): A step of contacting the transformant described in claim 13 with ellagic acid to produce urolithin M5 from ellagic acid.

16. 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 described in claim 13, whose host is a microorganism having the ability to produce urolithin C from urolithin M5, with ellagic acid, and producing urolithin M5 from the urolithin M5.

17. 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 described in claim 13, whose host is a microorganism having the ability to produce urolithin C from urolithin M5, with ellagic acid, and producing urolithin M5 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.

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