JP7473573B2 - New lactonase - Google Patents

Description

The present invention relates to a novel lactonase.

Urolithins, such as urolithin A and urolithin C, are known to be metabolites of ellagic acid derived from ellagitannins contained in pomegranates, raspberries, blackberries, cloudberries, strawberries, walnuts, etc.

Ellagitannins are classified as hydrolyzable tannins, and are known to be hydrolyzed in the body when ingested and converted into ellagic acid. They are also present in fruits as ellagic acid. For example, it has been reported that urolithins are produced in the body by ingesting ellagitannins such as geraniin to rats and then analyzing the urolithins in their urine (Non-Patent Document 1). It has also been reported that urolithins were detected in the urine of humans after ingesting a pomegranate extract containing ellagitannins, mainly punicalagin, and that urolithins A and C in particular are the main ellagic acid metabolites (Non-Patent Document 2).

These urolithins are known to have various physiological activities, and are expected to be used as ingredients in medicines, cosmetics, and food and beverages. For example, urolithin A has been reported to have functions such as antioxidant activity (Non-Patent Document 3), anti-inflammatory activity (Non-Patent Document 4), anti-glycation activity (Non-Patent Document 5), and mitophagy promotion activity (Non-Patent Document 6), and it is expected to be developed as a material with anti-aging functions.

One method reported for synthesizing these urolithins is to use 2-bromo-5-methoxybenzoic acid as a starting material, which is demethylated to produce 2-bromo-5-hydroxybenzoic acid, which is then reacted with resorcinol to obtain urolithin A (Non-Patent Document 7). However, chemical synthesis methods are not suitable for using urolithins as ingredients in functional foods (including beverages and supplements).

On the other hand, it is known that ellagitannins and ellagic acid are metabolized by the intestinal microflora and converted into urolithins after being ingested into the body. In recent years, Gordonibacter urolithinfaciens has been isolated and identified as an intestinal bacterium that produces urolithin C, a type of urolithin, from ellagic acid, and a method for producing urolithin C by fermenting ellagic acid using this intestinal bacterium has been reported (Patent Document 1, Non-Patent Document 8). However, the accumulation concentration of urolithin C in the fermentation liquid was about 2 mg/L. In addition to Gordonibacter urolithinfaciens, Gordonibacter pamelaeae has also been reported to produce urolithin C from ellagic acid (Patent Document 1, Non-Patent Document 8).
However, the pathway that acts on ellagic acid to metabolize urolithins has not been identified, and the enzymes involved and the genes encoding them have not been reported. Furthermore, the enzyme that produces urolithin M5 from ellagic acid, which is predicted to catalyze the initial reaction in ellagic acid metabolism, and the genes encoding it have not been reported.

International Publication No. 2014/147280

J. Agric. Food Chem., 56, 393-400 (2008) Mol. Nutr. Food Res., 58, 1199-1211 (2014) Biosci. Biotechnol. Biochem., 76, 395-399 (2012) J. Agric. Food Chem., 60, 8866-8876 (2012) Mol. Nutr. Food Res., 55, S35-S43 (2011) Nature Medicine, 22, 879-888 (2016) J. Agric. Food Chem., 56, 393-400 (2008) Food Func., 5, 8, 1779-1784 (2014)

The objective of the present invention is to provide a novel lactonase.

The present inventors searched for a novel lactonase involved in the metabolism of urolithins and discovered a lactonase that catalyzes the hydrolysis of at least one of the two ester (lactone) bonds present in ellagic acid, completing the present invention. Ellagic acid in which one of the two ester bonds has been hydrolyzed is a type of urolithin called urolithin M5, and lactonase with this activity is novel. The present invention is as follows.

[1] A lactonase having the following property (1):
(1) It has the activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid.
[2] The lactonase according to [1], further having the following properties (2) to (4):
(2) The optimum pH is 7.0 or more and 9.0 or less;
(3) The optimum temperature is 42°C or higher and 55°C or lower;
(4) The molecular weight is 37,800 or more and 46,200 or less.
[3] [1] or [
2. The lactonase according to claim 2.
[4] The lactonase according to [3], wherein the microorganism belonging to the genus Gordonibacter is selected from the group consisting of microorganisms belonging to Gordonibacter urolithinfaciens, microorganisms belonging to Gordonibacter pamelaeae, and microorganisms belonging to Gordonibacter faecihominis.
[5] A lactonase which is a protein represented by the following (A) or (B):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4;
(B) A protein having the activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, which consists of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, in which one to several amino acids have been substituted or deleted, or one to several amino acids have been inserted or added.
[6] A method for producing a microorganism comprising the step of culturing a microorganism belonging to the genus Gordonibacter,
A method for producing the lactonase described in [1] or [2].
[7] A polynucleotide represented by the following (a) or (b):
(a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2;
(b) a polynucleotide encoding a protein having a base sequence having 90% or more homology to the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and having the activity of catalyzing a reaction to hydrolyze at least one of the two ester bonds present in ellagic acid.
[8] A recombinant vector comprising the polynucleotide according to [7].
[9] A transformant which retains the polynucleotide according to [7] in an expressible manner, or which retains the vector according to [8] in an expressible manner.
[10] A method for producing a protein encoded by the polynucleotide according to [7], comprising the step of culturing the transformant according to [9].
[11] A method for producing urolithin M5, comprising the following step (I):
Step (I): A step of contacting ellagic acid with one or more selected from the following (i) to (iv):
(i) The lactonase according to any one of [1] to [5];
(ii) a protein encoded by the polynucleotide according to [7];
(iii) a microorganism producing the lactonase or the protein;
(iv) A treated product of the microorganism.
[12] A method for producing urolithin C, comprising the following steps (I) and (II), wherein the steps (I) and (II) are carried out in the same system:
Step (I): A step of producing urolithin M5 by contacting ellagic acid with one or more selected from the following (i) to (iv):
(i) The lactonase according to any one of [1] to [5];
(ii) a protein encoded by the polynucleotide according to [7];
(iii) a microorganism producing the lactonase or the protein;
(iv) A treated product of the microorganism.
Step (II): Allowing a microorganism having the ability to produce urolithin C from urolithin M5 to produce urolithin C from urolithin M5.
[13] A method for producing urolithin A, comprising the following steps (I) to (III), which are carried out in the same system:
Step (I): A step of producing urolithin M5 by contacting ellagic acid with one or more selected from the following (i) to (iv):
(i) The lactonase according to any one of [1] to [5];
(ii) a protein encoded by the polynucleotide according to [7];
(iii) a microorganism producing the lactonase or the protein;
(iv) A treated product of the microorganism.
Step (II): Allowing a microorganism having the ability to produce urolithin C from urolithin M5 to produce urolithin C from urolithin M5.
Step (III): Allowing a microorganism having the ability to produce urolithin A from urolithin C to produce urolithin A from urolithin C.
[14] A method for producing urolithin M6, comprising the following steps (I) and (IV), wherein the steps (I) and (IV) are carried out in the same system:
Step (I): contacting ellagic acid with one or more selected from the following (i) to (iv) to produce urolithin M5;
(i) The lactonase according to any one of [1] to [5];
(ii) a protein encoded by the polynucleotide according to [7];
(iii) a microorganism producing the lactonase or the protein;
(iv) A treated product of the microorganism.
Step (IV): Allowing a microorganism having the ability to produce urolithin M6 from urolithin M5 to produce urolithin M6 from urolithin M5.
[15] A method for producing urolithin D, comprising the following steps (I) and (V), wherein the steps (I) and (V) are carried out in the same system:
Step (I): A step of producing urolithin M5 by contacting ellagic acid with one or more selected from the following (i) to (iv):
(i) The lactonase according to any one of [1] to [5];
(ii) a protein encoded by the polynucleotide according to [7];
(iii) a microorganism producing the lactonase or the protein;
(iv) A treated product of the microorganism.
Step (V): Allowing a microorganism having the ability to produce urolithin D from urolithin M5 to produce urolithin D from urolithin M5.

According to the present invention, a novel lactonase can be provided. In a preferred embodiment, the lactonase can be allowed to act on ellagic acid to produce urolithin M5, and in another preferred embodiment, urolithin M6, urolithin A (wherein urolithin A is obtained via urolithin C), urolithin C, or urolithin D can be produced from urolithin M5.

FIG. 2 shows the pH dependence of the activity of lactonase according to one embodiment of the present invention. FIG. 2 shows the temperature dependence of the activity of lactonase according to one embodiment of the present invention.

1. Lactonase
One embodiment of the present invention is a lactonase having the following property (1):
(1) It has the activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid.
As shown below, ellagic acid has two ester bonds.

Urolithin M5 is a compound in which one of the two ester bonds present in ellagic acid is hydrolyzed. Urolithin M5 can also be said to be an intermediate produced during the hydrolysis of both of the two ester bonds present in ellagic acid.
The lactonase according to this embodiment has an optimum pH of preferably 6.0 or more and 9.5 or less, more preferably 7.0 or more and 9.0 or less.
The optimum temperature is preferably 28°C or higher and 60°C or lower, more preferably 42°C or higher and 55°C or lower.
The molecular weight thereof, as measured by SDS-PAGE, is from 37,800 to 46,200, preferably 42,000.

The lactonase according to this embodiment is preferably derived from a microorganism belonging to the genus Gordonibacter, and among these, it is more preferable to use a microorganism derived from Gordonibacter urolithinfaciens, Gordonibacter pamelaeae, Gordonibacter faecihominis, or the like.
In addition, Gordonibacter urolithinfaciens
ns), DSM 27213 strain is more preferable, and Gordonibacter pamelaeae, DSM 19378 is more preferable.
More preferably, the microorganism belongs to Gordonibacter faecihominis, and more preferably, the microorganism belongs to the genus Gordonibacter faecihominis, and the microorganism is JCM 16058. One or more kinds of the microorganism may be used.

In this specification, the accession numbers of strains beginning with the words "DSM" are numbers given to microorganisms preserved at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), and the accession numbers of strains beginning with the words "JCM" are numbers given to microorganisms preserved at the RIKEN BioResource Center.
In addition, the DSM 27213 strain, the DSM 19378 strain, and the JCM 16058 strain are not limited to the same strain as each of the strains, and may be substantially equivalent strains. A substantially equivalent strain is a microorganism whose 16S rRNA gene base sequence has a homology of 97.5% or more, preferably 98% or more, more preferably 99% or more with the 16S rRNA gene base sequence of the above strain. Furthermore, each of the strains may be a strain bred by mutation treatment, genetic recombination, selection of natural mutants, etc. from each of the strains or a strain substantially equivalent thereto. This applies equally to all of the microorganisms described in this specification.

The amino acid sequence of lactonase possessed by Gordonibacter urolithinfaciens DSM 27213 strain and the nucleotide sequence of the gene encoding same are set forth as SEQ ID NO: 3 and SEQ ID NO: 1, respectively.
The amino acid sequence of lactonase possessed by Gordonibacter pamelaeae DSM 19378 strain and the nucleotide sequence of the gene encoding same are shown as SEQ ID NO: 4 and SEQ ID NO: 2, respectively.

Furthermore, the lactonase according to this embodiment may be a protein having the activity of catalyzing a reaction to hydrolyze at least one of the two ester bonds present in ellagic acid, which is composed of amino acids in which one to several amino acids have been substituted or deleted, or one to several amino acids have been inserted or added, in the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
The term "1 to several" means preferably 1 to 35, more preferably 1 to 30, even more preferably 1 to 10, and particularly preferably 1 to 3, and the same applies when amino acids are added to the N-terminus and/or C-terminus.
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.
Conservative substitutions refer to substitutions between Ala, Arg, and His, between Asp and Glu in the case of acidic amino acids, and between Ser and Thr in the case of amino acids having a hydroxyl group. Specific examples of conservative substitutions include substitutions of Ala with Ser or Thr, Arg with Gln, His, or Lys, Asn with Glu, Gln, Lys, His, or Asp, Asp with Asn, Glu, or Gln, Cys with Ser or Ala, Gln with Asn, Glu, Lys, His, Asp, or Arg, Glu with Gly, Asn, Gln, Lys, or Asp, Gly with Pro, His with Asn, Lys, Gln, Arg, or Tyr, Ile with L substitution of eu, 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.
Furthermore, the lactonase of this embodiment may be a protein that has a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more to the full length of the amino acid sequence (e.g., the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4), so long as it has the activity of catalyzing the reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid.

Furthermore, the lactonase according to this embodiment may be a protein encoded by a polynucleotide that hybridizes under stringent conditions with a base sequence (e.g., the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2) so long as it has the activity of catalyzing the reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid. Examples of "stringent conditions" include conditions under which polynucleotides having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more hybridize with each other, and polynucleotides having a lower homology than that do not hybridize with each other.

The activity of the lactonase according to this embodiment to catalyze the reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid can be evaluated, for example, by using ellagic acid as a raw material (substrate) and measuring the amount of urolithin M5 produced, as in the Examples described below.

2. Method for producing lactonase
Another embodiment of the present invention is a method for producing a lactonase having the above-mentioned property (1), comprising a step of culturing a microorganism belonging to the genus Gordonibacter.
The detailed description of the properties of the microorganism belonging to the genus Gordonibacter and the lactonase used in this production method are the same as those described above.

Microorganisms belonging to the genus Gordonibacter are cultured in media commonly used for culturing anaerobic bacteria, such as Anaerobe Basal Broth (ThermoFisher Scientific, CM0957), Wilkins-Chalgren Anaerobe Broth (ThermoFisher Scientific, CM0643), GAM medium (Nissui Pharmaceutical Co., Ltd.), and modified GAM medium (Nissui Pharmaceutical Co., Ltd.).
The culture temperature is preferably 25°C or higher, more preferably 30°C or higher, and even more preferably 33°C or higher, while it is preferably 45°C or lower, more preferably 40°C or lower, and even more preferably 38°C or lower.

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

In addition to the above carbon sources, the medium may contain a nitrogen source, which may be any of various nitrogen compounds that can be used in normal culture or fermentation.
Preferred inorganic nitrogen sources 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 organic nitrogen sources are amino acids, yeast extract, peptones (milk-derived peptone, soybean-derived peptone, soybean-derived peptide, etc.), meat extracts (e.g., Lab-Remco powder, bonito extract, tuna extract, bonito extract, bouillon, shellfish extract, etc.), liver extract, digested serum powder, etc. More preferred organic nitrogen sources are arginine, cysteine, citrulline, lysine, tryptophan, yeast extract, and peptones.

In addition to the carbon source and nitrogen source, microbial growth factors such as extracts, vitamins, metal salts, and inorganic compounds can 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, thiooctoic 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 selenate, sodium selenite, ferrous ammonium sulfate, iron (II) citrate, sodium acetate trihydrate, magnesium sulfate heptahydrate, and manganese sulfate tetrahydrate. These metals can also be added in the form of mineral yeast.
Methods for producing a culture medium by adding growth cofactors derived from animals and plants, such as inorganic compounds and vitamins, are known. The medium can be liquid, semi-solid, or solid. The preferred form of the medium is a liquid medium.

The production of lactonase by microorganisms belonging to the genus Gordonibacter is
The induction is achieved by adding a raw material (substrate) of ellagic acid or a precursor of ellagic acid to the medium. Examples of precursors of ellagic acid include ellagitannins such as punicalagin and geraniin. The raw material (substrate) is preferably added so that the concentration in the medium is 0.01 g/L or more and 20 g/L or less.

To recover lactonase produced by microorganisms belonging to the genus Gordonibacter, the culture is recovered after the lactonase is produced, and the microorganisms are disrupted in a buffer containing a reducing agent such as cysteine, 2-mercaptoethanol, or dithiothreitol, or a protease inhibitor such as phenylmethanesulfonyl fluoride (PMFS), pepstatin A, or ethylenediaminetetraacetic acid to obtain a cell-free extract. The cell-free extract can be purified by appropriately combining fractionation based on the solubility of the protein and various types of chromatography. Either method can be used according to the conventional method.

3. Polynucleotides
Another embodiment of the present invention is a polynucleotide of the following (a) or (b), the details of which are as described above:
(a) a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2;
(b) a polynucleotide encoding a protein having a base sequence having 90% or more homology to the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 and having the activity of catalyzing a reaction to hydrolyze at least one of the two ester bonds present in ellagic acid.

4. Genetic Engineering
Another embodiment of the present invention is a method for producing a protein encoded by said polynucleotide, comprising the steps of: a recombinant vector containing said polynucleotide; a transformant carrying said polynucleotide in an expressible state or carrying said vector in an expressible state; and culturing said transformant.

The polynucleotide can be inserted into a known expression vector to produce a lactonase expression vector.Then, a transformant can be obtained by transforming a microorganism or the like using the expression vector, and lactonase can be obtained by culturing the transformant to produce lactonase.The microorganism can be, for example, an anaerobic bacterium including lactic acid bacteria. Either method can be used according to the usual method.
In addition to microorganisms, various host and vector systems have been developed for plants and animals. In particular, a system using silkworms (Nature 315, 592-594 (1985)) and systems for expressing large amounts of heterologous proteins in plants such as rapeseed, corn, and potato have been developed, and these may also be used.

<5. Method for producing Urolithin M5>
Another embodiment of the invention is a method for producing urolithin M5, comprising the step (I) of:
Step (I): A step of contacting ellagic acid with one or more selected from the following (i) to (iv):
(i) the lactonase;
(ii) a protein encoded by the polynucleotide;
(iii) a microorganism producing the lactonase or the protein;
(iv) A treated product of the microorganism.

(i) The lactonase and (ii) the protein encoded by the polynucleotide each hydrolyze at least one of the two ester bonds present in ellagic acid upon contact with ellagic acid to produce urolithin M5. In addition, (iii) the microorganism that produces the lactonase or the protein, and (iv) the processed product of the microorganism, the lactonase or the protein contained therein contacts ellagic acid to hydrolyze at least one of the two ester bonds present in ellagic acid to produce urolithin M5.

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

The contact of one or more selected from (i) to (iv) with ellagic acid can be carried out in water; in an organic solvent that is poorly soluble in water, such as ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, or the like; or in a two-phase mixture system of the organic solvent and an aqueous medium such as ethanol or acetone.
In addition, in order to increase the solubility of the raw material (substrate) ellagic acid and the product urolithin M5, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or derivatives thereof may be added.
This step can also be carried out using immobilized enzymes, membrane reactors, and the like.
The temperature in this step is preferably 4° C. or higher, more preferably 25° C. or higher, while it is preferably 60° C. or lower, more preferably 50° C. or lower.
The pH is preferably 5 or more, more preferably 6 or more, while it is preferably 10 or less, more preferably 9.5 or less.
The concentration of ellagic acid, which is the raw material (substrate), in the reaction solution is 0.01 g/L or more, preferably 0.1 g/L or more, and more preferably 1.0 g/L or more, while it is 100 g/L or less, preferably 50 g/L or less, and more preferably 20 g/L or less.

The production method may include a step of quantifying the obtained urolithin M5 (quantification step). The quantification method may follow a conventional method. For example, ethyl acetate, to which an acid such as formic acid is added as necessary, is added to the culture solution, and the mixture is vigorously stirred and centrifuged to remove the ethyl acetate layer. If necessary, the same operation is performed several times, and the ethyl acetate layers are combined to obtain a urolithin extract. This extract is concentrated and dried under reduced pressure using an evaporator or the like, and dissolved in methanol. This is filtered using a membrane such as a polytetrafluoroethylene (PTFE) membrane, and the insoluble matter is removed, and then quantified using high-performance liquid chromatography. Examples of conditions for high-performance liquid chromatography include, but are not limited to, the following.
[High performance liquid chromatography conditions]
Column: Inertsil ODS-3 (250 x 4.6 mm) (GL Science)
Eluent: water/acetonitrile/acetic acid = 74/25/1
Flow rate: 1.0 mL / min
Column temperature: 40°C
Detection: UV (305 nm)

The production method may also include a step of purifying the obtained urolithin M5 (purification step) or a step of concentrating the obtained urolithin M5 (concentration step). Examples of purification treatments in the purification step include sterilization of microorganisms by heat or the like; sterilization by centrifugation, microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), or the like; removal of solids and polymeric substances; extraction and crystallization using organic solvents, ionic liquids, or the like; chromatography such as adsorption using hydrophobic adsorbents, ion exchange resins, activated carbon columns, or the like; and decolorization. Examples of concentration treatments in the concentration step include concentration using an evaporator, a reverse osmosis membrane, or the like.
Furthermore, a solution containing urolithin M5 can be powdered by freeze-drying, spray-drying, etc. In powdering, excipients such as lactose, dextrin, corn starch, etc. can also be added.

6. Method for producing Urolithin C
Another embodiment of the present invention is a method for producing urolithin C, comprising the above-mentioned step (I) and the following step (II), wherein steps (I) and (II) are carried out in the same system.
Step (II): A step of allowing a microorganism having the ability to produce urolithin C from urolithin M5 to produce urolithin C from the urolithin M5.

In step (II), for example, as described in the Examples, a microorganism capable of producing urolithin C from urolithin M5 is cultured in a solution containing urolithin M5, and urolithin C is produced from the urolithin M5. Examples of such microorganisms include microorganisms belonging to the genus Gordonibacter. Preferred examples of microorganisms belonging to the genus Gordonibacter include the microorganisms belonging to the genus Gordonibacter described in the section <1. Lactonase> above. In addition, culture conditions for microorganisms belonging to the genus Gordonibacter include:
Gordonibacter, as described in the section "2. Method for producing lactonase" above.
The explanation of the culture conditions, etc. of the microorganisms belonging to the genus are cited below.

Steps (I) and (II) are "carried out in the same system" means that urolithin M5 produced in step (I) is used as is as urolithin M5 in step (II), and the series of steps up to the production of urolithin C in step (II) are carried out continuously in the same system. In other words, this means that there is no step between step (I) and step (II), such as a step of separating and/or purifying urolithin M5 produced in step (I).
This production method may include a step of quantifying the obtained urolithin C (quantification step), a step of purifying it (purification step), and a step of concentrating it (concentration step). For details thereof, refer to the explanation in the section <5. Production method of urolithin M5> above.
Furthermore, a solution containing urolithin C can be powdered by freeze-drying, spray-drying, etc. In powdering, excipients such as lactose, dextrin, corn starch, etc. can also be added.

7. Method for producing Urolithin A
Another embodiment of the present invention includes the above steps (I) and (II) and the following step (III), in which the above steps (I) and (II) are carried out in the same system, or the above step (II) and the following step (III) are carried out in the same system.
The method for producing urolithin A, wherein steps (I) and (III) are carried out in the same system, or steps (I) to (III) are carried out in the same system.
Step (III): Allowing a microorganism having the ability to produce urolithin A from urolithin C to produce urolithin A from the urolithin C.
Incidentally, the above definition of "carried out in the same system" is used.

In step (III), for example, as described in the Examples, a microorganism capable of producing urolithin A from urolithin C is cultured in a solution containing urolithin C, and urolithin A is produced from the urolithin C. Examples of the microorganism include microorganisms belonging to the genus Clostridium, and more specifically, microorganisms belonging to Clostridium bolteae, Clostridium asparagiforme, and Clostridium citroniae. One type or multiple types of the microorganism may be used.
Among the microorganisms belonging to Clostridium bolteae, D
SM 29485 strain, DSM 15670 strain, and JCM 12243 strain are more preferred, and DSM 15670 strain is even more preferred.
Among microorganisms belonging to Clostridium asparagiforme, the DSM 15981 strain is preferred.
Among microorganisms belonging to Clostridium citroniae, the DSM 19261 strain is preferred.
The above microorganisms may be used alone or in combination with one or more species, regardless of genus, species or strain.
In this specification, the accession numbers of strains beginning with the word DSM are DSMZ (Deutsche
The number is given to the microorganisms preserved in the Sammlung von Mikroorganismen und Zellkulturen GmbH. The accession number of the strain beginning with the word JCM is the number given to the microorganisms preserved in the RIKEN BioResource Center.

The culture conditions for a microorganism capable of producing urolithin A from urolithin C include the Gordonibacter
The explanation of the culture conditions, etc. of the microorganisms belonging to the genus are cited below.

This production method 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). For details thereof, the explanation in the section <5. Production method of urolithin M5> above is cited.
Furthermore, a solution containing urolithin A can be powdered by freeze-drying, spray-drying, etc. In powdering, excipients such as lactose, dextrin, corn starch, etc. can also be added.

8. Method for producing urolithin M6
Another embodiment of the present invention is a method for producing urolithin M6, comprising the above-mentioned step (I) and the following step (IV), wherein steps (I) and (IV) are carried out in the same system.
Step (IV): Allowing a microorganism having the ability to produce urolithin M6 from urolithin M5 to produce urolithin M6 from the urolithin M5.
Incidentally, the above definition of "carried out in the same system" is used.

In step (IV), for example, as described in the Examples, a microorganism capable of producing urolithin M6 from urolithin M5 is cultured in a solution containing urolithin M5, and urolithin M6 is produced from the urolithin M5. Examples of the microorganism include microorganisms belonging to the genus Gordonibacter. Preferred examples of microorganisms belonging to the genus Gordonibacter include those belonging to the genus Gordonibacter described in the above section <1. Lactonase>. In addition, the culture conditions, etc. for microorganisms belonging to the genus Gordonibacter are the same as those described in the above section <2. Method for producing lactonase>.

This production method may include a step of quantifying the obtained urolithin M6 (quantification step), a step of purifying it (purification step), and a step of concentrating it (concentration step). For details of these, the explanation in the section <5. Production method of urolithin M5> above is cited.
Furthermore, a solution containing urolithin M6 can be powdered by freeze-drying, spray-drying, etc. When powdering, excipients such as lactose, dextrin, corn starch, etc. can also be added.

9. Method for producing Urolithin D
Another embodiment of the present invention is a method for producing urolithin D, comprising the above-mentioned step (I) and the following step (V), wherein steps (I) and (V) are carried out in the same system.
Step (IV): Allowing a microorganism having the ability to produce urolithin D from urolithin M5 to produce urolithin D from the urolithin M5.
Incidentally, the above definition of "carried out in the same system" is used.

In step (V), for example, as described in the Examples, a microorganism capable of producing urolithin D from urolithin M5 is cultured in a solution containing urolithin M5, and urolithin D is produced from the urolithin M5. Examples of the microorganism include microorganisms belonging to the genus Gordonibacter. Preferred examples of microorganisms belonging to the genus Gordonibacter include the microorganisms belonging to the genus Gordonibacter described in the section <1. Lactonase> above.
In addition, the culture conditions, etc. for microorganisms belonging to the genus Gordonibacter are the same as those described in the section <2. Method for producing lactonase> above.

This production method may include a step of quantifying the obtained urolithin D (quantification step), a step of purifying it (purification step), and a step of concentrating it (concentration step). For details thereof, refer to the explanation in the section <5. Production method of urolithin M5> above.
Furthermore, a solution containing Urolithin D can be powdered by freeze-drying, spray-drying, etc. In powdering, excipients such as lactose, dextrin, corn starch, etc. can also be added.

The present invention will be described in more detail below with reference to specific examples, but the present invention is not limited to these examples.

[Example 1: Cloning of the lactonase gene]
Genomic DNA was prepared from Gordonibacter urolithinfaciens DSM 27213 strain in a conventional manner. The lactonase gene was amplified by PCR using this as a template and the following primer set, and inserted into the expression vector pET-21b(+) to construct pET-21b(+)_GuUroH+119.
5'- AATGGATCCGCGGAGCGCGAAGTTACACCGATCTACCGGTAA-3' (SEQ ID NO: 5)
5'-TATGAATTCCTACAGGTTGAACAGCTTCGCCGCGTTGCCGCC-3' (SEQ ID NO: 6)

Example 2: Expression of lactonase in E. coli
E. coli Rosetta2(DE3) strain was transformed with pET-21b(+)_GuUroH+119 by the Ca method.
The resulting transformant was cultured in 5 mL of LB medium containing 34 μg/mL chloramphenicol and 30 μg/mL kanamycin at 37° C. for 2 hours with shaking. IPTG was added to the culture solution to a concentration of 1 mM, and induction culture was further performed at 37° C. for 3 hours.
The resulting culture solution was centrifuged to prepare wet cells, which were then suspended in 0.3 mL of BugBuster Master Mix (Novagen) and gently shaken at room temperature to disrupt the cells.

[Example 3: Expression analysis of lactonase]
The cell lysate prepared in Example 2 was analyzed by SDS-PAGE. The acrylamide concentration was 12.5%, and AE-1440EzStandard (manufactured by ATTO Corporation) was used as a protein molecular weight marker. As a result, a band thought to be lactonase was observed, and its molecular weight was 42,400. It is generally believed that molecular weight measurements by SDS-PAGE include an error of about 10%.

Example 4: Production of urolithin M5
Using the cell lysate prepared in Example 2, a reaction was carried out at 37° C. for 24 hours in a reaction solution containing ellagic acid at an initial concentration of 1 mg/mL (approximately 3.3 mM).
100 μL of the reaction solution was sampled, and 200 mL of dimethylacetamide containing 1% formic acid was added and mixed. The resulting diluted solution was centrifuged, and the supernatant was analyzed by high performance liquid chromatography (HPLC). The conditions were as follows: HPLC Condition 1.
As a result, 0.267 mM urolithin M5 was detected.

<HPLC condition 1>
Column: Cosmosil 5C18-AR-II (4.6 x 150)
Eluent A: 1% formic acid B: acetonitrile containing 1% formic acid Flow rate: 1 mL/min
Column temperature: 40°C
Detection: UV (349 nm)

[Example 5: Construction of a plasmid expressing lactonase as a His-tagged protein]
Similarly to Example 1, the lactonase gene was cloned by PCR using genomic DNA derived from Gordonibacter urolithinfaciens DSM 27213 strain as a template and the primer set shown below, and inserted into pET-28a(+) to construct an expression plasmid pET-28a_GuUroH-Histag(N) capable of expressing lactonase as a protein with a His-tag added to the N-terminus thereof.
5'-GCCGGATCCATGGCAGACAACAAGGTCATCGACATCAACATG-3' (SEQ ID NO: 7)
5'-TATGAATTCCTACAGGTTGAACAGCTTCGCCGCGTTGCCGCC-3' (SEQ ID NO: 8)

Example 6: Expression of lactonase in E. coli
E. coli Rosetta2 (DE3) strain was transformed with pET-28a_GuUroH-Histag(N) by the Ca method.
The resulting transformant was cultured in 5 mL of LB medium containing 34 μg/mL chloramphenicol and 30 μg/mL kanamycin at 37° C. for 2.5 hours with shaking. IPTG was added to the culture solution to a final concentration of 0.1 mM, and the resulting mixture was further cultured at 30° C. for 4 hours for induction.
The resulting culture solution was centrifuged to prepare wet cells. The resulting wet cells were suspended in 0.3 mL of BugBuster MasteMix (Novagen) and gently shaken at room temperature to disrupt the cells. The resulting disrupted solution was centrifuged to prepare a soluble fraction as the supernatant and an insoluble fraction as the precipitate.

[Example 7: Expression analysis of lactonase]
The soluble and insoluble fractions prepared in Example 6 were analyzed by SDS-PAGE. The acrylamide concentration was 12.5%, and AE-1440EzStandard (manufactured by ATTO Corporation) was used as a protein molecular weight marker. As a result, a band thought to be lactonase was observed in both the soluble and insoluble fractions, and the molecular weight was 43,800 (including His-tag). It is generally believed that molecular weight measurement by SDS-PAGE contains an error of about 10%.

[Example 8-1: Confirmation of lactonase activity]
As in Example 6, the cells induced with IPTG at a final concentration of 0.1 mM at 30°C for 7 hours were disrupted to prepare a cell-free extract. The obtained cell-free extract was used for a reaction at 37°C for 7 hours, and urolithin M5 produced from the raw material (substrate) ellagic acid was quantified by HPLC. The conditions were the same as those of HPLC condition 1 above.
As a result, the specific activity of the cell-free extract was 15.7 mU/mg protein, where 1 U refers to the activity that catalyzes the production of 1 μmol of urolithin M5 per minute under the above conditions.

[Example 8-2: Confirmation of optimal pH for lactonase activity]
The same experiment as in Example 8-1 was carried out using the following buffer solution to confirm the optimum pH for lactonase activity, except that the temperature was 37° C. and the reaction time was 3 hours.
pH 2.5, 3, 3.5: 20 mM citrate buffer pH 3.5, 4, 4.5, 5, 5.5: 20 mM acetate buffer pH 6, 6.5, 7, 7.5, 8: 20 mM potassium phosphate buffer pH 7.5, 8, 8.5, 9: 20 mM Tris-HCl buffer pH 9.5, 10, 10.5, 11: 20 mM carbonate buffer pH 11.5, 12: 20 mM sodium phosphate buffer The results are shown in Figure 1. Figure 1 shows the relative activity of lactonase at each pH, with the lactonase activity at pH 9 taken as 100. In the vicinity of the pH range of 7.0 to 9.0, the lactonase activity showed 80% or more of the maximum activity.

[Example 8-3: Confirmation of optimal temperature for lactonase activity]
The same experiment as in Example 8-1 was carried out using 20 mM potassium phosphate buffer (pH 6.5) to confirm the optimal temperature for lactonase activity. However, the reaction time was set to 3 hours. The temperatures used were 10, 15, 20, 28, 32, 37, 42, 50, 55, 60, and 70°C.
The results are shown in Figure 2. Figure 2 shows the relative activity of lactonase at each temperature, assuming that the lactonase activity at a temperature of 50°C is 100. When the temperature was in the range of 42°C or higher and 55°C or lower, the lactonase activity was 80% or more of the maximum activity.

Example 9: Purification of lactonase
The cell-free extract obtained by the method of Example 6 was purified using a His-trap column.
HisTrap was equilibrated with washing solution A (20 mM KPB (pH 7.4), 0.5 M NaCl, 20 mM imidazole).
The cell-free extract was added to HP (5 mL) (GE Healthcare) to adsorb lactonase, and the column was washed with the same washing solution A. Then, lactonase was eluted with elution solution B (20 mM KPB (pH 7.4), 0.5 M NaCl, 500 mM imidazole).
The purified enzyme showed a single band on SDS-PAGE and had a specific activity of 444 mU/mg protein.

[Example 10: Production of urolithin M5 from ellagic acid]
The transformant obtained in Example 6 was cultured in 5 mL of LB medium containing 34 μg/mL chloramphenicol and 30 μg/mL kanamycin at 37° C. for 15 hours with shaking. The culture solution was inoculated into 7 mL of the same medium freshly and cultured at 37° C. for 38 hours, after which 0.2 mg of ellagic acid (final concentration: approximately 0.1 mM) was added and the culture was continued for another 12 hours.
100 μL of the culture solution was sampled, and 200 mL of dimethylacetamide containing 1% formic acid was added and mixed. The resulting diluted solution was centrifuged, and the supernatant was analyzed by HPLC under the same conditions as those of HPLC condition 1 above.
As a result, 0.076 mM urolithin M5 and 0.008 mM ellagic acid were detected.

[Comparative Example 1]
The same procedure was carried out as in Example 10, except that a plasmid-free host (Escherichia coli Rosetta2 (DE3) strain) was used instead of the transformant.
As a result, urolithin M5 was not detected.

[Example 11: Production of urolithin M6 from urolithin M5]
Urolithin M5 was added to ABB medium (Oxoid) to a final concentration of 3.3 mM, followed by heat sterilization and replacement of the gas phase with _N2 : _CO2 : _H2 (80%/10%/10%) gas to prepare a basic medium. Gordonibacter urolithifaciens DSM 27213 strain was inoculated into the basic medium and cultured anaerobically at 37°C. After the culture was completed, an equal amount of DMSO was added to 1 mL of the culture solution to dissolve the urolithins, and quantitative analysis of the urolithins was performed by HPLC. The conditions were as follows: HPLC condition 2.
As a result, 0.406 mM urolithin M6 was produced after 14 days of culture.

<HPLC condition 2>
Column: Inertsil ODS-3 (φ4.6 mm×250 mm, 5 μm) (GL Science)
Eluent A: 1% formic acid B: acetonitrile containing 1% formic acid Flow rate: 1 mL/min
Column temperature: 40°C
Detection: UV (305 nm)

[Example 12: Production of urolithin C from urolithin M5]
Urolithin M5 was added to ABB medium (Oxoid) to a final concentration of 3.3 mM, followed by heat sterilization and replacement of the gas phase with _N2 : _CO2 : _H2 (80%/10%/10%) gas to prepare a basic medium. Gordonibacter urolithifaciens DSM 27213 strain was inoculated into the basic medium and cultured anaerobically at 37°C. After completion of the culture, an equal amount of DMSO was added per 1 mL of the culture solution to dissolve the urolithins, and quantitative analysis of the urolithins was performed by HPLC. The conditions were as described in HPLC condition 2 above.
As a result, 0.0707 mM urolithin C was produced after 14 days of culture.

[Example 13: Production Example 1 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, followed by heat sterilization and replacing the gas phase with _N2 : _CO2 : _H2 (80%/10%/10%) gas to prepare a basic medium. Clostridium volteae JCM 12243, DSM 15670, or DSM 29485 was inoculated into the basic medium and cultured anaerobically at 37°C. After the culture was completed, urolithins were extracted with an equal amount of ethyl acetate per 5 mL of culture solution, and the resulting ethyl acetate phase was concentrated under reduced pressure and dried. The thus obtained dried product was redissolved in 0.5 mL of methanol, and quantitative analysis of urolithins was performed by HPLC. The conditions were as described in HPLC condition 2 above. Furthermore, urolithins manufactured by Dalton Pharma were used as standards and dissolved in DMSO.
As a result, after two weeks of culture, 89% of the added Urolithin C was
100% and 89% were converted to urolithin A.

[Example 14: Production Example 2 of Urolithin A from Urolithin C]
The same procedure was followed as in Example 13, except that Clostridium asparagiforme DSM 15981 strain was used and cultured for 5 days. As a result, 95% of the added urolithin C was converted to urolithin A.

[Example 15: Production Example 3 of Urolithin A from Urolithin C]
The same procedure was followed as in Example 13, except that Clostridium citronellae DSM 19261 strain was used and cultured for 5 days. As a result, 82% of the added urolithin C was converted to urolithin A.

[Example 16: Production Example 4 of Urolithin A from Urolithin C]
Clostridium volteae JCM 12243 strain and Gordonibacter pamelaeae DSM 19378 strain were inoculated into ABB medium (Oxoid) containing 0.1% ellagic acid (SIGMA) and cultured in the same manner as in Example 13. As a result, after 2 weeks of culture, 67% of the added ellagic acid was converted to urolithin A.

[Example 17: Production Example 5 of Urolithin A from Urolithin C]
Cultivation was performed in the same manner as in Example 16 except that Clostridium volteae JCM 12243 strain and Gordonibacter urolithifaciens DSM 27213 strain were used. As a result, after 2 weeks of culture, 62% of the added ellagic acid was converted to urolithin A.

[Example 18: Production Example 6 of Urolithin A from Urolithin C]
Cultivation was performed in the same manner as in Example 16 except that Clostridium asparagiforme strain DSM 15981 and Gordonibacter urolithifaciens strain DSM 27213 were used. As a result, after 5 days of culture, 60% of the added ellagic acid was converted to urolithin A.

[Example 19: Production Example 7 of Urolithin A from Urolithin C]
Cultivation was performed in the same manner as in Example 16 except that Clostridium citronniae strain DSM 19261 and Gordonibacter urolithifaciens strain DSM 27213 were used. As a result, after 5 days of culture, 60% of the added ellagic acid was converted to urolithin A.

[Example 20: Production of urolithin D from urolithin M5]
Urolithin M5 was added to ABB medium (Oxoid) to a final concentration of 3.3 mM, followed by heat sterilization and replacement of the gas phase with _N2 : _CO2 : _H2 (80%/10%/10%) gas to prepare a basic medium. Gordonibacter urolithifaciens DSM 27213 strain was inoculated into the basic medium and cultured anaerobically at 37°C. After the culture was completed, an equal amount of DMSO was added to 1 mL of the culture solution to dissolve the urolithins, and quantitative analysis of the urolithins was performed by HPLC. The conditions were as described in HPLC condition 2 above.
As a result, 0.011 mM urolithin M6 was produced after 14 days of culture.

The present invention provides a lactonase that is advantageous for the industrial production of urolithins. The lactonase enables efficient production of urolithin M5, and enables the production of urolithins via urolithin M5. Urolithins are useful as ingredients for pharmaceuticals, cosmetics, and functional foods, and the present invention is extremely useful industrially.

Claims (12)

A method for producing urolithin M5, comprising the following step (I):
Step (I): A step of contacting ellagic acid with one or more selected from the following (i) to (iii):
(i) a protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added, and which has an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid ;
(ii) A transformant which retains in an expressible manner the following polynucleotide (a) or (b), or which retains in an expressible manner a recombinant vector containing the following polynucleotide (a) or (b):
(a) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4;
(b) a polynucleotide encoding a protein having an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, the protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added;
(iii) A processed product of the transformant. A method for producing urolithin C, comprising the following steps (I) and (II), wherein the steps (I) and (II) are carried out in the same system:
Step (I): contacting ellagic acid with one or more selected from the following (i) to (iii) to produce urolithin M5;
(i) a protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added, and which has an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid ;
(ii) A transformant comprising the following polynucleotide (a) or (b) in an expressible state, or a recombinant vector comprising the following polynucleotide (a) or (b) in an expressible state:
(a) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4;
(b) a polynucleotide encoding a protein having an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, the protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added;
(iii) A processed product of the transformant.
Step (II): Allowing a microorganism having the ability to produce urolithin C from urolithin M5 to produce urolithin C from urolithin M5. A method for producing urolithin A, comprising the following steps (I) to (III), which are carried out in the same system:
Step (I): contacting ellagic acid with one or more selected from the following (i) to (iii) to produce urolithin M5;
(i) a protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added, and which has an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid ;
(ii) A transformant comprising the following polynucleotide (a) or (b) in an expressible state, or a recombinant vector comprising the following polynucleotide (a) or (b) in an expressible state:
(a) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4;
(b) a polynucleotide encoding a protein having an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, the protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added;
(iii) A processed product of the transformant.
Step (II): Allowing a microorganism having the ability to produce urolithin C from urolithin M5 to produce urolithin C from urolithin M5.
Step (III): Allowing a microorganism having the ability to produce urolithin A from urolithin C to produce urolithin A from urolithin C. A method for producing urolithin M6, comprising the following steps (I) and (IV), wherein the steps (I) and (IV) are carried out in the same system:
Step (I): contacting ellagic acid with one or more selected from the following (i) to (iii) to produce urolithin M5;
(i) a protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added, and which has an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid ;
(ii) A transformant comprising the following polynucleotide (a) or (b) in an expressible state, or a recombinant vector comprising the following polynucleotide (a) or (b) in an expressible state:
(a) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4;
(b) a polynucleotide encoding a protein having an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, the protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added;
(iii) A processed product of the transformant.
Step (IV): Allowing a microorganism having the ability to produce urolithin M6 from urolithin M5 to produce urolithin M6 from urolithin M5. A method for producing urolithin D, comprising the following steps (I) and (V), wherein the steps (I) and (V) are carried out in the same system:
Step (I): A step of producing urolithin M5 by contacting one or more selected from the following (i) to (iii) with ellagic acid;
(i) a protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added, and which has an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid ;
(ii) A transformant comprising the following polynucleotide (a) or (b) in an expressible state, or a recombinant vector comprising the following polynucleotide (a) or (b) in an expressible state:
(a) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4;
(b) a polynucleotide encoding a protein having an activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, the protein consisting of an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added;
(iii) A processed product of the transformant.
Step (V): Allowing a microorganism having the ability to produce urolithin D from urolithin M5 to produce urolithin D from urolithin M5. In the step (I)(ii)(a),
When the polynucleotide is a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO:3, the transformant is a transformant in which the host does not belong to Gordonibacter urolithinfaciens,
When the polynucleotide is a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4, the transformant is a transformant in which the host does not belong to Gordonibacter pamelaeae.
The method according to any one of claims 1 to 5. The method according to any one of claims 1 to 6 , wherein the lactonase (i) in step (I) further has the following properties (2) to (4):
(2) The optimum pH is 7.0 or more and 9.0 or less;
(3) The optimum temperature is 42°C or higher and 55°C or lower;
(4) The molecular weight is 37,800 or more and 46,200 or less. Lactonase, which is a protein represented by the following (A) or (B):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO:4;
(B) A protein having the activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, which consists of an amino acid sequence represented by SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added. A polynucleotide encoding a lactonase protein represented by the following (A) or (B):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO:4;
(B) A protein having the activity of catalyzing a reaction of hydrolyzing at least one of the two ester bonds present in ellagic acid, which consists of an amino acid sequence represented by SEQ ID NO: 4 in which 1 to 3 amino acids have been substituted or deleted, or 1 to 3 amino acids have been inserted or added. A recombinant vector comprising the polynucleotide of claim 9. A transformant comprising the polynucleotide according to claim 9 in an expressible state, or the vector according to claim 10 in an expressible state. A method for producing a protein encoded by the polynucleotide of claim 9, comprising the step of culturing the transformant of claim 11.

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