An enzyme that dehydrates and oxidizes the hydroxyl group at position 9 of urolithin compounds.

Abstract

The present disclosure addresses the problem of providing an enzyme that dehydroxylates a hydroxyl group at the 9-position of a urolithin compound, said urolithin compound having the hydroxyl group at least at the 9-position. This problem can be solved by an enzyme that has the following properties (1) and (2). (1) The enzyme dehydroxylates the hydroxyl group at the 9-position of a urolithin compound. (2) The enzyme is activated by reduced nicotinamide adenine dinucleotide (NADH) and / or reduced nicotinamide adenine dinucleotide phosphate (NADPH).

Description

Technical Field

[0001] The present disclosure relates to an enzyme that dehydrates and oxidizes the hydroxyl group at the 9-position 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 pomegranates, raspberries, blackberries, cloudberries, strawberries, and walnuts.

[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 and demethylated to 2-bromo-5-hydroxybenzoic acid, and then 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 after being ingested into the body and converted into urolithins. In recent years, microorganisms belonging to Gordonibacter urolithinfaciens and microorganisms belonging to Gordonibacter pamelaeae, which are intestinal bacteria that produce urolithin C, a kind of urolithins, from ellagic acid, have been found, and a method for producing urolithin C by fermenting ellagic acid using these intestinal bacteria 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] J. Agric. Food Chem., 56, 393-400 (2008) [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of this disclosure is to provide an enzyme that dehydrates and oxidizes the hydroxyl group at the 9th position of urolithin compounds having a hydroxyl group at the 9th position. [Means for solving the problem]

[0008] The inventors have discovered, through proteomic analysis of specific strains of Clostridium bolteae, that certain proteins are expressed at significantly higher levels when cultured in a medium containing urolithin C than when cultured in a medium without urolithin C. This disclosure includes the following aspects.

[0009] [1] An enzyme having the properties of (1) and (2) below. (1) The hydroxyl group at position 9 of urolithin is dehydrated and oxidized. (2) It is activated by reduced nicotinamide adenine dinucleotide (NADH) and / or reduced nicotinamide adenine dinucleotide phosphate (NADPH). [2] The enzyme described in [1], having the properties of (3) to (5) below. (3) The SDS-PAGE results include a band with a relative molecular mass between 77,000 and 95,000. (4) The optimal pH is between 6.0 and 8.5. (5) The optimal temperature is between 37°C and 60°C. [3] The enzyme described in [1] or [2], derived from a microorganism belonging to the genus Clostridium. [4] The enzyme according to [3], wherein the microorganism belonging to the genus Clostridium is one or more selected from the group consisting of microorganisms belonging to Clostridium bolteae, microorganisms belonging to Clostridium asparagiforme, microorganisms belonging to Clostridium citroniae, and Clostridium sp. [5] The microorganism belonging to Clostridium bolteae is one or more selected from the group consisting of Clostridium bolteae JCM 12243 strain, Clostridium bolteae DSM 29485 strain, and Clostridium bolteae DSM 15670 strain, The microorganism belonging to the aforementioned Clostridium asparagiforme is Clostridium asparagiforme strain DSM 15981. The microorganism belonging to Clostridium citroniae mentioned above is Clostridium citroniae strain DSM 19261. The aforementioned Clostridium sp. is Clostridium sp. DC3656 (NITE BP-02708) strain. The enzyme described in [4]. [6] Containing the amino acid sequence represented by Sequence ID No. 3, Containing the amino acid sequence represented by Sequence ID No. 6, or The amino acid sequence represented by SEQ ID NO: 9 is included. The enzyme described in any of [1] to [5]. [7] Including 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 It includes the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, and the amino acid sequence represented by SEQ ID NO: 6, or The amino acid sequence includes the amino acid sequence represented by SEQ ID NO: 7, the amino acid sequence represented by SEQ ID NO: 8, and the amino acid sequence represented by SEQ ID NO: 9. The enzyme described in any of [1] to [6].

[0010] [8] The base sequence represented by Sequence ID No. 12, Includes the base sequence represented by Sequence ID No. 15, or The base sequence represented by Sequence ID No. 18 is included. Polynucleotide. [9] Including 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, The sequence includes the base sequence represented by SEQ ID NO: 13, the base sequence represented by SEQ ID NO: 14, and the base sequence represented by SEQ ID NO: 15, or The sequence includes the base sequence represented by SEQ ID NO: 16, the base sequence represented by SEQ ID NO: 17, and the base sequence represented by SEQ ID NO: 18. Polynucleotide.

[0011] A recombinant vector comprising the polynucleotide described in

[10] , [8], or [9]. A transformant having a polynucleotide described in

[11] , [8], or [9] in an expressible form, or a vector described in

[10] in an expressible form. A method for producing a polynucleotide-encoded protein according to [8] or [9], comprising the step of culturing the transformant described in

[12] or

[11] .

[0012]

[13] A method for dehydrating and oxidizing the hydroxyl group at position 9 of urolithin compounds, comprising the following step (I): Step (I): A step of contacting urolithin having a hydroxyl group at the 9-position with one or more of the following (i) to (iv) to dehydrate and oxidize the hydroxyl group at the 9-position. (i) An enzyme according to any one of [1] to [7]; (ii) A protein encoded by the polynucleotide according to [8] or [9]; (iii) A microorganism that produces the enzyme according to (i) above or the protein according to (ii) above; (iv) A processed product of the microorganism according to (iii) above.

[14] The urolithins are urolithin M5, urolithin M6, urolithin C, or isourolithin A, The products formed by dehydrating and oxidizing the hydroxyl group at the 9-position of the urolithins are urolithin E, urolithin M7, urolithin A, and urolithin B, respectively, The method according to

[13] .

[0013]

[15] A method for producing urolithin A, comprising the following steps (I) and (II): Step (I): A step of causing a microorganism having the ability to produce urolithin C from ellagic acid to produce urolithin C from ellagic acid. Step (II): A step of contacting the urolithin C with one or more selected from the following (i) to (iv) to produce urolithin A. (i) An enzyme according to any one of [1] to [7]; (ii) A protein encoded by the polynucleotide according to [8] or [9]; [[ID=2⑦]](iii) A microorganism that produces the enzyme according to (i) above or the protein according to (ii) above; (iv) A processed product of the microorganism according to (iii) above.

[0014]

[16] A method for producing urolithin B, comprising the following steps (I) to (III): Step (I): A step of causing a microorganism having the ability to produce urolithin C from ellagic acid to produce urolithin C from ellagic acid. Step (II): A step of causing a microorganism having the ability to produce isourolithin A from urolithin C to produce isourolithin A from the urolithin C. Step (III): A step of contacting isouroroline A with one or more of the following (i) to (iv) to produce uroline B. (i) An enzyme listed in any of [1] to [7]; (ii) Proteins encoded by polynucleotides as described in [8] or [9]; (iii) Microorganisms that produce the enzyme described in (i) above, or the protein described in (ii) above; (iv) The treated product of the microorganism described in (iii) above. [Effects of the Invention]

[0015] This disclosure may have the effect of providing an enzyme that dehydrates and oxidizes the hydroxyl group at the 9th position of urolithin compounds having a hydroxyl group at the 9th position. [Brief explanation of the drawing]

[0016] [Figure 1] Results of SDS-PAGE in one experimental example of this disclosure (photograph used as a substitute for drawing). [Figure 2] A graph showing the temperature dependence of an enzyme in one experimental example of this disclosure. [Figure 3] A graph showing the pH dependence of an enzyme in one experimental example of this disclosure. [Figure 4] A graph showing the temperature stability of an enzyme in one experimental example of this disclosure. [Figure 5] A graph showing the pH stability of an enzyme in one experimental example of this disclosure. [Figure 6] Results of SDS-PAGE in one experimental example of this disclosure (photograph used as a substitute for drawing). [Modes for carrying out the invention]

[0017] The details of this disclosure are described below. Each configuration and combination thereof in each embodiment is an example, and additions, omissions, substitutions, and other modifications are permitted as appropriate, without departing from the spirit of this disclosure. This disclosure is not limited by its embodiments, but is limited only by the scope of its claims. Furthermore, each aspect disclosed herein can be combined with any other features disclosed herein.

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

[0019] Urolithins are represented by the following general formula (1). [ka] (In the formula, R1 to R6 each independently represent a hydroxyl group, a hydrogen atom, or a methoxy group, and at least one of R1 to R6 is a hydroxyl group.) The hydroxyl group at position 9 of uroritins described herein refers to the hydroxyl group of uroritins in which R4 of the above general formula (1) is a hydroxyl group.

[0020] <1. Dehydrating the hydroxyl group at position 9 of urolithin compounds with a dehydrating enzyme> One aspect of this disclosure is a dehydrating oxidase for the hydroxyl group at position 9 of urolithins. In this specification, this may be referred to as "the enzyme according to this aspect," etc. It is presumed that the enzyme is composed of one subunit, two subunits, or three subunits.

[0021] The enzyme according to this embodiment is an enzyme having the following properties (1) and (2). (1) The hydroxyl group at position 9 of urolithin is dehydrated and oxidized. (2) It is activated by reduced nicotinamide adenine dinucleotide (NADH) and / or reduced nicotinamide adenine dinucleotide phosphate (NADPH).

[0022] In this embodiment, the uroritins are preferably uroritin M5, uroritin M6, uroritin C, or isouroritin A. When the hydroxyl group at position 9 of these is dehydrated and oxidized by the enzyme according to this embodiment, uroritin E, uroritin M7, uroritin A, and uroritin B are produced, respectively.

[0023] The enzyme according to this embodiment is activated by a cofactor. Examples of cofactors include reduced nicotinamide adenine dinucleotide (NADH) and / or reduced nicotinamide adenine dinucleotide phosphate (NADPH). In embodiments where NADH and / or NADPH are used in combination, flavin mononucleotide (FMN) may also be used as a cofactor.

[0024] The enzyme according to this embodiment preferably has the following properties (3) to (5). (3) In the SDS-PAGE results, the relative molecular mass preferably includes a band of 77,000 or more, more preferably 80,000 or more, and even more preferably 83,000 or more, while the relative molecular mass preferably includes a band of 95,000 or less, more preferably 92,000 or less, and even more preferably 89,000 or less.

[0025] (4) The optimal pH is preferably 6.0 or higher, more preferably 6.5 or higher, and even more preferably 7.0 or higher, while preferably 8.5 or lower, and more preferably 8.0 or lower.

[0026] (5) The optimal temperature is preferably 37°C or higher, more preferably 45°C or higher, and even more preferably 50°C or higher, while preferably 60°C or lower, more preferably 58°C or lower, and even more preferably 56°C or lower.

[0027] Furthermore, it is preferable that the enzyme according to this embodiment has the following properties (3-1). (3-1) In the SDS-PAGE results, the relative molecular mass preferably includes bands with a value of 25,000 or more, more preferably 27,000 or more, and even more preferably 29,000 or more, while the relative molecular mass preferably includes bands with a value of 37,000 or less, more preferably 35,000 or less, and even more preferably 33,000 or less.

[0028] Furthermore, it is preferable that the enzyme according to this embodiment has the following properties (3-2). (3-2) In the SDS-PAGE results, the relative molecular mass preferably includes bands with a value of 14,000 or more, more preferably 15,000 or more, and even more preferably 16,000 or more, while the relative molecular mass preferably includes bands with a value of 21,000 or less, more preferably 20,000 or less, and even more preferably 19,000 or less.

[0029] The enzyme according to this embodiment is preferably derived from a microorganism belonging to the genus Clostridium.

[0030] More preferably, it is one or more selected from the group consisting of microorganisms belonging to Clostridium bolteae, microorganisms belonging to Clostridium asparagiforme, microorganisms belonging to Clostridium citroniae, and Clostridium sp.

[0031] The preferred microorganisms belonging to Clostridium bolteae are one or more selected from the group consisting of Clostridium bolteae JCM 12243 strain, Clostridium bolteae DSM 29485 strain, and Clostridium bolteae DSM 15670 strain.

[0032] Of the microorganisms belonging to the genus Clostridium asparagiforme, the preferred strain is Clostridium asparagiforme DSM 15981.

[0033] Of the microorganisms belonging to Clostridium citroniae, the preferred strain is Clostridium citroniae DSM 19261.

[0034] The preferred strain of Clostridium sp. is Clostridium sp. DC3656 (NITE BP-02708). Clostridium sp. DC 3656 (NITE BP-02708) strain was deposited domestically on May 8, 2018, at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) under accession number NITE P-02708. Subsequently, on July 15, 2020, a request for transfer of the bacterial strain to international deposit under the Budapest Convention was made to the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NITE) [Address: Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818], and it was assigned the accession number NITE BP-02708.

[0035] The Clostridium bolteae JCM 12243 strain is not limited to the same strain, but may be a bacterium substantially equivalent to the deposited strain. A substantially equivalent bacterium is a microorganism belonging to Clostridium bolteae that can exhibit the effects of this embodiment, such as expressing the enzyme according to this embodiment, and whose 16S rRNA gene sequence has preferably 98% or more, more preferably 99% or more, and more preferably 100% homology to the 16S rRNA gene sequence of the deposited strain, and preferably has the same mycological properties as the deposited strain. In addition, as long as the effects of this embodiment are not impaired, the microorganism may be a strain bred from the deposited strain or a substantially equivalent strain by mutation treatment, genetic modification, selection of a naturally occurring mutant, etc. This also applies to the other strains mentioned above.

[0036] Microorganisms belonging to the genus Clostridium are cultured in common culture media used for anaerobic bacteria, such as Anaerobe Basal Broth (ThermoFisher Scientific CM0957), Wilkins-Chalgren Anaerobe Broth (ThermoFisher Scientific CM0643), GAM medium (Nissui Pharmaceutical Co., Ltd.), and Modified GAM medium (Nissui Pharmaceutical Co., Ltd.). The culture temperature is preferably 25°C or higher, more preferably 30°C or higher, and even more preferably 33°C or higher, while preferably 45°C or lower, more preferably 42°C or lower, and even more preferably 40°C or lower.

[0037] Furthermore, for example, water-soluble organic substances can be added as a carbon source. Examples of water-soluble organic substances include sugars such as sorbose, fructose, glucose, dextrin, and soluble starch; alcohols such as methanol; organic acids such as valeric acid, butyric acid, propionic acid, acetic acid, formic acid, and succinic acid; and amino acids such as arginine, methionine, phenylalanine, valine, and glutamic acid. The concentration of organic matter added to the culture medium as a carbon source can be adjusted as needed to ensure efficient growth. Typically, it can be selected within the range of 0.1 to 10 wt / vol%.

[0038] In addition to the carbon source mentioned above, a nitrogen source may be added to the culture medium. Various nitrogen compounds that can be used in normal culture and fermentation can be used as the nitrogen source. Preferred inorganic nitrogen sources are ammonium salts and nitrates. More preferably, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, triammonium citrate, potassium nitrate, and sodium nitrate. On the other hand, preferred sources of organic nitrogen include amino acids, yeast extract, peptones (milk-derived peptones, soy-derived peptones, soy-derived peptides, etc.), meat extracts (e.g., rabu-remko powder, bonito extract, tuna extract, bonito extract, bouillon, shellfish extract, etc.), liver extract, and digested serum powder. More preferred sources of organic nitrogen include arginine, cysteine, citrulline, lysine, tryptophan, yeast extract, and peptones.

[0039] Furthermore, in addition to carbon and nitrogen sources, microbial growth factors such as extracts, vitamins, metal salts, and inorganic compounds can also be added to the culture medium. Examples of extracts include hemin, heme iron, digested serum powder, liver extract, and blood digestate. Examples of vitamins include biotin, folic acid, pyridoxal, thiamine, riboflavin, nicotinic acid, nicotinamide, pantothenic acid, vitamin B12, thiooctic acid, p-aminobenzoic acid, and vitamin K. 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 selenite, sodium selenite, ammonium ferrous sulfate, ferric 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 preparing culture media by adding these inorganic compounds and vitamins, as well as other growth adjuncts derived from plants and animals, are well known. The culture medium can be liquid, semi-solid, or solid. The preferred form of culture medium is liquid.

[0040] The production of the enzyme according to this embodiment by microorganisms belonging to the genus Clostridium is induced by adding urolithin, which has a hydroxyl group at position 9, as a raw material (substrate) to the culture medium. It is preferable to add the raw material (substrate) at a concentration of 0.01 g / L or more and 20 g / L or less in the culture medium.

[0041] To recover the enzyme according to this embodiment produced by microorganisms belonging to the genus Clostridium, the culture is collected after the enzyme is produced, and the microorganisms are disrupted in a buffer containing reducing agents such as cysteine, 2-mercaptoethanol, or dithiothreitol, or protease inhibitors such as phenylmethanesulfonyl fluoride (PMFS), pepstatin A, or ethylenediaminetetraacetic acid to obtain a cell-free extract. This cell-free extract can then be purified by appropriate combinations of fractionation based on protein solubility and various chromatography methods. All of these methods can be followed according to conventional procedures.

[0042] The first, second, or third subunits of the Clostridium bolteae JCM 12243 strain, which are presumed to constitute the enzyme according to this embodiment, are preferably selected from a combination of CbUroC1, CbUroC2, and CbUroC3. The amino acid sequences of CbUroC1, CbUroC2, and CbUroC3 are represented by sequence numbers 1-3, respectively. The nucleotide sequences of the genes encoding CbUroC1, CbUroC2, and CbUroC3 are represented by sequences 10-12, respectively. Furthermore, the base sequences of the genes encoding CbUroC1, CbUroC2, and CbUroC3 may be modified, such as by optimization, for gene expression using a different host (heterogeneic expression). For example, they may be modified, such as by optimization, for expression in microorganisms belonging to the genus Rhodococcus. Examples of base sequences of the genes encoding CbUroC1, CbUroC2, and CbUroC3 optimized for expression in microorganisms belonging to the genus Rhodococcus are, for example, the sequences represented by SEQ ID NOs. 19 to 21, respectively.

[0043] The first, second, or third subunits of the Clostridium asparagiforme DSM 15981 strain, which are presumed to constitute the enzyme according to this embodiment, are preferably selected from a combination of CaUroC1, CaUroC2, and CaUroC3. The amino acid sequences of CaUroC1, CaUroC2, and CaUroC3 are the sequences represented by SEQ ID NOs: 4-6, respectively. The nucleotide sequences of the genes encoding CaUroC1, CaUroC2, and CaUroC3 are represented by sequences 13-15, respectively.

[0044] The first, second, or third subunits of the Clostridium citroniae DSM 19261 strain, which are presumed to constitute the enzyme according to this embodiment, are preferably selected from a combination of CcUroC1, CcUroC2, and CcUroC3. The amino acid sequences of CcUroC1, CcUroC2, and CcUroC3 are the sequences represented by SEQ ID NOs. 7-9, respectively. The nucleotide sequences of the genes encoding CcUroC1, CcUroC2, and CcUroC3 are represented by sequences 16-18, respectively.

[0045] Therefore, the enzyme according to this embodiment is preferably, An enzyme comprising one or more amino acid sequences 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; An enzyme comprising one or more amino acid sequences selected from the group consisting of the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, and the amino acid sequence represented by SEQ ID NO: 6; or, The enzyme comprises one or more amino acid sequences selected from the group consisting of the amino acid sequence represented by Sequence ID No. 7, the amino acid sequence represented by Sequence ID No. 8, and the amino acid sequence represented by Sequence ID No. 9.

[0046] Furthermore, CbUroC1 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, insofar as it has the activity to dehydrate and oxidize the hydroxyl group at position 9 of urolithin when it is alone as a subunit or when it forms a unit with the other subunits CbUroC2 and / or CbUroC3. One or more amino acids is preferably 1 to 29, more preferably 1 to 28, even more preferably 1 to 20, and particularly preferably 1 to 10, and the same applies when amino acids are added to the N-terminal and / or C-terminal side. This also applies to CaUroC1 and CcUroC1.

[0047] Conservative substitutions are preferred, and a conservative substitution refers to the substitution between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid, between Gln and Asn when the substitution site is a polar amino acid, between Lys, Arg, and His when the substitution site is a basic amino acid, between Asp and Glu when the substitution site is an acidic amino acid, and between Ser and Thr when the amino acid has a hydroxyl group. Conservative substitutions include, specifically, substitutions from Ala to Ser or Thr, Arg to Gln, His or Lys, Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, and Ile to L Examples of substitutions include substitutions of eu, Met, Val, or Phe; substitution of Leu to Ile, Met, Val, or Phe; substitution of Lys to Asn, Glu, Gln, His, or Arg; substitution of Met to Ile, Leu, Val, or Phe; substitution of Phe to Trp, Tyr, Met, Ile, or Leu; substitution of Ser to Thr or Ala; substitution of Thr to Ser or Ala; substitution of Trp to Phe or Tyr; substitution of Tyr to His, Phe, or Trp; and substitution of Val to Met, Ile, or Leu. This also applies to CaUroC1 and CcUroC1.

[0048] Furthermore, CbUroC1 may be a protein having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homology to the full length of the amino acid sequence represented by Sequence ID No. 1, as long as it has the activity to dehydrate and oxidize the hydroxyl group at position 9 of urolithin when it is alone as a subunit or when it forms a unit with the other subunits CbUroC2 and / or CbUroC3. This also applies to CaUroC1 and CcUroC1.

[0049] Furthermore, CbUroC1 may be a protein encoded by a polynucleotide that hybridizes under stringent conditions with the base sequence represented by Sequence ID No. 10, as long as it has the activity to dehydrate and oxidize the hydroxyl group at position 9 of urolithin, either as a single subunit or when it forms a unit with the other subunits CbUroC2 and / or CbUroC3. "Stringent conditions" include, for example, conditions in which polynucleotides having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homology hybridize with each other, and polynucleotides with lower homology do not hybridize with each other. This also applies to CaUroC1 and CcUroC1.

[0050] The aforementioned amino acid substitutions, protein homology, and stringent conditions also apply to CbUroC2. However, with respect to CbUroC2, "1 to more" means preferably 1 to 17, more preferably 1 to 16, even more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3. This also applies to CaUroC2 and CcUroC2. Furthermore, the aforementioned amino acid substitutions, protein homology, and stringent conditions also apply to CbUroC3. However, with respect to CbUroC3, the "1 to more" refers preferably to 1 to 79, more preferably to 1 to 78, even more preferably to 1 to 50, even more preferably to 1 to 30, and particularly preferably to 1 to 10. This also applies to CaUroC3 and CcUroC3.

[0051] The activity of the enzyme according to this embodiment in dehydrating and oxidizing the hydroxyl group at position 9 of urolithin compounds can be evaluated, for example, as shown in Experimental Example 5 described later.

[0052] <2. Polynucleotides> Another aspect of this disclosure is a polynucleotide encoding an enzyme according to the aforementioned aspect. A specific example 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; A polynucleotide comprising one or more selected from the group consisting of the base sequence represented by SEQ ID NO: 13, the base sequence represented by SEQ ID NO: 14, and the base sequence represented by SEQ ID NO: 15; or This is a polynucleotide comprising one or more selected from the group consisting of the base sequence represented by SEQ ID NO: 16, the base sequence represented by SEQ ID NO: 17, and the base sequence represented by SEQ ID NO: 18. The details of each base sequence are as described in the previously mentioned section, "1. Dehydration enzymes of the hydroxyl group at position 9 of urolithins."

[0053] The base sequence of the gene encoding GbUroC1 may be a base sequence that hybridizes under stringent conditions with the base sequence represented by Sequence ID No. 10, as long as GbUroC1 itself, as a single subunit, or when it forms a unit with the other subunits GbUroC2 and / or GbUroC3, has the activity to dehydrate and oxidize the hydroxyl group at position 9 of urolithin. "Stringent conditions" include, for example, conditions in which polynucleotides having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more homology hybridize with each other, and polynucleotides with lower homology do not hybridize with each other. This also applies to the nucleotide sequences of the genes encoding GbUroC2 and GbUroC3. Furthermore, it applies to the nucleotide sequences of the genes encoding GaUroC1, GaUroC2, and GaUroC3. Additionally, it applies to the nucleotide sequences of the genes encoding GcUroC1, GcUroC2, and GcUroC3.

[0054] <3. Genetic Engineering Aspects> Other aspects of the present disclosure include a recombinant vector comprising the polynucleotide; a transformant expressible with respect to the polynucleotide or the vector; and a method for producing a protein encoded by the polynucleotide, comprising the step of culturing the transformant. The protein is an enzyme that dehydrates and oxidizes the hydroxyl group at position 9 of urolithin. By inserting the aforementioned polynucleotide into a known expression vector, an expression vector that expresses the enzyme can be obtained. Then, by transforming microorganisms or the like using this expression vector, a transformant can be obtained, and by culturing the transformant to produce the enzyme, the enzyme can be acquired.

[0055] Examples of host vector systems include, for example, lactic acid bacteria such as 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 (JP-A-6-253861), Lactococcus lactis subsp. lactis / Lactobacillus delbruxii Examples of Bifidobacteria include Bifidobacterium delbrueckii, etc. -pSYE1 (Japanese Patent Publication No. 5-176776), Bifidobacterium adolescentis -pKKT427 (Nucleic Acids Research, 2009, Vol. 37, No. 1 e3 doi:10.1093 / nar / gkn884), a wide range of Bifidobacterium including Bifidobacterium longum -pNC7 (Res. Microbiol., 147, 133-143 (1996)), and Bifidobacterium longum -pBS423 (Appl. Environ. Microbiol., 78, 4984-4994 (2012)). Examples of anaerobic bacteria include Clostridium acetobutylicum-pTY10 (Agric. Biol. Chem., 54 (2), 437-441 (1990)) / pMTL500E (Microbiol. Sci. 5:310-315 (1988)) and ACE vector (Anaerobe, 41, 104-112 (2016)). While the expression of the enzyme in question requires an anaerobic environment, heterologous gene expression does not necessarily require an anaerobic host-vector system. For example, the Rhodococcus erythropolis-pTip, pNit, pCpi Vector series (Hokkaido System Science, Biotechnol. Bioeng., 86, 136-148 (2004)) is suitably usable. A specific host strain is L88, and a specific vector is pTipQC1. In addition, the Escherichia coli-pET Vector is suitably usable. A specific host strain is Rosetta2 (DE3), and a specific vector is pET21b. In addition to microorganisms, various host and vector systems have been developed in plants and animals. For example, systems using silkworms (Nature 315, 592-594 (1985)) and systems for expressing large amounts of heterologous proteins in plants such as rapeseed, corn, and potatoes have been developed and may also be used.

[0056] <4. Method for dehydrating the hydroxyl group at position 9 of urolithin compounds> Another aspect of this disclosure is a method for dehydrating the hydroxyl group at position 9 of urolithin compounds. More specifically, a method for dehydrating the hydroxyl group at position 9 of urolithin compounds having a hydroxyl group at position 9. This embodiment is a method for dehydrating and oxidizing the hydroxyl group at position 9 of urolithin, comprising the following step (I): Step (I): A step of contacting urolithin having a hydroxyl group at the 9-position with one or more of the following (i) to (iv) to dehydrate and oxidize the hydroxyl group at the 9-position. (i) Enzymes described in the section "1. Enzymes that dehydrate and oxidize the hydroxyl group at position 9 of urolithin compounds" above (ii) Proteins encoded by the polynucleotides listed in the "2. Polynucleotides" section above; (iii) Microorganisms that produce the enzyme described in (i) above, or the protein described in (ii) above; (iv) The treated product of the microorganism described in (iii) above.

[0057] (Urolithins) Details of the urolithins in this embodiment are as described in the previously mentioned section "1. Enzymes that dehydrate and oxidize the hydroxyl group at position 9 of urolithins".

[0058] (Process (I)) In step (I), (i) the enzyme and (ii) the protein encoded by the polynucleotide come into contact with urolithin compounds having a hydroxyl group at position 9, thereby dehydrating and oxidizing the hydroxyl group at position 9 present on the urolithin compounds. Furthermore, in (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii), and (iv) a product of the microorganism described in (iii), the enzyme or protein contained in the microorganism or the product of the microorganism comes into contact with urolithin compounds having a hydroxyl group at position 9, thereby dehydrating and oxidizing the hydroxyl group at position 9 present on the urolithin compounds.

[0059] (iii) Microorganisms that produce the enzyme described in (i) above, or the protein described in (ii) above; (iv) The microorganisms in the processed product of the microorganism described in (iii) above may be microorganisms obtained by genetic engineering methods. Examples of such microorganisms include those belonging to the genus Clostridium. Preferred specific examples of microorganisms belonging to the genus Clostridium are the same as those described in the previously mentioned section "1. Enzymes that dehydrate and oxidize the hydroxyl group at position 9 of urolithins."

[0060] (i) The enzyme and (ii) the protein encoded by the polynucleotide are not limited to purified products, but include partially purified products. Furthermore, (iv) examples of treated products of the microorganism include microorganisms whose cell membrane permeability has been altered by treatment with surfactants or organic solvents such as toluene, and cell-free extracts obtained by disrupting bacterial cells by treatment with glass beads or enzymes, or partially purified extracts thereof.

[0061] When one or more of the substances selected from (i) to (iv) above are brought into contact with urolithin compounds having a hydroxyl group at the 9-position, this can be done in water; in an organic solvent that is poorly soluble in water, such as ethyl acetate, butyl acetate, toluene, chloroform, or n-hexane; or in a two-phase mixed system of the organic solvent and an aqueous medium such as ethanol or acetone. It is also possible to immobilize one or more of the substances selected from (i) to (iv) above, or to use a membrane reactor or the like. Furthermore, if one or more of the items selected from (i) to (iv) above are microorganisms that produce the enzyme described in (i) above or the protein described in (ii) above, the procedure may be carried out in a solution commonly used in culturing such microorganisms, such as a culture medium or phosphate buffer.

[0062] The temperature in step (I) is preferably 25°C or higher, more preferably 30°C or higher, and even more preferably 33°C or higher, while preferably 55°C or lower, more preferably 50°C or lower, and even more preferably 42°C or lower. The pH in step (I) is preferably 4.0 or higher, more preferably 5.0 or higher, and even more preferably 5.5 or higher, while preferably 9.0 or lower, more preferably 8.0 or lower, and even more preferably 7.0 or lower. The concentration of the raw material (substrate) in step (I), which is urolithin having a hydroxyl group at position 9, in the reaction solution is 0.001 g / L or more, preferably 0.01 g / L or more, more preferably 0.1 g / L or more, while on the other hand, it is 100 g / L or less, preferably 20 g / L or less, more preferably 10 g / L or less.

[0063] This embodiment may include a step for quantifying the obtained product (quantification step). The quantification method can follow a conventional method. For example, ethyl acetate, to which an acid such as formic acid may be added as needed, is added to the culture medium, vigorously stirred, and then centrifuged to remove the ethyl acetate layer. The same operation is repeated several times as needed, and the ethyl acetate layers are combined to obtain an extract of the product. This extract is concentrated under reduced pressure using an evaporator or the like, dried to dryness, and dissolved in methanol. This can be filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane to remove insoluble matter, and the resulting product can be quantified using high-performance liquid chromatography.

[0064] Furthermore, this embodiment may include a step of recovering the obtained product. This recovery step may include a purification step and a concentration step. Purification treatments in the purification step may include sterilization of microorganisms by heat, etc.; disinfection by microfiltration (MF), ultrafiltration (UF), etc.; removal of solids and polymeric substances; extraction with organic solvents or ionic liquids, etc.; and adsorption and decolorization using hydrophobic adsorbents, ion exchange resins, activated carbon columns, etc. Concentration treatments in the concentration step may include concentration using an evaporator, reverse osmosis membrane, etc. Furthermore, the solution containing the obtained product can be powdered by freeze-drying, spray-drying, or other methods. Excipients such as lactose, dextrin, and corn starch can also be added during the powdering process.

[0065] <5. Method for producing urolithin A> Another aspect of this disclosure is a method for producing urolithin A (first aspect). This embodiment is a method for producing urolithin A, comprising the following steps (I) and (II): Step (I): A step in which a microorganism capable of producing uroritin C from ellagic acid is used to produce uroritin C from ellagic acid. Step (II): A step of contacting uroritin C with one or more of the following (i) to (iv) to produce uroritin A. (i) The enzymes listed in the section "1. Enzymes that dehydrate and oxidize the hydroxyl group at position 9 of urolithin" above; (ii) Proteins encoded by the polynucleotides listed in the "2. Polynucleotides" section above; (iii) Microorganisms that produce the enzyme described in (i) above, or the protein described in (ii) above; (iv) The treated product of the microorganism described in (iii) above.

[0066] (Process (I)) In step (I), microorganisms capable of producing uroritin C from ellagic acid are used to produce uroritin C from ellagic acid.

[0067] Examples of microorganisms capable of producing urolithin C from ellagic acid include those belonging to the genus Gordonibacter and those belonging to the genus Eggerthella. Among microorganisms belonging to the genus Gordonibacter, microorganisms belonging to Gordonibacter pamelaeae, Gordonibacter urolithinfaciens, and Gordonibacter faecihominis are preferred. Furthermore, if the microorganism belongs to the genus Gordonibacter pamelaeae, strain DSM 19378 is more preferred, and if the microorganism belongs to the genus Gordonibacter urolithinfaciens, strain DSM 27213 is more preferred. Among microorganisms belonging to the genus Eggerthella, strain Eggerthella sp. DC 3563 (NITE BP-02376) is preferred. The above microorganisms may be used individually or in combination of two or more species, regardless of genus, species, or strain. Furthermore, the strain assigned accession number Eggerthella sp. DC 3563 (NITE BP-02376) was internationally deposited on November 11, 2016, at the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) in accordance with the Budapest Convention.

[0068] Conventional methods can be used to produce uroritin C from ellagic acid using microorganisms capable of producing uroritin C from ellagic acid. Alternatively, it may be produced by a method similar to the culture method for microorganisms belonging to the genus Clostridium described in section 1, "Dehydrating oxidases for the hydroxyl group at position 9 of uroritins."

[0069] (Step (II)) In step (II), one or more of the following (i) to (iv) are brought into contact with the uroritin C to generate uroritin A. (i) The enzymes listed in the section "1. Enzymes that dehydrate and oxidize the hydroxyl group at position 9 of urolithin" above; (ii) Proteins encoded by the polynucleotides listed in the "2. Polynucleotides" section above; (iii) Microorganisms that produce the enzyme described in (i) above, or the protein described in (ii) above; (iv) The treated product of the microorganism described in (iii) above.

[0070] In step (II), (i) the enzyme and (ii) the protein encoded by the polynucleotide come into contact with urolithin C having a hydroxyl group at position 9, thereby dehydrating and oxidizing the hydroxyl group at position 9 present on the urolithin C. Furthermore, in (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii), and (iv) a product of the microorganism described in (iii), the enzyme or protein contained in the microorganism or the product of the microorganism come into contact with urolithin C having a hydroxyl group at position 9, thereby dehydrating and oxidizing the hydroxyl group at position 9 present on the urolithin C. The method is the same as described in detail in the previously mentioned section "4. Method for dehydrating and oxidizing the hydroxyl group at position 9 of urolithin compounds."

[0071] Process (I) and process (II) may or may not be carried out in the same system. The phrase "performed in the same system" means that the entire process, from the production of uroritin C in step (I) to the production of uroritin A in step (II), is carried out continuously in the same system. In other words, it does not include steps such as separating and / or purifying the uroritin C produced in step (I). Furthermore, if microorganisms are used in each step, the microorganisms used in each step may be the same or different.

[0072] This embodiment may include other steps. For example, it may include a step of quantifying the obtained urolithin A (quantification step), a step of purifying it (purification step), and a step of concentrating it (concentration step). The details of these steps are the same as those described in section 4, "Method for dehydrating and oxidizing the hydroxyl group at position 9 of urolithins." Furthermore, it may include a step of powdering the solution containing urolithin A by freeze-drying, spray-drying, etc. Excipients such as lactose, dextrin, and corn starch may be added during powdering.

[0073] <6. Method for producing urolithin B> Another aspect of this disclosure is a method for producing urolithin B. This embodiment is a method for producing urolithin B, comprising the following steps (I) to (III): Step (I): A step in which a microorganism capable of producing uroritin C from ellagic acid is used to produce uroritin C from ellagic acid. Step (II): A step of causing a microorganism capable of producing isourorotin A from uroritin C to produce isourorotin A from uroritin C. Step (III): A step of contacting isouroroline A with one or more of the following (i) to (iv) to produce uroline B. (i) The enzymes listed in the section "1. Enzymes that dehydrate and oxidize the hydroxyl group at position 9 of urolithin" above; (ii) Proteins encoded by the polynucleotides listed in the "2. Polynucleotides" section above; (iii) Microorganisms that produce the enzyme described in (i) above, or the protein described in (ii) above; (iv) The treated product of the microorganism described in (iii) above.

[0074] (Process (I)) For step (I), refer to the content described in "5. Method for producing urolithin A" above.

[0075] (Step (II)) In step (II), a microorganism capable of producing isourorotin A from uroritin C is used to produce isourorotin A from uroritin C.

[0076] Examples of microorganisms capable of producing isourorotin A from uroritin C include those belonging to the genus Slackia. Among microorganisms belonging to the genus Slackia, those belonging to Slackia heliotrinireducens are preferred. Furthermore, among the microorganisms belonging to the genus Slackia heliotrinireducens, strain DSM 20476 of Slackia heliotrinireducens is more preferred.

[0077] In step (III), one or more of the following (i) to (iv) are brought into contact with the isouroroline A to generate uroline B. (i) The enzymes listed in the section "1. Enzymes that dehydrate and oxidize the hydroxyl group at position 9 of urolithin" above; (ii) Proteins encoded by the polynucleotides listed in the "2. Polynucleotides" section above; (iii) Microorganisms that produce the enzyme described in (i) above, or the protein described in (ii) above; (iv) The treated product of the microorganism described in (iii) above.

[0078] In step (III), (i) the enzyme and (ii) the protein encoded by the polynucleotide come into contact with isourorolithin A having a hydroxyl group at position 9, thereby dehydrating and oxidizing the hydroxyl group at position 9 present on the isourorolithin A. Furthermore, in (iii) a microorganism that produces the enzyme described in (i) or the protein described in (ii), and (iv) a product of the microorganism described in (iii), the enzyme or protein contained in the microorganism or the product of the microorganism comes into contact with isourorolithin A having a hydroxyl group at position 9, thereby dehydrating and oxidizing the hydroxyl group at position 9 present on the isourorolithin A. The method is the same as described in detail in the previously mentioned section "4. Method for dehydrating and oxidizing the hydroxyl group at position 9 of urolithin compounds."

[0079] Processes (I) through (III) may or may not be carried out in the same system. The phrase "performed in the same system" means that the entire process, from the production of uroritin C in step (I) to the production of isouroritin A in step (II), and from the production of isouroritin A in step (III) to the production of uroritin B in step (III), is carried out continuously in the same system. In other words, it does not include steps such as separating and / or purifying the uroritin C produced in step (I). Furthermore, if microorganisms are used in each step, the microorganisms used in each step may be the same or different.

[0080] This embodiment may include other steps. The details thereof are the same as those described in section 5, "Method for Producing Uroritin A." [Examples]

[0081] Examples are described below, but none of these examples should be interpreted as being limited in meaning.

[0082] [Experimental Example 1] Preparation of bacterial cells for proteomic analysis Clostridium bolteae strain JCM 12243 was inoculated into 10 mL of ABB medium (ANAEROBE BASAL BROTH, Oxoid) containing 0.1% uroritin C or without uroritin C, and incubated at 37°C for 48 hours under anaerobic conditions (N2 / H2 / CO2 = 80:10:10). The culture medium was centrifuged to collect the bacterial cells, which were then used for proteomic analysis.

[0083] [Experimental Example 2] Proteome Analysis The bacterial cells prepared in Experimental Example 1 were lysed using a lysis solution (7 M urea, 2 M thiourea, 2% CHAPS (3-[3-Cholamidopropyl)dimethylammonio]propanesulfonate), 10 mM DTT (dithiostreitol), and 50 mM Tris-HCl buffer (pH 7.0), and proteomic analysis was performed according to the method described in AMB Express, 2:37 (2012). As a result, proteins whose expression levels significantly increased when cultured in a medium containing uroritin C compared to when cultured in a medium without uroritin C were identified, i.e., proteins whose expression is induced by uroritin C. Based on genome analysis information, EDP14314, EDP14313, and EDP14312 were identified. Genetic information analysis, such as homology analysis, was performed using GENETYX (Genetics Co., Ltd.).

[0084] [Experimental Example 3] Prediction of Protein Function The functions of EDP14314, EDP14313, and EDP14312 were predicted by homology searches of their amino acid sequences against known proteins. The results are as follows: EDP14314 is presumed to be "carbon monoxide dehydrogenase". EDP14313 is presumed to be a protein containing an Fe-S (iron-sulfur) cluster. EDP14312 is presumed to be an enzyme that catalyzes the redox reaction between hydroxybenzoyl-CoA and ferredoxin. These genes, EDP14314, EDP14313, and EDP14312, are thought to be subunits that constitute an enzyme that dehydrates and oxidizes the hydroxyl group at position 9 of urolithin. These genes were named CburoC1, CburoC2, and CburoC3, respectively, encoding CbUroC1, CbUroC2, and CbUroC3.

[0085] [Experimental Example 4] Enzyme Purification (Culturing of microorganisms) As a pre-culture, 10 mL of GAM medium (Nippon Pharmaceutical Co., Ltd.) containing 0.1 mg / mL of urolithin C was added to a screw-cap test tube (φ16.5 × 110 mm), and Clostridium bolteae strain JCM 12243 was inoculated. The culture was then incubated in an anaerobic chamber for 48 hours at 37°C and 80 rpm with shaking. The pre-culture solution was inoculated into a 2 L Erlenmeyer flask containing 2.0 L of the same culture medium, and the cells were cultured in an anaerobic chamber for 48 hours at 37°C and 120 rpm with shaking. The resulting culture medium was centrifuged (8,000 rpm, 10 minutes, 4°C) to prepare the bacterial cells as a precipitate fraction.

[0086] (Preparation of ultracentrifugation supernatant) The obtained bacterial cells were suspended in 20 mM potassium phosphate buffer (KPB, pH 6.5) containing 1 mM DTT, and the bacteria were disrupted using a KUBOTA Insonator Model 201M (Kubota Shoji Co., Ltd.). The sonicated solution was centrifuged at 20,000 × g, 4°C, for 60 minutes to obtain the supernatant, which was used as a cell-free extract. The obtained cell-free extract was ultracentrifuged at 100,000 × g, 4°C, for 90 minutes, and the supernatant fraction was obtained.

[0087] (Enzyme purification (ammonium sulfate fractionation)) Ammonium sulfate was added to the obtained supernatant fraction to a final concentration of 30%, and the mixture was stirred in an anaerobic chamber for 1 hour. The mixture was then centrifuged at 20,000 × g, 4°C, for 10 minutes to obtain the supernatant fraction (ammonium sulfate fraction (i)). Ammonium sulfate was added to ammonium sulfate fraction (i) to a final concentration of 50%, and the mixture was stirred in an anaerobic chamber for 1 hour. The mixture was then centrifuged at 20,000 × g, 4°C, for 10 minutes, and the precipitate fraction was suspended in 20 mM KPB (pH 6.5). Desalting of this solution was performed using PD-10D Desaultinf Columns containing 8.3 ml of Sephadex® G-25 Medium (GE Healthcare). After applying a 3 mL sample, the process of washing with 5 mL of 20 mM KPB (pH 6.5) and desalting was repeated, and the desalted solution was concentrated to obtain ammonium sulfate fraction (ii).

[0088] (Enzyme purification (anion exchange chromatography)) The obtained ammonium sulfate fraction (ii) was subjected to anion exchange chromatography using a Hiprep DEAE 16 10 ff column. Elution A: 20 mM KPB (pH 6.5) containing 1 mM DTT Elution B: 20 ​​mM KPB (pH 6.5) containing 1 mM DTT, and 1 M NaCl Flow rate: 3 mL / min Gradient elution was performed from eluate A to B, and the active fraction of the enzyme that dehydrates and oxidizes the hydroxyl group at position 9 of urolithin was recovered and designated as the DEAE fraction.

[0089] (Enzyme purification (hydrophobic column)) The obtained DEAE fraction was fractionated using HiTrap™ Butyl Sepharose HP 5 mL. Elution A: 20 mM KPB (pH 6.5) containing 1 mM DTT, and (NH4)2SO4 Elution B: 20 ​​mM KPB (pH 6.5) containing 1 mM DTT Flow rate: 3.0 mL / min Gradient elution from eluate A to B was performed to recover the active fraction of the enzyme that dehydrates and oxidizes the 9-hydroxy group of urolithins, which was designated as the Butyl fraction.

[0090] (Enzyme purification (gel filtration)) The obtained Butyl fraction was subjected to gel filtration chromatography using Super 200 Increase 10 / 300. Eluate A: 20 mM KPB (pH 6.5) containing 1 mM DTT and 150 mM NaCl Flow rate: 1.0 mL / min The active fraction of the enzyme that dehydrates and oxidizes the 9-hydroxy group of urolithins was recovered and designated as the Increase fraction.

[0091] [Experimental Example 5] Measurement of enzyme activity For each fraction obtained in Experimental Example 4, the activity of dehydrating and oxidizing the 9-hydroxy group of urolithins was measured. Urolithin C was used as the urolithin having a hydroxy group at the 9-position. When the 9-hydroxy group is dehydrated and oxidized, urolithin A is produced. A reaction solution for measuring the enzyme activity of each fraction equivalent to 40 mL of the medium was adjusted to 500 μL with 0.05 mg / mL urolithin C, 5 mM NADH, 100 mM Gly / NaOH buffer (pH 9.5) containing 1 mM DTT, and placed in a 1.5 mL tube. The gas phase was set as the gas phase in an anaerobic chamber (COY vinyl anaerobic chamber), and the reaction was carried out at 45 °C for 13 - 18 hours. After the reaction, 200 μL of N,N-dimethylacetamide containing 10% formic acid was added, mixed, and centrifuged (10,000 rpm, 4 °C, 3 minutes), and the obtained supernatant was subjected to HPLC analysis.

[0092] HPLC was performed under the following conditions to quantify the produced urolithin A (detection wavelengths: urolithin C: 337 nm, urolithin A: 354 nm). <HPLC conditions> Column: COSMOSIL Packed Column 5C18-AR II (inner diameter 4.6 mm × length 15 mm, manufactured by Nacalai Tesque) Elutate: Ultrapure water (Milli-Q water) / Acetonitrile / Formic acid = 80:20:1 Column temperature: 40℃ Flow rate: 1.0 mL / min Detector: PDA (Photodiode Array) Enzyme activity of 1 U was defined as the amount of enzyme that catalyzes the production of 1 μmol of urolithin A per minute.

[0093] The results are shown in Table 1.

[0094] [Table 1]

[0095] [Experimental Example 6] SDS-PAGE Using a 5-20% gradient gel (e-PAGEL, E-R520L, ATTO), the purified enzyme fraction was subjected to sodium dodecyl sulfate electrophoresis (SDS-PAGE) to evaluate its molecular weight. The Protein Molecular Weight Marker (Broad) from Takara Bio was used as the molecular weight marker. The obtained bands were semi-dry-rotted onto a PVDF membrane (Immobilom-P Transfer membrane, Millipore) using a polarize plot AE6677G (Atto), and each band was excised and its sequence was analyzed using an amino acid sequencer.

[0096] The results are shown in Figure 1. In Figure 1, lane 1 is the molecular weight marker lane, lane 2 is the ultracentrifugation supernatant fraction lane, lane 3 is the ammonium sulfate fraction (ii) lane, lane 4 is the DEAE fraction lane, lane 5 is the butyl fraction lane, and lane 6 is the increase fraction lane. Analysis of the amino acid sequences of bands I, II, III, and IV obtained from the Increase fraction in lane 6 yielded four different amino acid sequences. Band I was identified as CbUroC3, band II as CbUroC1, and band III as CbUroC2. The molecular weights of each band are estimated to be 86.7, 31.1, and 17.8 kDa, respectively. Based on its amino acid sequence, band IV is presumed to be Shaperonin GroEL.

[0097] [Experimental Example 7] Effect of cofactors on enzyme activity In the reaction solution for enzyme activity measurement described in Experimental Example 5, the types and concentrations of cofactors were prepared as shown in Table 2, and the amount of urolithin A produced after the reaction was quantified. The DEAE fraction was used as the enzyme.

[0098] The results are shown in Table 2. It was confirmed that activity increased when NADH or NADPH was added individually. Note that No. 1 is a control sample that contains the enzyme but does not contain cofactors. FAD stands for flavin adenine dinucleotide.

[0099] [Table 2]

[0100] [Experimental Example 8] Temperature Dependence of Enzymes Activity measurements were performed at varying reaction temperatures in solutions containing 0.1 mg / mL urolithin C, 5 mM NADH, 1 mM DTT, 100 mM Tris / HCl buffer (pH 8.0), and the DEAE fraction.

[0101] The results are shown in Figure 2. The optimal temperature was 55°C, and the activity at that temperature was defined as 100%. In the range of 37-60°C, activity of 50% or more was observed.

[0102] [Experimental Example 9] pH Dependence of Enzymes Under the activity measurement conditions described in Experimental Example 8, the activity was measured by changing the pH using the following buffer solution. Citrate buffer: 100 mM citrate-sodium citrate buffer Fiscal acetate: 100 mM Sodium acetate Fiscal acetate Potassium phosphate buffer: 100 mM potassium dihydrogen phosphate-potassium hydrogen phosphate buffer Tris-HCl buffer: 100 mM Tris-HCl buffer Glycine buffer: 100 mM glycine-sodium hydroxide buffer Carbonate buffer: 100 mM NaHCO3-Na2CO3 buffer Sodium phosphate buffer: 100 mM Na2HPO4-Na3PO4 buffer

[0103] The results are shown in Figure 3. The optimal pH was 8.0, and activity was observed at over 60% of the optimal conditions within the pH range of 6.0 to 8.5.

[0104] [Experimental Example 10] Temperature Stability of Enzymes The enzyme was kept at 0-80°C for 24 hours, and then its activity was measured under the activity measurement conditions described in Experimental Example 8.

[0105] The results are shown in Figure 4. The optimal temperature was 0°C, and if the enzyme activity before 24 hours of storage is considered 100%, then more than 60% residual activity was observed at temperatures between 0 and 20°C.

[0106] [Experimental Example 11] pH stability of enzymes To 56 μL of purified enzyme solution (20 mM phosphate buffer, pH 6.5), 1 mL of each of the buffers listed below (100 mM) was added and the mixture was held at 0°C for 24 hours. After concentration by ultrafiltration, 100 mM Tris / HCl buffer (pH 8.0) was added to restore the pH, and then the activity was measured under the activity measurement conditions described in Experimental Example 8. Citrate buffer: 100 mM citrate-sodium citrate buffer Fiscal acetate: 100 mM sodium acetate phylloacetate Potassium phosphate buffer: 100 mM potassium dihydrogen phosphate-potassium hydrogen phosphate buffer Tris-HCl buffer: 100 mM Tris-HCl buffer Glycine buffer: 100 mM glycine-sodium hydroxide buffer Carbonate buffer: 100 mM NaHCO3-Na2CO3 buffer Sodium phosphate buffer: 100 mM Na2HPO4-Na3PO4 buffer

[0107] The results are shown in Figure 5. The most stable pH was 8.5 (glycine buffer). Taking the enzyme activity before 24 hours as 100%, over 80% residual activity was observed in the pH range of 8.0 to 10.0.

[0108] [Example 12] Construction of a vector expressing an enzyme The gene sequences of CbUroC1, CbUroC2, and CbUroC3, which are presumed to constitute enzymes that dehydrate and oxidize the hydroxyl group at position 9 of urolithin from Clostridium bolteae strain JCM 12243, were optimized for heterologous expression using microorganisms belonging to the genus Rhodococcus, and the nucleotide sequences represented by SEQ ID NOs. 19-21 were designed for each gene.

[0109] Each nucleotide sequence represented by Sequence ID No. 22, including each nucleotide sequence represented by Sequence ID No. 19-21, was artificially synthesized and inserted into the Rhodococcus genus expression vector pTipQC1 (Hokkaido System Science Co., Ltd.), which had been cleaved with Nco I, using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain a heterologous expression plasmid (pTipQC1_uroC123_opt.).

[0110] For the in-fusion reaction, primers with the following base sequence were used. SYN6606 F01: 5'-TAAGAAGGAGATATACATGGTCCTCCCGCAGTTCGA-3'(Sequence ID 23) SYN6606 R02: 5'-GTGATGGTGGCCCATGTCACTTCGAGTTGCGGTCGT-3'(Sequence ID 24)

[0111] [Example 13] Construction of the transformant The prepared recombinant vector was introduced into Rhodococcus erythropolis L88 strain (Hokkaido System Science Co., Ltd.) by electroporation. Chloramphenicol was used as a selection marker. The formed colonies were cultured in LB medium (20 μg / mL chloramphenicol) at 28°C, and a glycerol stock (30% glycerol) was prepared and stored from the culture medium at -80°C, which was later used for enzyme expression.

[0112] [Example 14] Heterogeneous expression of enzymes As a pre-culture, 5 mL of LB medium containing 20 μg / mL chloramphenicol was added to a 10 mL test tube, the transformed strain was inoculated, and the culture was incubated with shaking at 28°C and 300 rpm for 2-3 days. A 1 L Erlenmeyer flask containing 400 mL of LB medium with 20 μg / mL chloramphenicol was inoculated with 1% of the pre-culture solution, and the mixture was cultured at 28°C and 120 rpm for 9 hours with shaking. Then, 0.2 μg / mL thiostrepton was added, and the enzyme was induced under an anaerobic atmosphere for 48 hours. Bacterial cells were collected by centrifugation (8,000 rpm, 20 minutes, 4°C) and washed twice with physiological saline to obtain resting cells.

[0113] [Example 15] Preparation of cell-free extract Washed bacterial cells were suspended in 20 mM KPB (pH 6.5) containing 1 mM DTT, and the cells were disrupted using glass beads (2,700 rpm, 60 seconds on / 60 seconds off for 6 cycles, 4°C). The resulting bacterial cell lysate was centrifuged (20,000 g, 60 minutes, 4°C), and the supernatant was used as a cell-free extract.

[0114] [Example 16] SDS-PAGE The cell-free extract (soluble fraction), which is the supernatant obtained by centrifugation of the bacterial cell lysate described in Example 15, was subjected to sodium dodecyl sulfate electrophoresis (SDS-PAGE) using a 5-20% gradient gel (e-PAGEL, E-R520L, ATTO Corporation), and its molecular weight was evaluated. The Protein Molecular Weight Marker (Broad) manufactured by Takara Bio was used as the molecular weight marker.

[0115] The results are shown in Figure 6. In Figure 6, lane 1 is the molecular weight marker lane, lane 2 is the lane loaded with 1.0 μL of cell-free extract (soluble fraction) prepared using pTipQC1 that does not contain any of the genes encoding CbUroC1, CbUroC2, or CbUroC3, and lane 3 is the lane loaded with 1.0 μL of cell-free extract (soluble fraction) from Example 15 prepared using pTipQC1_uroC123_opt. In lanes 2 and 3, CbUroC3 was identified in band I, CbUroC1 in band II, and CbUroC2 in band III. The molecular weights of each band agreed well with the calculated values ​​(86.7, 31.1, and 17.8 kDa, respectively).

[0116] [Example 17] Measurement of enzyme activity obtained by heterologous expression The activity of dehydrating the hydroxyl group at position 9 of uroritins was measured. Uroritin C was used as the uroritin with a hydroxyl group at position 9. Enzyme activity was measured by quantifying uroritin A, which is produced by dehydrating the hydroxyl group at position 9. A 50 μL reaction solution for enzyme activity measurement, containing 0.5 mg / mL urolithin C, 5 mM NADH (reduced nicotinamide adenine diphosphate), 1 mM DTT (dithiothreitol), 20 mM KPB (potassium phosphate buffer, pH 6.5), and 15 μL of the cell-free extract obtained in Example 15, was placed in a 200 μL tube for PCR. The gas phase was set in an anaerobic chamber (COY vinyl anaerobic chamber), and the reaction was carried out at 45°C for 24 hours (200 rpm). 100 μL of N,N-dimethylacetamide containing 1% formic acid was added to the reaction solution, and after mixing, the supernatant was analyzed by HPLC after centrifugation (10,000 rpm, 4°C, 3 minutes).

[0117] In control samples prepared using cell-free extracts with pTipQC1 that does not contain any of the genes encoding CbUroC1, CbUroC2, or CbUroC3, the amount of uroritin A produced was 0 (below the detection limit). However, in the sample prepared using the cell-free extract of Example 15, which was prepared using pTipQC1_uroC123_opt., 4.33 mM of uroritin A was produced.

Claims

1. It was isolated from a microorganism belonging to Clostridium bolteae. Furthermore, an enzyme having the following properties (1) and (2). (1) The hydroxyl group at position 9 of urolithin C is dehydrated and oxidized. (2) It is activated by reduced nicotinamide adenine dinucleotide (NADH) and / or reduced nicotinamide adenine dinucleotide phosphate (NADPH).

2. The enzyme according to claim 1, having the properties (3) to (5) below. (3) The SDS-PAGE results include a band with a relative molecular mass between 77,000 and 95,000. (4) The optimal pH is 6.0 or higher and 8.5 or lower. (5) The optimal temperature is 37°C or higher and 60°C or lower.

3. The microorganisms belonging to Clostridium bolteae mentioned above are One or more strains selected from the group consisting of Clostridium bolteae JCM 12243, Clostridium bolteae DSM 29485, and Clostridium bolteae DSM 15670. The enzyme according to claim 1 or 2.

4. The amino acid sequence includes 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. The enzyme according to any one of claims 1 to 3.

5. A polynucleotide encoding a protein consisting of an amino acid sequence in which 1 to 28 amino acids are substituted, deleted, inserted, or added in the amino acid sequence represented by SEQ ID NO:

1. A polynucleotide encoding a protein consisting of an amino acid sequence in which 1 to 16 amino acids are substituted, deleted, inserted, or added in the amino acid sequence represented by Sequence ID No. 2, and The polynucleotide encoding a protein comprising an amino acid sequence in which 1 to 78 amino acids are substituted, deleted, inserted, or added in the amino acid sequence represented by Sequence ID No. 3, A polynucleotide that encodes a protein having the activity to dehydrate and oxidize the hydroxyl group at position 9 of urolithin C.

6. A recombinant vector comprising the polynucleotide described in claim 5.

7. A transformant that can express the polynucleotide described in claim 5, or that can express the vector described in claim 6.

8. A method for producing a polynucleotide-encoded protein according to claim 5, comprising the step of culturing the transformant according to claim 7.

9. A method for dehydrating and oxidizing the hydroxyl group at position 9 of urolithin C, including the following step (I): Step (I): A step of contacting urolithin C with one or more substances selected from (i) to (iv) below to dehydrate and oxidize the hydroxyl group at position 9. (i) The enzyme according to any one of claims 1 to 4; (ii) The polynucleotide-encoded protein according to claim 5; (iii) Microorganisms that produce the protein described in (ii) above; (iv) A product of the microorganism described in (iii) above, wherein the product is processed by a treatment comprising one or more treatments selected from toluene treatment and cell disruption treatment.

10. The product produced when the hydroxyl group at position 9 of urolithin C is dehydrated and oxidized is urolithin A. The method according to claim 9.

11. A method for producing urolithin A, comprising the following steps (I) and (II): Step (I): A step in which a microorganism capable of producing uroritin C from ellagic acid produces uroritin C from ellagic acid. Step (II): A step of contacting uroritin C with one or more of the following (i) to (iv) to produce uroritin A. (i ) The enzyme according to any one of claims 1 to 4; (ii) The polynucleotide-encoded protein according to claim 5; (iii) Microorganisms that produce the protein described in (ii) above; (iv) A product of the microorganism described in (iii) above, wherein the product is processed by a treatment comprising one or more treatments selected from toluene treatment and cell disruption treatment.

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