Novel microorganisms, cultures or extracts thereof, and methods for producing ergothioneine

Abstract

This invention relates to a microorganism (NITE BP-03572) belonging to Rhodosporidiobolus azoricus or a microorganism (NITE BP-03573) belonging to the Vanrija genus.

Description

[Technical Field]

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

[0002] Ergothioneine is a sulfur-containing amino acid that has antioxidant properties superior to those of vitamin E, and is attracting attention as a valuable compound in the fields of 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 a transformed microorganism of the genus Methylobacrium with enhanced ergothioneine-producing ability, and also describes that microorganisms of the genera Aureobasidium and Rhodotorula have ergothioneine-producing ability.

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

[0006] Patent Document 2 describes that microorganisms of the genera Methylobacterium and Rhodotorula are capable of producing ergothioneine. Patent Document 3 describes that microorganisms of the genus Moniliella are capable of producing ergothioneine. Patent Document 4 describes that microorganisms of the genera Dirkmeia, Papiliotrema, and Apiotrichum are capable of producing ergothioneine. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2016 / 121285 [Patent Document 2] International Publication No. 2016 / 121285 [Patent Document 3] International Publication No. 2019 / 004234 [Patent Document 4] International Publication No. 2021 / 140693 [Non-patent literature]

[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) Summary of the Invention [Problem to be solved by the invention]

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

[0010] Using genetic engineering techniques, microorganisms can be modified to enhance ergothioneine production. However, the ergothioneine produced by these microorganisms cannot be used in the food industry, etc. Therefore, there is a strong demand for unmodified, non-genetically modified microorganisms that produce high amounts of ergothioneine.

[0011] The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel microorganism that produces ergothioneine in high amounts. [Means for solving the problem]

[0012] As a result of screening, the present inventors discovered a novel microorganism that produces ergothioneine in high amounts, and thus completed the present invention.

[0013] A microorganism according to one embodiment of the present invention is a microorganism belonging to Rhodosporidiobolus azolicus (NITE BP-03572) or a microorganism belonging to Vanrija sp. (NITE BP-03573), which is a species closely related to Vanrija humicola.

[0014] Furthermore, a method for producing ergothioneine according to one embodiment of the present invention is a method for producing ergothioneine, comprising the steps of culturing a microorganism belonging to the genus Rhodosporidiobolus or Wanria to obtain a culture containing ergothioneine. [Effects of the Invention]

[0015] According to one aspect of the present invention, a microorganism capable of producing ergothioneine in high amounts can be provided. DETAILED DESCRIPTION OF THE INVENTION

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

[0017] [Novel microorganisms] A microorganism according to one embodiment of the present invention is a microorganism belonging to the genus Rhodosporidiobolus that has the ability to produce ergothioneine, or a microorganism belonging to the genus Wanria that has the ability to produce ergothioneine.

[0018] A microorganism according to one embodiment of the present invention produces a high amount of ergothioneine. Ergothioneine is a sulfur-containing amino acid with excellent antioxidant properties. Furthermore, since the microorganism according to one embodiment of the present invention has not been modified by genetic recombination technology or the like, it can also be used in the food industry.

[0019] (1. Rhodosporidiobolus azolicus EB152) Rhodosporidiobolus azoricus EB152 (hereinafter sometimes abbreviated as "yeast EB152") is the first microorganism isolated from plant leaves as an isolation source.

[0020] The nucleotide sequences of the D1 / D2 and ITS regions of the 26S rDNA ribosomal RNA gene were determined. A BLAST homology search was performed against the TechnoSuruga Laboratory Microbial Identification System (TechnoSuruga Laboratory, Japan) database DB-FU13.0 and the international nucleotide sequence databases (DDBJ / ENA (EMBL) / GenBank). As a result, EB152 was assigned to Rhodosporidiobolus azolicus. Furthermore, EB152 exhibits physiological and biochemical properties similar to those of Rhodosporidiobolus azolicus, except for its ability to utilize maltose and soluble starch as carbon sources and its vitamin requirement.

[0021] Yeast EB152 was deposited at the National Patent Microorganism Depositary (NPMD) of the National Institute of Technology and Evaluation (hereinafter abbreviated as “NITE”), Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture (original deposit date: December 15, 2021, accession number: NITE BP-03572).

[0022] Yeast EB152 may be cultured according to a general culture method used for microorganisms of the genus Rhodosporidiobolus. The culture method is batch culture using a liquid medium or fed-batch culture in which a carbon source and / or an organic nitrogen source is continuously added to the culture system, preferably with aeration and agitation. The medium may contain a carbon source, a nitrogen source, or a necessary nutrient source such as inorganic salts that can be assimilated by microorganisms belonging to the genus Rhodosporidiobolus. The culture pH is preferably 3 to 8, the culture temperature is preferably 20 to 30°C, and the culture time is preferably 2 to 14 days.

[0023] (2. Wanriya SP EB891) Vanrija sp. EB891 (hereinafter sometimes abbreviated as "yeast EB891") is the first microorganism isolated from a basidiomycete fruiting body.

[0024] The nucleotide sequences of the D1 / D2 and ITS regions of the 26S rDNA ribosomal RNA gene were determined. A BLAST homology search was performed against the TechnoSuruga Laboratory Microbial Identification System (TechnoSuruga Laboratory, Japan) database DB-FU13.0 and the international sequence databases (DDBJ / ENA (EMBL) / GenBank). The results indicated that EB891 is closely related to Vanrija humicola. Furthermore, EB891 exhibits physiological and biochemical properties similar to Vanrija humicola, except for differences in its ability to utilize inulin and soluble starch as carbon sources, its ability to utilize nitrate as a nitrogen source, its vitamin requirements, and its growth in 50% D-glucose and 10% NaCL / 5% glucose.

[0025] Yeast EB891 was deposited at the National Patent Microorganism Depositary (NPMD) of the National Institute of Technology and Evaluation (hereinafter abbreviated as “NITE”), Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (original deposit date: December 15, 2021, accession number: NITE BP-03573).

[0026] Yeast EB891 can be cultured according to general culture methods used for microorganisms of the genus Wanria. The culture method is batch culture using a liquid medium or fed-batch culture in which a carbon source and / or an organic nitrogen source is continuously added to the culture system, preferably with aeration and agitation. The culture medium may contain a carbon source, a nitrogen source, or a necessary nutrient source such as inorganic salts that can be assimilated by microorganisms belonging to the genus Wanria. The culture pH is preferably 3 to 8, the culture temperature is preferably 20 to 30°C, and the culture time is preferably 2 to 14 days.

[0027] [Culture] A culture according to one embodiment of the present invention is a culture of yeast EB152 or yeast EB891. The culture according to one embodiment of the present invention includes a culture supernatant, a culture precipitate, a medium, cultured bacterial cells, a cultured bacterial cell lysate, a processed cultured bacterial cell product such as a lyophilized cultured bacterial cell product, and the like. The culture according to one embodiment of the present invention contains ergothioneine.

[0028] [Extract] An extract according to one embodiment of the present invention is an extract of yeast EB152 or yeast EB891. As used herein, the term "microorganism extract" refers to an extract obtained by subjecting a microorganism to an extraction treatment, or an extract obtained by subjecting a culture of a microorganism to an extraction treatment. Thus, an extract according to one embodiment of the present invention can be obtained, for example, by subjecting yeast EB152 or yeast EB891 to an extraction treatment, or by subjecting a culture of yeast EB152 or yeast EB891 to an extraction treatment. An extract according to one embodiment of the present invention contains ergothioneine.

[0029] Examples of extraction methods include hot water extraction, solvent extraction using organic solvents, etc., pressure extraction, chemical extraction using enzymes and surfactants, ultrasonic extraction, alkaline extraction, acid extraction, extraction using osmotic pressure, extraction by pulverization, extraction by grinding, extraction by freezing and thawing, extraction using liquid nitrogen, and extraction by high-speed stirring. Hot water extraction is preferred as the extraction method, as it exhibits excellent plant growth promoting effects. One type of extraction method may be used, or two or more types of extraction methods may be used.

[0030] Hot water extraction involves contacting or immersing the material to be extracted 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, more preferably 60°C or higher.

[0031] The extract according to one embodiment of the present invention may be a hot water extract, a solvent extract using an organic solvent or the like; a pressurized extract; a chemical extract using an enzyme, a surfactant or the like; an ultrasonic extract; an alkaline extract; an acid extract; an extract using osmotic pressure; an extract by crushing; an extract by grinding; an extract by freezing and thawing; an extract using liquid nitrogen; or an extract by high-speed stirring, of a microorganism that is yeast EB152 or yeast EB891.

[0032] The culture or extract according to one embodiment of the present invention can be used as a plant growth regulator containing the culture or extract as an active ingredient.

[0033] [Method for producing ergothioneine] A method for producing ergothioneine according to one embodiment of the present invention includes a step of culturing a microorganism belonging to the genus Rhodosporidiobolus or Wanria to obtain a culture containing ergothioneine.

[0034] In the method for producing ergothioneine according to one embodiment of the present invention, the microorganism belonging to the genus Rhodosporidiobolus is preferably a microorganism belonging to Rhodosporidiobolus azolicus, and more preferably yeast EB152, in terms of high ergothioneine production.Furthermore, the microorganism belonging to the genus Wanria is preferably a microorganism belonging to Wanria sp., a species closely related to Wanria humicola, and more preferably yeast EB891, in terms of high ergothioneine production.

[0035] Ergothioneine can be recovered from a culture containing ergothioneine by, for example, a method for recovering and purifying ergothioneine from a conventional microbial culture. For example, the culture is centrifuged or the like to recover the bacterial cells. The recovered bacterial cells are then subjected to hot water extraction or the like to obtain an extract containing ergothioneine. Ergothioneine can then be recovered by purifying the 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 device and a mass spectrometer such as LCMS.

[0036] 〔summary〕 The microorganism according to the first aspect of the present invention is a microorganism belonging to Rhodosporidiobolus azolicus (NITE BP-03572) or a microorganism belonging to Wanliya sp. (NITE BP-03573), which is a species closely related to Wanliya humicola.

[0037] The culture according to the second aspect of the present invention is a culture of the microorganism.

[0038] The extract according to aspect 3 of the present invention is an extract of the microorganism.

[0039] A method for producing ergothioneine according to a fourth aspect of the present invention includes the step of culturing a microorganism belonging to the genus Rhodosporidiobolus or Wanria to obtain a culture containing ergothioneine.

[0040] A fifth aspect of the present invention provides a method for producing ergothioneine in the fourth aspect, wherein the microorganism belonging to the genus Rhodosporidiobolus is Rhodosporidiobolus azolicus.

[0041] A sixth aspect of the present invention provides a method for producing ergothioneine in the fourth or fifth aspect, wherein the microorganism belonging to the genus Wanria is Wanria sp., which belongs to a species closely related to Wanria humicola.

[0042] A seventh aspect of the present invention relates to a method for producing ergothioneine in any one of the above-mentioned fourth to sixth aspects, wherein the microorganism belonging to the genus Rhodosporidiobolus is a microorganism belonging to Rhodosporidiobolus azolicus (NITE BP-03572).

[0043] A method for producing ergothioneine according to an eighth aspect of the present invention may be such that, in any one of the above-mentioned fourth to seventh aspects, the microorganism belonging to the genus Wanria is a microorganism belonging to Wanria sp. (NITE BP-03573), which is a species closely related to Wanria humicola.

[0044] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents cited in this specification are incorporated by reference. [Example]

[0045] In the following examples, % means % by mass unless otherwise specified.

[0046] (1) Enrichment culture of isolated strains collected from the environment First, microbial samples were collected from plants, soil, and other environmental sources in two separate batches, resulting in a total of 150 samples (90 in the first batch and 60 in the second batch).

[0047] Next, each sample was immersed in a 15 mL plastic tube containing 2 mL of screening medium and cultured at 200 rpm for 3 to 5 days at 25°C. 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.

[0048] Then, 126 samples (70 in the first run, 56 in the second run) in which the medium was visually cloudy (microorganisms had proliferated) were selected.

[0049] (2) Sample selection by oxidative stress The culture fluids of the 126 samples selected in (1) above were diluted 100-fold or 100,000-fold with YM medium. The diluted culture fluids were then spread onto YM agar medium and YM agar medium supplemented with 3 mM HO (hereinafter referred to as HO-containing YM agar medium), and cultured at 25°C for 2 to 5 days.

[0050] The number of colonies growing on YM agar medium and on YM agar medium containing HO was counted, and 112 samples that showed colonies growing on both YM agar medium and YM agar medium containing HO were selected.

[0051] Furthermore, the morphology and color of the colonies grown on the agar medium of the 112 selected samples were visually observed, and 181 yeast-like colonies of different types (106 in the first round, 75 in the second round) were selected.

[0052] (3) Culture of selected colonies in 96-well plates 181 colonies selected in (2) above were inoculated into a 96-well plate containing 1 mL of YM medium and cultured at 1600 rpm at 25°C for 3 to 4 days. After culture, the collected culture solution 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 centrifuged again.

[0053] 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 centrifuged to remove the bacterial cell residue, yielding an extract.

[0054] (4) Quantitative analysis of ergothioneine in extracts by LCMS A solution obtained by mixing 0.15 mL of the extract obtained in (3) above with 0.35 mL of acetonitrile was filtered through a 0.45 μm PVDF filter, and the obtained filtrate was used as a sample for LCMS measurement.

[0055] The LCMS analysis was performed using an LCMS-2020 manufactured by Shimadzu Corporation. The LC column used was an Asahipak NH2P-40 2D+ guard column manufactured by SHODEX. The LC mobile phase used was a mixture of 10 mM ammonium formate and acetonitrile (10 mM ammonium formate / acetonitrile = 30 / 70 (v / v)). The flow rate was 0.1 mL / min, and the analysis was performed at 25°C.

[0056] For MS detection, ionization was performed in DUIS mode, which simultaneously performs ESI and APCI ionization, and in SIM mode at m / z = 230(+), which allows the detection of ergothioneine.

[0057] Extracts from 181 colonies selected in (2) above were analyzed, and 22 colonies (7 in the first round and 15 in the second round) with high ergothioneine production were selected.

[0058] (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 200 rpm at 25° C. for 7 days (n=1).

[0059] The culture medium was appropriately collected on days 3 to 7 of the culture. As in (3) above, the cells were centrifuged and washed, and then the extract was collected by hot water extraction.

[0060] The obtained extract was analyzed by LCMS in the same manner as in (4) above, and two strains (EB152 and EB891) with high ergothioneine production were selected.

[0061] Colonies of the two selected strains were inoculated into 50 mL of YM medium in a 300 mL flask and cultured at 200 rpm for 5 days at 25°C (n=3). The amount of ergothioneine produced on the 5th day was measured by LCMS.

[0062] Table 1 shows the production amount and production rate of ergothioneine in colonies of the two selected strains. Table 2 shows the production amount and production rate of known microorganisms. In Tables 1 and 2, unless otherwise specified, the unit of ergothioneine (EGT) production is mg / L, and the EGT production rate is mg / L / d (ergothioneine production amount per day). Furthermore, the EGT production amount in Table 1 indicates the ergothioneine production amount on the fifth day of culture.

[0063] Regarding the "EGT content in the extract" in Table 1, the extract was obtained by aerobically culturing the microorganism in question for 5 days at 25°C in a 5L jar fermenter containing 2L of YM medium, and then extracting the dried cells with hot water. The EGT content in the extract was then measured by LCMS.

[0064] [Table 1]

[0065] [Table 2]

[0066] Tables 1 and 2 show that the ergothioneine production amounts of the two selected strains were equal to or greater than those of known ergothioneine-producing microorganisms. It was also found that the ergothioneine production rates of the two selected strains were equal to or greater than those of known ergothioneine-producing microorganisms. It was also confirmed that extracts from the two selected strains contained abundant ergothioneine.

[0067] (6) Identification of the two selected strains The taxonomic group of the two selected strains was estimated by analyzing the base sequences of the D1 / D2 and ITS regions of the 26S rDNA ribosomal RNA gene.

[0068] (7) Molecular phylogenetic position and physiological properties of strain EB152 A BLAST homology search was performed on the 26S rDNA D1 / D2 region nucleotide sequence and ITS-5.8 rDNA nucleotide sequence of strain EB152 against the International Nucleotide Sequence Database. The results showed 98.4-100% identity with multiple nucleotide sequences of Rhodosporidiobolus azolicus, a type of basidiomycete yeast. Furthermore, in a molecular phylogenetic tree analyzed based on the obtained nucleotide sequences, strain EB152 was included in the phylogenetic group consisting of the genus Rhodosporidiobolus. Furthermore, Rhodosporidiobolus azolicus JCM11251 T It showed the same molecular phylogenetic position as

[0069] The EB152 strain was cultured on a YM agar plate at 25°C for 3 days, and the colonies formed were observed. The colonies had entire margins and were cushion-shaped. The colony surfaces were smooth. The colonies were buttery and moist, and pink to pale orange in color.

[0070] In addition, the EB152 strain was cultured on YM agar plates at 25°C for 3 days, and then its cell morphology was observed. The vegetative cells were elliptical to oval, and proliferation was confirmed to be by budding. No sexual reproductive organ formation was observed on plates cultured for more than 6 weeks.

[0071] The morphological characteristics of strain EB152 described above were largely consistent with those of R. azolicus assigned by DNA sequence analysis of the D1 / D2 and ITS regions. The physiological characteristics of strain EB152 are shown in Table 3.

[0072] In Table 3, "+" indicates a positive result. "-" indicates a negative result. "W" indicates a weak positive result. "D" indicates that the result gradually became positive over a period of one week or more after the start of the test, and "L" indicates that the result rapidly became positive two weeks or more after the start of the test.

[0073] [Table 3]

[0074] Based on its molecular phylogenetic position, physiological properties, and ergothioneine production, strain EB152 was determined to be a novel microorganism belonging to Rhodosporidiobolus azolicus.

[0075] (8) Molecular phylogenetic position and physiological properties of strain EB891 A BLAST homology search was performed on the 26S rDNA D1 / D2 region of strain EB891 against the international sequence database. The results showed 99.0-99.8% identity with multiple sequences from Vanrija humicola, a basidiomycete yeast. Furthermore, a molecular phylogenetic tree analyzed based on the obtained sequences showed that strain EB891 is included in the phylogenetic group consisting of the genus Vanrija, and Vanrija humicola CBS571 TThe EB891 strain clustered with Vanrija humicola, indicating its close relationship to the genus Vanrija. Furthermore, a BLAST homology search was performed on the ITS-5.8 rDNA sequence of the EB891 strain against the International Nucleotide Sequence Database. The results showed 98.5-100% identity with multiple sequences from Vanrija humicola, a basidiomycete yeast. Furthermore, a molecular phylogenetic tree analyzed based on the resulting sequences showed that the EB891 strain was included in the phylogenetic group consisting of Vanrija. It clustered with multiple Vanrija humicola sequences, supported by a high bootstrap value of 99%, indicating its possible affiliation with Vanrija humicola.

[0076] The EB891 strain was cultured on a YM agar plate at 25°C for 3 days, and the colonies formed were observed. The colonies had entire margins and were flat to cushion-like in elevation. The colony surfaces ranged from smooth to slightly rough. The colonies were buttery and moist, and white to cream in color.

[0077] In addition, the EB891 strain was cultured on YM agar plates at 25°C for 3 days, and then its cell morphology was observed. The vegetative cells were oval to club-shaped, and proliferation was confirmed to be by budding. No sexual reproductive organ formation was observed on plates cultured for more than 6 weeks.

[0078] The morphological characteristics of strain EB891 described above were largely consistent with those of Wanliya humicola assigned by DNA sequence analysis of the D1 / D2 and ITS regions. The physiological properties of strain EB891 are shown in Table 3.

[0079] In Table 4, "+" indicates a positive result. "-" indicates a negative result. "W" indicates a weak positive result. "D" indicates that the result gradually became positive over a period of one week or more after the start of the test, and "L" indicates that the result rapidly became positive two weeks or more after the start of the test.

[0080] [Table 4]

[0081] Based on its molecular phylogenetic position, physiological properties, and ergothioneine production, strain EB891 was determined to be a novel microorganism belonging to Wanliya humicola or a closely related species.

[0082] Nucleotide sequence analysis suggested that strain EB152 belonged to Rhodosporidiobolus azolicus, and strain EB891 to Wanliya humicola or a closely related species. [Industrial Applicability]

[0083] The microorganism of the present invention is a high producer of ergothioneine and can be used in fields such as health and beauty. [Accession number]

[0084] NITE BP-03572 NITE BP-03573

Claims

1. A microorganism belonging to Rhodosporidiobolus azolicus deposited under accession number NITE BP-03572 or a microorganism belonging to the genus Wanlia deposited under accession number NITE BP-03573.

2. A culture of the microorganism of claim 1.

3. A method for producing ergothioneine, comprising the step of culturing a microorganism belonging to the genus Rhodosporidiobolus or Wanria to obtain a culture containing ergothioneine.

4. 4. The method for producing ergothioneine according to claim 3, wherein the microorganism belonging to the genus Rhodosporidiobolus is Rhodosporidiobolus azolicus.

5. 4. The method for producing ergothioneine according to claim 3, wherein the base sequence of the D1 / D2 region of 26S rDNA of the microorganism belonging to the genus Wanria has an identity of 99% or more with the base sequence of the D1 / D2 region of 26S rDNA of Wanria humicola.

6. 4. The method for producing ergothioneine according to claim 3, wherein the microorganism belonging to the genus Rhodosporidiobolus is a microorganism belonging to Rhodosporidiobolus azolicus deposited under accession number NITE BP-03572.

7. The method for producing ergothioneine according to claim 3, wherein the microorganism belonging to the genus Wanria is a microorganism deposited under accession number NITE BP-03573.

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