Novel microorganisms, cultures or extracts of novel microorganisms, and methods for producing ergothioneine.

Abstract

The microorganism of the present disclosure is a microorganism belonging to Dirkmeia churashimaensis (accession number: NITE BP-03707, accession number: NITE BP-03708, accession number: NITE BP-03709, accession number: NITE BP-03711, or accession number: NITE BP-03712), Aureobasidium melanogenum (assignment number: NITE BP-03706), or a microorganism belonging to a species (Ustilago sp.) closely related to Ustilago sporoboli-indici (accession number: NITE BP-03710).

Description

Technical Field

[0001] The present invention relates to novel microorganisms, cultures or extracts of novel microorganisms, and a method for producing ergothioneine.

Background Art

[0002] Ergothioneine is a kind of sulfur-containing amino acid. Ergothioneine has an antioxidant effect superior to that of vitamin E and has attracted attention as a highly valuable compound in fields such as health and beauty.

[0003] For example, Patent Document 1 and Non-Patent Document 1 describe transformed filamentous fungi with enhanced ergothioneine-producing ability.

[0004] Non-Patent Document 2 describes transformed microorganisms of the genus Methylobacrium with enhanced ergothioneine-producing ability. Non-Patent Document 2 describes that microorganisms of the genus Aureobasidium and the genus Rhodotorula have the ability to produce ergothioneine.

[0005] Non-Patent Document 3 describes that microorganisms of the genus Pleurotus have the ability to produce ergothioneine.

[0006] [[ID=URL=28]]Patent Document 2 describes that microorganisms of the genus Methylobactrium and the genus Rhodotorula have the ability to produce ergothioneine. Patent Document 3 describes that microorganisms of the genus Moniliella have the ability to produce ergothioneine. Patent Document 4 describes that microorganisms of the genus Dirkmeia, the genus Papiliotrema, and the genus Apiotrichum have the ability to produce ergothioneine.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

[0008] [Non-Patent Document 1] S. Takusagawa, Biosci. Biotechnol. Biochem., 83, 181-184 (2019) [Non-Patent Document 2] Y. Fujitani et al., J. Biosci. Bioeng., 126, 715-722 (2018) [Non-Patent Document 3] SY. Lin, Int. J. Med. Mushrooms, 17, 749-761 (2015) [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] Ergothioneine is not biosynthesized in the human body, but is known to be biosynthesized by some microorganisms. Therefore, as described in the prior art literature mentioned above, research and development is underway to search for microorganisms that produce ergothioneine, and to modify microorganisms to enhance ergothioneine production.

[0010] Genetic engineering technology can be used to modify microorganisms to increase ergothioneine production. However, the ergothioneine produced by these microorganisms cannot be used in the food industry, etc. Therefore, there is a strong desire to search for unmodified microorganisms that have high ergothioneine production.

[0011] The present invention has been made in view of the above problems, and an object thereof is to provide a novel microorganism having a high production amount of ergothioneine.

Means for Solving the Problems

[0012] As a result of screening, the inventors of the present invention have found a novel microorganism having a high production amount of ergothioneine and have completed the present invention.

[0013] The microorganism according to one aspect of the present invention is a microorganism belonging to Dilomyces churasimaensis (Accession Number: NITE BP-03707, Accession Number: NITE BP-03708, Accession Number: NITE BP-03709, Accession Number: NITE BP-03711, or Accession Number: NITE BP-03712), Aureobasidium melanogenum (Accession Number: NITE BP-03706), or a microorganism belonging to a species closely related to Ustilago sporoboli-indici (Ustilago sp.) (Accession Number: NITE BP-03710).

Effects of the Invention

[0014] According to one aspect of the present invention, a microorganism having a high production amount of ergothioneine can be provided.

Brief Description of the Drawings

[0015] [Figure 1] It is a figure which shows the simple molecular phylogenetic tree based on the base sequence of the 26S rDNA D1 / D2 region of EB682 strain. [Figure 2] It is a figure which shows the simple molecular phylogenetic tree based on the base sequence of the ITS region of the rDNA of EB682 strain. [Figure 3] It is a figure which shows the simple molecular phylogenetic tree based on the base sequence of the 26S rDNA D1 / D2 region of EC431 strain. [Figure 4] It is a figure which shows the simple molecular phylogenetic tree based on the base sequence of the ITS region of the rDNA of EC431 strain. [Figure 5]It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the 26S rDNA D1 / D2 region of the EC171 strain. [Figure 6] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of the EC171 strain. [Figure 7] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the 26S rDNA D1 / D2 region of the EC581 strain. [Figure 8] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of the EC581 strain. [Figure 9] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the 26S rDNA D1 / D2 region of the EC592 strain. [Figure 10] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of the EC592 strain. [Figure 11] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the 26S rDNA D1 / D2 region of the EB761 strain. [Figure 12] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of the EB761 strain. [Figure 13] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the 26S rDNA D1 / D2 region of the EC021 strain. [Figure 14] It is a diagram showing a simple molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of the EC021 strain.

Mode for Carrying Out the Invention

[0016] Unless otherwise specified in this specification, "A~B" representing a numerical range is intended to mean "A or more (including A and greater than A) and B or less (including B and less than B)".

[0017] 〔Novel Microorganism〕 A microorganism according to one aspect of the present invention is a microorganism belonging to Dirkmeia churasimaensis or Aureobasidium melanogenum that has the ability to produce ergothioneine, or a microorganism belonging to the genus Ustyrago that has the ability to produce ergothioneine.

[0018] The microorganism according to one aspect of the present invention has a high production rate of ergothioneine. Ergothioneine is a type of sulfur-containing amino acid and has excellent antioxidant properties. Furthermore, since the microorganism according to one aspect of the present invention has not been modified by genetic engineering or other technologies, it can be used in the food industry.

[0019] (1. Aureobasidium melanogenum EB682) Aureobasidium melanogenum EB682 (hereinafter sometimes abbreviated as "yeast EB682") is the first microorganism isolated from hibiscus leaves.

[0020] The D1 / D2 and ITS regions of the 26S rDNA of the ribosomal RNA gene were sequenced. Then, a BLAST homology search was performed against the TechnoSuruga Laboratory, Japan database DB-FU13.0 and the international nucleotide sequence database (DDBJ / ENA(EMBL) / GenBank). As a result, EB682 was assigned to Aureobasidium melanogenum.

[0021] Yeast EB682 was deposited at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture (original deposit date: August 5, 2022, accession number: NITE BP-03706).

[0022] The culture method for yeast EB152 should follow the general culture methods used for microorganisms of the genus Aureobasidium. The culture method should be batch culture using liquid medium or fed-batch culture with continuous addition of a carbon source and / or organic nitrogen source to the culture system, and aeration and stirring are desirable. The culture medium may contain necessary nutrients such as a carbon source, nitrogen source, or inorganic salts that can be assimilated by microorganisms belonging to the genus Aureobasidium. The culture pH is preferably 3 to 8, the culture temperature is preferably 20°C to 30°C, and the culture time is preferably 2 to 14 days.

[0023] (2. Ustillago SP EC431) Ustilago sp. EC431 (hereinafter sometimes abbreviated as "yeast EC431") is the first microorganism isolated from the peel of the sudachi citrus fruit.

[0024] The D1 / D2 and ITS regions of the 26S rDNA of the ribosomal RNA gene were sequenced. Then, a BLAST homology search was performed against the TechnoSuruga Laboratory, Japan database DB-FU13.0 and the international nucleotide sequence database (DDBJ / ENA(EMBL) / GenBank). As a result, EC431 was shown to be closely related to Ustilago sporoboli-indici.

[0025] Yeast EC431 was deposited at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture (original deposit date: August 5, 2022, accession number: NITE BP-03710).

[0026] The culture method for yeast EC431 should follow the general culture methods used for microorganisms of the genus Ustylago. The culture method should be batch culture using liquid medium or fed-batch culture with continuous addition of a carbon source and / or organic nitrogen source to the culture system, and aeration and stirring are desirable. The culture medium may contain necessary nutrients such as a carbon source, nitrogen source, or inorganic salts that microorganisms belonging to the genus Ustylago can utilize. The culture pH is preferably 3 to 8, the culture temperature is preferably 20°C to 30°C, and the culture time is preferably 2 to 14 days.

[0027] (3. Dirkmeia churasimaensis EC171) Dirkmeia churashimaensis EC171 (hereinafter sometimes abbreviated as "yeast EC171") is the first microorganism isolated from the leaves of Japanese mustard spinach.

[0028] The D1 / D2 and ITS regions of the 26S rDNA of the ribosomal RNA gene were sequenced. Then, a BLAST homology search was performed against the TechnoSuruga Laboratory, Japan database DB-FU13.0 and the international nucleotide sequence database (DDBJ / ENA(EMBL) / GenBank). As a result, EC171 was assigned to Dirkmeia churasimaensis.

[0029] Yeast EC171 was deposited at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture (Original deposit date: August 5, 2022, Accession number: NITE BP-03709).

[0030] The culture method for yeast EC171 should follow the general culture methods used for microorganisms of the genus Dirkmeia. The culture method should be batch culture using liquid medium or fed-batch culture with continuous addition of a carbon source and / or organic nitrogen source to the culture system, and aeration and stirring are desirable. The culture medium may contain necessary nutrients such as a carbon source, nitrogen source, or inorganic salts that microorganisms belonging to the genus Dirkmeia can utilize. The culture pH is preferably 3 to 8, the culture temperature is preferably 20°C to 30°C, and the culture time is preferably 2 to 14 days.

[0031] (4. Dirkmeia churasimaensis EC581) Dirkmeia churashimaensis EC581 (hereinafter sometimes abbreviated as "yeast EC581") is the first microorganism isolated from kale leaves.

[0032] The D1 / D2 and ITS regions of the 26S rDNA of the ribosomal RNA gene were sequenced. Then, a BLAST homology search was performed against the TechnoSuruga Laboratory, Japan database DB-FU13.0 and the international nucleotide sequence database (DDBJ / ENA(EMBL) / GenBank). As a result, EC581 was assigned to Dirkmeia churasimaensis.

[0033] Yeast EC581 was deposited at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture (original deposit date: August 5, 2022, accession number: NITE BP-03711).

[0034] The culture method for yeast EC581 is the same as that for yeast EC171.

[0035] (5. Dirkmeia churasimaensis EC592) Dirkmeia churashimaensis EC592 (hereinafter sometimes abbreviated as "yeast EC592") is the first microorganism isolated from kale leaves.

[0036] The D1 / D2 and ITS regions of the 26S rDNA of the ribosomal RNA gene were sequenced. Then, a BLAST homology search was performed against the TechnoSuruga Laboratory, Japan database DB-FU13.0 and the international nucleotide sequence database (DDBJ / ENA(EMBL) / GenBank). As a result, EC592 was assigned to Dirkmeia churasimaensis.

[0037] Yeast EC592 was deposited at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture (Original deposit date: August 5, 2022, Accession number: NITE BP-03712).

[0038] The culture method for yeast EC592 is the same as that for yeast EC171.

[0039] (6. Dirkmeia churasimaensis EB761) Dirkmeia churashimaensis EB761 (hereinafter sometimes abbreviated as "yeast EB761") is the first microorganism isolated from herbaceous plant leaves.

[0040] The D1 / D2 and ITS regions of the 26S rDNA of the ribosomal RNA gene were sequenced. Then, a BLAST homology search was performed against the TechnoSuruga Laboratory, Japan database DB-FU13.0 and the international nucleotide sequence database (DDBJ / ENA(EMBL) / GenBank). As a result, EB761 was assigned to Dirkmeia churasimaensis.

[0041] Yeast EB761 was deposited at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture (Original deposit date: August 5, 2022, Accession number: NITE BP-03707).

[0042] The culture method for yeast EB761 is the same as that for yeast EC171.

[0043] (7. Dirkmeia churasimaensis EC021) Dirkmeia churashimaensis EC021 (hereinafter sometimes abbreviated as "yeast EC021") is the first microorganism isolated from the flower buds of molokhia.

[0044] The D1 / D2 and ITS regions of the 26S rDNA of the ribosomal RNA gene were sequenced. Then, a BLAST homology search was performed against the TechnoSuruga Laboratory, Japan database DB-FU13.0 and the international nucleotide sequence database (DDBJ / ENA(EMBL) / GenBank). As a result, EC021 was assigned to Dirkmeia churasimaensis.

[0045] Yeast EC021 was deposited at the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located at Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture (Original deposit date: August 5, 2022, Accession number: NITE BP-03708).

[0046] The culture method for yeast EC021 is the same as that for yeast EC171.

[0047] [Culture] A culture according to one aspect of the present invention is a culture of yeast EB682, EC431, EC171, EC581, EC592, EB761, or EC021. A culture according to one aspect of the present invention includes culture supernatant, culture precipitate, culture medium, cultured cells, cultured cell lysates, freeze-dried cultured cells, and other processed cultured cell products. A culture according to one aspect of the present invention contains ergothioneine.

[0048] [Extract] An extract according to one aspect of the present invention is an extract of yeast EB682, EC431, EC171, EC581, EC592, EB761, or EC021. In this specification, "microbial extract" refers to an extract obtained by performing an extraction treatment on a microorganism, and an extract obtained by performing an extraction treatment on a microbial culture. Therefore, an extract according to one aspect of the present invention can be obtained, for example, by performing an extraction treatment on yeast EB682, EC431, EC171, EC581, EC592, EB761, or EC021, or by performing an extraction treatment on a culture of yeast EB682, EC431, EC171, EC581, EC592, EB761, or EC021. An extract according to one aspect of the present invention contains ergothioneine.

[0049] Extraction methods include hot water extraction; solvent extraction with organic solvents, etc.; pressurized extraction; chemical extraction with enzymes and surfactants, etc.; ultrasonic extraction; alkaline extraction; acid extraction; osmotic extraction; extraction by grinding; extraction by crushing; extraction by freeze-thaw cycle; extraction with liquid nitrogen; and extraction by high-speed stirring. Hot water extraction is preferred because it exhibits excellent plant growth effects. One type of extraction method may be used, or two or more types of extraction methods may be used.

[0050] Hot water extraction is an extraction method in which the object to be extracted is brought into contact with or immersed in hot water for a certain period of time. The temperature of the water used for hot water extraction is preferably 40°C or higher, and more preferably 60°C or higher.

[0051] An extract according to one aspect of the present invention may be a hot water extract, a solvent extract with an organic solvent such as EC581, EC592, EC761, or EC021 of a microorganism, and may be a pressurized extract, a chemical extract with enzymes and surfactants such as ultrasonic extract, an alkaline extract, an acid extract, an osmotic extract, an extract by grinding, an extract by freeze-thaw, an extract with liquid nitrogen, or an extract by high-speed stirring.

[0052] One application of the culture or extract according to one aspect of the present invention is a plant growth regulator containing the culture or extract as an active ingredient.

[0053] [Ergothioneine production method] A method for producing ergothioneine according to one aspect of the present invention includes the step of culturing the above-mentioned microorganism to obtain a culture containing ergothioneine. In this production method, one type of microorganism may be cultured, or multiple types of microorganisms may be cultured.

[0054] The recovery of ergothioneine from a culture containing ergothioneine can be carried out, for example, by the same method used to recover and purify ergothioneine from a typical microbial culture. For example, the culture can be centrifuged to collect the microbial cells. Next, the collected microbial cells can be subjected to hot water extraction to obtain an extract containing ergothioneine. The ergothioneine can then be recovered by purifying this extract. The amount of ergothioneine produced by the microorganism can be quantified, for example, by measuring the obtained extract using a high-performance liquid chromatography (HCM) system and a mass spectrometer such as LCMS.

[0055] 〔summary〕 One aspect of the present invention relates to a microorganism belonging to Dirkmeia churasimaensis (accession number: NITE BP-03707, NITE BP-03708, NITE BP-03709, NITE BP-03711, or NITE BP-03712), Aureobasidium melanogenum (accession number: NITE BP-03706), or a microorganism belonging to a species closely related to Ustillago sporobori-indisi (Ustillago sp.) (accession number: NITE BP-03710).

[0056] A culture according to one aspect of the present invention is a culture of the above-mentioned microorganism.

[0057] An extract according to one aspect of the present invention is an extract of the above-mentioned microorganism.

[0058] A method for producing ergothioneine according to one aspect of the present invention includes the step of culturing the above-mentioned microorganism to obtain a culture containing ergothioneine.

[0059] The embodiments of the present invention will be further described in detail below with reference to some examples. Of course, the present invention is not limited to the following embodiments, and it goes without saying that various forms are possible in terms of details. Furthermore, the present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims, and embodiments obtained by appropriately combining the disclosed technical means are also included in the technical scope of the present invention. In addition, all references cited herein are incorporated by reference. [Examples]

[0060] In the following examples, unless otherwise specified, % represents mass %.

[0061] [Evaluation Example 1] Screening of ergothioneine-producing microorganisms (1) Accumulation culture in isolation sources collected from the environment First, microbial samples were collected from plants and other environmental sources in two separate collections. As a result, a total of 150 samples were collected (90 in the first collection and 60 in the second).

[0062] Next, each sample was immersed in a 15 mL plastic tube containing 2 mL of screening medium and incubated at 25°C for 3–7 days at 200 rpm. The screening medium used was YM medium containing antibiotics. Specifically, the medium contained 1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, 0.01% streptomycin sulfate, and 0.005% chloramphenicol.

[0063] Then, 126 samples (70 in the first round, 56 in the second round) were selected based on visual inspection, indicating that the culture medium had become cloudy (indicating microbial growth).

[0064] (2) Selection of samples by oxidative stress loading The culture media of the 126 samples selected in (1) above were diluted 100-fold or 10,000-fold with YM medium. The diluted culture media were then spread onto YM agar medium and YM agar medium to which 3 mM H2O2 had been added (hereinafter abbreviated as H2O2-containing YM agar medium), and incubated at 25°C for 2 to 7 days.

[0065] The number of colonies that grew on YM agar medium and the number of colonies that grew on H2O2-containing YM agar medium were counted. Then, 112 samples (69 in the first sample and 43 in the second sample) in which colonies grew on both YM agar medium and H2O2-containing YM agar medium were selected.

[0066] Furthermore, the colonies grown on agar plates from the 112 selected samples were visually observed for their morphology and color, and 181 different types of yeast-like colonies were selected (106 in the first round and 75 in the second round).

[0067] (3) Culture of selected colonies in 96 wells The 181 colonies selected in (2) above were inoculated into a 96-well plate containing 1 mL of YM medium and cultured at 1600 rpm for 3-4 days at 25°C. After culturing, the collected culture medium was centrifuged at 2000 rpm for 10 minutes at 4°C. The bacterial pellet obtained by centrifugation was washed with 1 mL of pure water and subjected to centrifugation again.

[0068] The bacterial cell pellet obtained by centrifugation was suspended in 0.1 mL of pure water. The resulting suspension was heated at 96°C for 10 minutes to extract the intracellular components. The extracted intracellular components were then subjected to centrifugation to remove the bacterial residue, and an extract was obtained.

[0069] (4) Quantitative analysis of ergothioneine in the extract by LC-MS The solution obtained by mixing 0.15 mL of the extract from (3) above with 0.35 mL of acetonitrile was filtered through a 0.45 μm PVDF filter. The resulting filtrate was used as a sample for LCMS measurement.

[0070] For LC-MS analysis, a Shimadzu LCMS-2020 was used. A SHODEX Asahipak NH2P-40 2D+ guard column was used for the LC column. A mixture of 10 mM ammonium formate and acetonitrile (10 mM ammonium formate / acetonitrile = 30 / 70 (v / v)) was used as the mobile phase. The flow rate was 0.1 mL / min, and the analysis was performed at 25°C.

[0071] For MS detection, ionization was performed using the DUIS mode, which simultaneously performs ESI ionization and APCI ionization. Furthermore, detection of ergothioneine was carried out using the SIM mode with m / z = 230(+), which is suitable for detection.

[0072] Based on the analysis of the extracts from the 181 colonies selected in (2) above, 22 colonies with high ergothioneine production (7 in the first round, 15 in the second round) were selected.

[0073] (5) Scale-up culture of ergothioneine-producing microorganisms in flasks The 22 colonies selected in (4) above were inoculated into a 300 mL flask containing 50 mL of YM medium and cultured at 25°C for 7 days at 200 rpm (n=1).

[0074] Culture media were collected as needed from days 3 to 7 of incubation. As in (3) above, the bacterial cells were centrifuged and washed, and the extract was collected by hot water extraction.

[0075] The obtained extracts were analyzed by LCMS in the same manner as in (4) above, and seven strains (EB682, EC431, EC171, EC581, EC592, EB761, and EC021) that produced high levels of ergothioneine were selected.

[0076] [Evaluation Example 2] Measurement of Ergothioneine Production EB682, EC431, EC171, EC581, and EC592 were inoculated into 300 mL flasks containing 50 mL of YM medium and cultured at 200 rpm for 5 days at 25°C (n=3). The amount of ergothioneine (EGT) produced on day 5 was measured by LC-MS.

[0077] Furthermore, extracts of each microorganism were obtained by culturing each microorganism aerobically at 25°C for 5 days in a 5L jar fermenter containing 2L of YM medium, and then extracting the dried microbial cells with hot water. The amount of EGT (mg / L) in the extract was then measured by LCMS.

[0078] Tables 1 and 2 show the ergothioneine production volume, production rate, and EGT content in the extract for each strain. In Table 1, the EGT production volume (mg / L-culture medium) is the EGT production volume per liter of culture medium on day 5 of cultivation. The EGT production rate (mg / L / d) is the EGT production volume per day (mg / L). In Table 2, the EGT production volume (mg / g-dried cells) is the EGT production volume per gram of dried cells.

[0079] Tables 3 and 4 show the production volume and production rate of known microorganisms. In Table 3, the EGT production volume for Aspergillus oryzae NSAR1 is the EGT production volume per 1 kg of culture medium, and the EGT production rate is the EGT production volume per day (mg / kg). A dash (-) in Tables 3 and 4 indicates that the data was not measured.

[0080] [Table 1]

[0081] [Table 2]

[0082] [Table 3]

[0083] [Table 4]

[0084] As shown in Tables 1 and 2, Aureobasidium melanogenum EB682 was found to produce more EGT than Aureobasidium pullulans kz25. Ustilago sp. EC431 was found to produce more EGT than Ustilago maydis UM521. Dirkmeia churashimaensis EC171, Dirkmeia churashimaensis EC581, and Dirkmeia churashimaensis EC592 were each found to produce more EGT than Dirkmeia churashimaensis S111. Furthermore, it was confirmed that all five strains evaluated in Evaluation Example 2 were rich in EGT.

[0085] [Evaluation Example 3] Measurement of Ergothioneine Production For EB761 and EC021, the amount of EGT produced on day 5 was measured by LCMS, similar to Evaluation Example 2. The amount of EGT in the extracts of each microorganism was also measured, similar to Evaluation Example 2. The measurement results are shown in Table 5.

[0086] [Table 5]

[0087] As shown in Tables 4-5, Dirkmeia churashimaensis EB761 and Dirkmeia churashimaensis EC021 were found to produce higher EGT compared to Dirkmeia churashimaensis S111. Furthermore, it was confirmed that the extracts from both strains evaluated in Evaluation Example 3 were rich in EGT.

[0088] [Evaluation Example 4] Identification of Microorganisms The taxonomic classification of the seven selected strains was estimated by analyzing the nucleotide sequences of the D1 / D2 and ITS regions of the 26S rDNA of the ribosomal RNA gene.

[0089] (Molecular phylogenetic position and morphological properties of strain EB682) BLAST homology searches using the microbial identification system "ENKI" against DB-FU and international nucleotide sequence databases revealed that the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EB682 (SEQ ID NO: 1) showed 100% identity with multiple nucleotide sequences of Aureobasidium melanogenum, a type of ascomycete yeast (Tables 6 and 7). In the molecular phylogenetic tree (Figure 1) analyzed based on the nucleotide sequences obtained from the homology search against DB-FU, strain EB682 showed the same molecular phylogenetic position as Aureobasidium melanogenum CBS105.22T (accession number FJ150926).

[0090] Table 6 shows the BLAST search results for DB-FU, specifically the D1 / D2 region sequencing data of the top 30 26S rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0091] Table 7 shows the BLAST search results for the international nucleotide sequence database, specifically the D1 / D2 region of 26S rDNA found in the top 30 results based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis. Aureobasidium pullulans var. melanigenum is considered to correspond to the currently known name, Aureobasidium melanogenum.

[0092] Figure 1 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EB682. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. "T" at the end of the strain name indicates the type strain of that species, and "NT" indicates the new type strain of that species.

[0093] [Table 6]

[0094] [Table 7]

[0095] BLAST homology searches of DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the nucleotide sequence of the ITS region of the rDNA of strain EB682 (SEQ ID NO: 2) showed 99.8–100% identity with multiple nucleotide sequences of Aureobasidium melanogenum, a type of ascomycete yeast (Tables 8 and 9). In a molecular phylogenetic tree (Figure 2) analyzed based on the nucleotide sequences obtained from the homology search against DB-FU, strain EB682 formed clusters with multiple nucleotide sequences of Aureobasidium melanogenum.

[0096] Table 8 shows the BLAST search results for DB-FU, which are the nucleotide sequence analysis data for the ITS regions of the top 30 rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0097] Table 9 shows the BLAST search results for the international nucleotide sequence database, specifically the sequence analysis data of the ITS regions of the top 30 rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis. Aureobasidium pullulans var. melanigenum is considered to correspond to the current name, Aureobasidium melanogenum.

[0098] Figure 2 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of strain EB682. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. "T" at the end of the strain name indicates the type strain of that species, and "NT" indicates the new type strain of that species.

[0099] [Table 8]

[0100] [Table 9]

[0101] Based on the above, the EB682 strain was identified as Aureobasidium melanogenum based on the nucleotide sequence analysis results of the D1 / D2 region of the 26S rDNA and the ITS region of the rDNA.

[0102] The morphological characteristics of strain EB682 were investigated by observing the characteristics and morphology of the colonies. After culturing on YM agar plates for two days, the colonies exhibited a creamy to light brown color, a moist surface, and a filamentous appearance. On the third day of culture, the formation of colorless, thin-walled, broadly oval to lemon-shaped yeast-like budding cells was observed. Furthermore, the formation of broadly oval to lemon-shaped, colorless, smooth budding conidia from short projections on the vegetative hyphae was observed.

[0103] Based on its molecular phylogenetic position, morphological characteristics, and ergothioneine production, strain EB682 was determined to be a novel microorganism belonging to Aureobasidium melanogenum.

[0104] (Molecular phylogenetic position and morphological properties of strain EC431) BLAST homology searches using the microbial identification system "ENKI" against DB-FU and international nucleotide sequence databases revealed that the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EC431 (SEQ ID NO: 3) showed 99.7% identity with multiple nucleotide sequences of the basidiomycetes (basidiomycete yeasts) Ustilago shanxiensis, Ustilago calamagrostidis, and Ustilago sporoboli-indici (Tables 10, 11). In the molecular phylogenetic tree (Figure 3) analyzed based on the nucleotide sequences obtained from the homology searches against DB-FU and international nucleotide sequence databases, strain EC431 formed a separate phylogenetic branch within the group of phylogenetic groups composed of the genus Ustilago.

[0105] Table 10 shows the BLAST search results for DB-FU, specifically the nucleotide sequence analysis data for the D1 / D2 regions of 26S rDNAs that were found in the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0106] Table 11 shows the BLAST search results for the international nucleotide sequence database, specifically the D1 / D2 region of 26S rDNA, which were the top 30 search results in terms of homology score. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0107] Figure 3 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EC431. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0108] [Table 10]

[0109] [Table 11]

[0110] BLAST homology searches against DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the nucleotide sequence of the ITS region of the rDNA of strain EC431 (SEQ ID NO: 4) showed 95.5–97.9% identity with multiple nucleotide sequences of Ustilago sporoboli-indici, a species of basidiomycete (basidiomycete yeast) (Tables 12, 13). In a molecular phylogenetic tree (Figure 4) analyzed based on the nucleotide sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC431 was included in a phylogenetic group composed of the genus Ustilago and formed a cluster supported by multiple nucleotide sequences of Ustilago sporoboli-indici with a bootstrap value of 99%. However, since there are differences of more than 13 base pairs in the ITS region of the rDNA of SIID35092-03 and Ustilago sporoboli-indici, it is difficult to determine the species-level classification, and it was considered appropriate to classify it as Ustilago sp., which is closely related to Ustilago sporoboli-indici.

[0111] Table 12 shows the BLAST search results for DB-FU, which are the nucleotide sequence analysis data for the ITS regions of the top 30 rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0112] Table 13 shows the BLAST search results for the international nucleotide sequence database, specifically the sequence analysis data for the ITS regions of rDNA that were found to be among the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0113] Figure 4 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of strain EC431. The line in the upper left indicates the scale bar. The numbers at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0114] [Table 12]

[0115] [Table 13]

[0116] Based on the above, the nucleotide sequence analysis of the D1 / D2 region of the 26S rDNA and the ITS region of the rDNA identified strain EC431 as a Ustilago sp. closely related to Ustilago sporoboli-indici.

[0117] The morphological characteristics of the EC431 strain were investigated by observing the characteristics and morphology of the colonies. After culturing on YM agar plates for 4 days, the colonies exhibited a creamy to yellowish-orange color, a smooth to wrinkled surface, a buttery texture, and a moist appearance. Vegetative cells were oval to cylindrical, and proliferation was confirmed to occur via budding from the short stalks at the cell poles. Furthermore, no formation of sexual reproductive organs was observed on the agar plates approximately 3 weeks after the start of culture.

[0118] Based on its molecular phylogenetic position, morphological characteristics, and ergothioneine production, strain EC431 was determined to be a novel microorganism belonging to Ustilago sp., closely related to Ustilago sporoboli-indici.

[0119] (Molecular phylogenetic position and morphological properties of strain EC171) BLAST homology searches against DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EC171 (SEQ ID NO: 5) showed 99.7–100% identity with multiple nucleotide sequences of Dirkmeia churashimaensis, a species of basidiomycete yeast (Tables 14 and 15). In a molecular phylogenetic tree (Figure 5) analyzed based on the nucleotide sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC171 showed the same molecular phylogenetic position as multiple nucleotide sequences of Dirkmeia churashimaensis.

[0120] Table 14 shows the BLAST search results for DB-FU, specifically the nucleotide sequence analysis data for the D1 / D2 regions of 26S rDNAs that were found in the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0121] Table 15 shows the BLAST search results for the international nucleotide sequence database, specifically the D1 / D2 region of 26S rDNA, which were the top 30 search results in terms of homology score. * indicates sequence data used for simplified molecular phylogenetic analysis. In Table 15, " a The sequence indicated by "[...] was not derived from the reference strain, and therefore was excluded from the analysis because it suggested a possible error in the registration information."

[0122] Figure 5 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EC171. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0123] [Table 14]

[0124] [Table 15]

[0125] BLAST homology searches against DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the nucleotide sequence of the ITS region of the rDNA of strain EC171 (SEQ ID NO: 6) showed 98.4–100% identity with multiple nucleotide sequences of Dirkmeia churashimaensis, a type of basidiomycete yeast (Tables 16 and 17). In a molecular phylogenetic tree (Figure 6) analyzed based on the nucleotide sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC171 formed a cluster supported by multiple nucleotide sequences of Dirkmeia churashimaensis with high bootstrap values ​​of 100%.

[0126] Table 16 shows the BLAST search results for DB-FU, which are the nucleotide sequence analysis data for the ITS regions of the top 30 rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0127] Table 17 shows the BLAST search results for the international nucleotide sequence database, specifically the sequence analysis data for the ITS regions of rDNA that were found to be among the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0128] Figure 6 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of strain EC171. The line in the upper left indicates the scale bar. The numbers at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0129] [Table 16]

[0130] [Table 17]

[0131] Based on the above, the EC171 strain was identified as Dirkmeia churashimaensis based on the nucleotide sequence analysis results of the D1 / D2 region of the 26S rDNA and the ITS region of the rDNA.

[0132] The morphological characteristics of the EC171 strain were investigated by observing the characteristics and morphology of the colonies. After culturing on YM agar plates for 4 days, the colonies exhibited yellow-orange to cream-colored, smooth, buttery surfaces, and a moist, viscous texture. Vegetative cells were oval to oval-shaped, and proliferation was confirmed to occur via budding from short stalks at the cell poles. Furthermore, no formation of sexual reproductive organs was observed on the agar plates approximately 3 weeks after the start of culture.

[0133] Based on its molecular phylogenetic position, morphological characteristics, and ergothioneine production, strain EC171 was determined to be a novel microorganism belonging to Dirkmeia churashimaensis.

[0134] (Molecular phylogenetic position and morphological properties of strain EC581) BLAST homology searches using the microbial identification system "ENKI" against DB-FU and international nucleotide sequence databases revealed that the D1 / D2 region of the 26S rDNA of strain EC581 (SEQ ID NO: 7) showed 99.7-100% identity with multiple sequences of Dirkmeia churashimaensis, a species of basidiomycete yeast (Tables 18 and 19). In a molecular phylogenetic tree (Figure 7) analyzed based on the sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC581 showed the same molecular phylogenetic position as multiple sequences of Dirkmeia churashimaensis.

[0135] Table 18 shows the BLAST search results for DB-FU, specifically the nucleotide sequence analysis data for the D1 / D2 regions of 26S rDNAs that were found in the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0136] Table 19 shows the BLAST search results for the international nucleotide sequence database, specifically the D1 / D2 region of 26S rDNA, which were the top 30 search results in terms of homology score. * indicates sequence data used for simplified molecular phylogenetic analysis. In Table 19, " a The sequence indicated by "[...] was not derived from the reference strain, and therefore was excluded from the analysis because it suggested a possible error in the registration information."

[0137] Figure 7 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EC581. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0138] [Table 18]

[0139] [Table 19]

[0140] BLAST homology searches against DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the nucleotide sequence of the ITS region of the rDNA of strain EC581 (SEQ ID NO: 8) showed 98.4–100% identity with multiple nucleotide sequences of Dirkmeia churashimaensis, a type of basidiomycete yeast (Tables 20, 21). In a molecular phylogenetic tree (Figure 8) analyzed based on the nucleotide sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC581 formed a cluster supported by multiple nucleotide sequences of Dirkmeia churashimaensis with high bootstrap values ​​of 100%.

[0141] Table 20 shows the BLAST search results for DB-FU, which are the nucleotide sequence analysis data for the ITS region of the top 30 rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0142] Table 21 shows the BLAST search results for the international nucleotide sequence database, specifically the sequence analysis data for the ITS regions of rDNA that were found to be among the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0143] Figure 8 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of strain EC581. The line in the upper left indicates the scale bar. The numbers at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0144] [Table 20]

[0145] [Table 21]

[0146] Based on the above, the EC581 strain was identified as Dirkmeia churashimaensis based on the nucleotide sequence analysis results of the D1 / D2 region of the 26S rDNA and the ITS region of the rDNA.

[0147] The morphological characteristics of the EC581 strain were investigated by observing the characteristics and morphology of the colonies. After culturing on YM agar plates for 4 days, the colonies exhibited yellow-orange to cream-colored, smooth, buttery surfaces, and a moist, viscous texture. Vegetative cells were oval to oval-shaped, and proliferation was confirmed to occur via budding from short stalks at localized cell locations. Furthermore, no formation of sexual reproductive organs was observed on the agar plates approximately 3 weeks after the start of culture.

[0148] Based on its molecular phylogenetic position, morphological characteristics, and ergothioneine production, strain EC581 was determined to be a novel microorganism belonging to Dirkmeia churashimaensis.

[0149] (Molecular phylogenetic position and morphological properties of strain EC592) BLAST homology searches using the microbial identification system "ENKI" against DB-FU and international nucleotide sequence databases revealed that the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EC592 (SEQ ID NO: 9) showed 99.7-100% identity with multiple nucleotide sequences of Dirkmeia churashimaensis, a species of basidiomycete yeast (Tables 22 and 23). In a molecular phylogenetic tree (Figure 9) analyzed based on the nucleotide sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC592 showed the same molecular phylogenetic position as multiple nucleotide sequences of Dirkmeia churashimaensis.

[0150] Table 22 shows the BLAST search results for DB-FU, specifically the nucleotide sequence analysis data for the D1 / D2 regions of 26S rDNAs that were found in the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0151] Table 23 shows the BLAST search results for the international nucleotide sequence database, specifically the D1 / D2 region of 26S rDNA, which were the top 30 searched based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis. In Table 23, " a The sequence indicated by "[...] was not derived from the reference strain, and therefore was excluded from the analysis because it suggested a possible error in the registration information."

[0152] Figure 9 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EC592. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0153] [Table 22]

[0154] [Table 23]

[0155] BLAST homology searches against DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the nucleotide sequence of the ITS region of the rDNA of strain EC592 (SEQ ID NO: 10) showed 98.4–100% identity with multiple nucleotide sequences of Dirkmeia churashimaensis, a type of basidiomycete yeast (Tables 24, 25). In a molecular phylogenetic tree (Figure 10) analyzed based on the nucleotide sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC592 formed a cluster supported by multiple nucleotide sequences of Dirkmeia churashimaensis with high bootstrap values ​​of 100%.

[0156] Table 24 shows the BLAST search results for DB-FU, which are the nucleotide sequence analysis data for the ITS regions of the top 30 rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0157] Table 25 shows the BLAST search results for the international nucleotide sequence database, specifically the sequence analysis data for the ITS regions of rDNA that were found to be among the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0158] Figure 10 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of strain EC592. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0159] [Table 24]

[0160] [Table 25]

[0161] Based on the above, the EC592 strain was identified as Dirkmeia churashimaensis based on the nucleotide sequence analysis results of the D1 / D2 region of the 26S rDNA and the ITS region of the rDNA.

[0162] The morphological characteristics of the EC592 strain were investigated by observing the characteristics and morphology of the colonies. After culturing on YM agar plates for 4 days, the colonies exhibited yellow-orange to cream-colored, smooth, buttery surfaces, and a moist, viscous texture. Vegetative cells were oval to oval-shaped, and proliferation was confirmed to occur via budding from short stalks at the cell poles. Furthermore, no formation of sexual reproductive organs was observed on the agar plates approximately 3 weeks after the start of culture.

[0163] Based on its molecular phylogenetic position, morphological characteristics, and ergothioneine production, strain EC592 was determined to be a novel microorganism belonging to Dirkmeia churashimaensis.

[0164] (Molecular phylogenetic position and morphological properties of strain EB761) BLAST homology searches using the microbial identification system "ENKI" against DB-FU and international nucleotide sequence databases revealed that the D1 / D2 region of the 26S rDNA of strain EB761 (SEQ ID NO: 11) showed 99.7–100% identity with multiple sequences of Dirkmeia churashimaensis, a species of basidiomycete yeast (Tables 26, 27). In a molecular phylogenetic tree (Figure 11) analyzed based on the sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EB761 showed the same molecular phylogenetic position as multiple sequences of Dirkmeia churashimaensis.

[0165] Table 26 shows the BLAST search results for DB-FU, specifically the D1 / D2 region sequencing data of the top 30 26S rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0166] Table 27 shows the BLAST search results for the international nucleotide sequence database, specifically the D1 / D2 region of 26S rDNA, which were the top 30 searched based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis. In Table 27, " a The sequence indicated by "[...] was not derived from the reference strain, and therefore was excluded from the analysis because it suggested a possible error in the registration information."

[0167] Figure 11 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EB761. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0168] [Table 26]

[0169] [Table 27]

[0170] BLAST homology searches against DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the nucleotide sequence of the ITS region of the rDNA of strain EB761 (SEQ ID NO: 12) showed 98.3–100% identity with multiple nucleotide sequences of Dirkmeia churashimaensis, a type of basidiomycete yeast (Tables 28, 29). In a molecular phylogenetic tree (Figure 12) analyzed based on the nucleotide sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EB761 formed a cluster supported by multiple nucleotide sequences of Dirkmeia churashimaensis with high bootstrap values ​​of 100%.

[0171] Table 28 shows the BLAST search results for DB-FU, which are the nucleotide sequence analysis data for the ITS regions of the top 30 rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0172] Table 29 shows the BLAST search results for the international nucleotide sequence database, specifically the sequence analysis data of the ITS regions of rDNA that were found to be among the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0173] Figure 12 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of strain EB761. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0174] [Table 28]

[0175] [Table 29]

[0176] Based on the above, the EB761 strain was identified as Dirkmeia churashimaensis based on the nucleotide sequence analysis results of the D1 / D2 region of the 26S rDNA and the ITS region of the rDNA.

[0177] The morphological characteristics of the EB761 strain were investigated by observing the characteristics and morphology of the colonies. After two days of culture on YM agar plates, the colonies exhibited yellow-orange to cream-colored, smooth, buttery surfaces, and a moist texture. On the third day of culture, the vegetative cells were oval to oval-shaped, and proliferation was confirmed to occur via budding from the short stalks at the cell poles. Furthermore, no formation of sexual reproductive organs was observed on the agar plates approximately five weeks after the start of culture.

[0178] Based on its molecular phylogenetic position, morphological characteristics, and ergothioneine production, strain EB761 was determined to be a novel microorganism belonging to Dirkmeia churashimaensis.

[0179] (Molecular phylogenetic position and morphological properties of strain EC021) BLAST homology searches against DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the D1 / D2 region of the 26S rDNA of strain EC021 (SEQ ID NO: 13) showed 99.7-100% identity with multiple sequences of Dirkmeia churashimaensis, a species of basidiomycete yeast (Tables 30, 31). In a molecular phylogenetic tree (Figure 13) analyzed based on the sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC021 showed the same molecular phylogenetic position as multiple sequences of Dirkmeia churashimaensis.

[0180] Table 30 shows the BLAST search results for DB-FU, specifically the nucleotide sequence analysis data for the D1 / D2 regions of 26S rDNA, which were the top 30 searched based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0181] Table 31 shows the BLAST search results for the international nucleotide sequence database, specifically the D1 / D2 region of 26S rDNA, which were the top 30 searched based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis. In Table 31, " a The sequence indicated by "[...] was not derived from the reference strain, and therefore was excluded from the analysis because it suggested a possible error in the registration information."

[0182] Figure 13 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the D1 / D2 region of the 26S rDNA of strain EC021. The line in the upper left indicates the scale bar. The numbers located at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0183] [Table 30]

[0184] [Table 31]

[0185] BLAST homology searches against DB-FU and international nucleotide sequence databases using the microbial identification system "ENKI" revealed that the nucleotide sequence of the ITS region of the rDNA of strain EC021 (SEQ ID NO: 14) showed 98.4–100% identity with multiple nucleotide sequences of Dirkmeia churashimaensis, a species of basidiomycete yeast (Tables 32, 33). In a molecular phylogenetic tree (Figure 14) analyzed based on the nucleotide sequences obtained from homology searches against DB-FU and international nucleotide sequence databases, strain EC021 formed a cluster supported by multiple nucleotide sequences of Dirkmeia churashimaensis with high bootstrap values ​​of 100%.

[0186] Table 32 shows the BLAST search results for DB-FU, which are the nucleotide sequence analysis data for the ITS regions of the top 30 rDNAs found based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0187] Table 33 shows the BLAST search results for the international nucleotide sequence database, specifically the sequence analysis data of the ITS regions of rDNA that were found to be among the top 30 based on homology scores. * indicates sequence data used for simplified molecular phylogenetic analysis.

[0188] Figure 14 shows a simplified molecular phylogenetic tree based on the nucleotide sequence of the ITS region of the rDNA of strain EC021. The line in the upper left indicates the scale bar. The numbers at the branching points of the phylogenetic branches indicate the bootstrap values. The "T" at the end of the strain name indicates the type strain of that species.

[0189] [Table 32]

[0190] [Table 33]

[0191] Based on the above, the EC021 strain was identified as Dirkmeia churashimaensis based on the nucleotide sequence analysis results of the D1 / D2 region of the 26S rDNA and the ITS region of the rDNA.

[0192] The morphological characteristics of the EC021 strain were investigated by observing the characteristics and morphology of the colonies. After culturing on YM agar plates for 3 days, the colonies exhibited yellow-orange to cream-colored, smooth, buttery surfaces, and a moist, viscous texture. Vegetative cells were oval to oval-shaped, and proliferation was confirmed to occur via budding from short stalks at the cell sites. Furthermore, no formation of sexual reproductive organs was observed on the agar plates approximately 3 weeks after the start of culture.

[0193] Based on its molecular phylogenetic position, morphological characteristics, and ergothioneine production, strain EC021 was determined to be a novel microorganism belonging to Dirkmeia churashimaensis. [Industrial applicability]

[0194] The microorganisms of this invention have a high production rate of ergothioneine and can be used in fields such as health and beauty. [Accession Number]

[0195] NITE BP-03706 NITE BP-03707 NITE BP-03708 NITE BP-03709 NITE BP-03710 NITE BP-03711 NITE BP-03712

Claims

1. Microorganisms belonging to Dirkmeia churasimaensis (accession numbers: NITE BP-03707, NITE BP-03708, NITE BP-03709, NITE BP-03711, or NITE BP-03712), Aureobasidium melanogenum (accession number: NITE BP-03706), or microorganisms belonging to species closely related to Ustillago sporobori-indisi (Ustillago sp) (accession number: NITE BP-03710).

2. A culture of microorganisms according to claim 1.

3. A method for producing ergothioneine, comprising the step of culturing the microorganism described in claim 1 to obtain a culture containing ergothioneine.

Images