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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/165—Yeast isolates
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/145—Fungi isolates
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/10—Nitrogen as only ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi

Definitions

• the present invention relates to a novel microorganism, a culture or extract of the novel microorganism, and a method for producing ergothioneine.
• Ergothioneine is a type of sulfur-containing amino acid. Ergothioneine has an antioxidant effect superior to that of vitamin E, and is attracting attention as a highly useful compound in the fields of health and beauty.
• Patent Document 1 and Non-Patent Document 1 describe transformed filamentous fungi with enhanced ergothioneine-producing ability.
• Non-Patent Document 2 describes a transformed microorganism of the genus Methylobacrium with enhanced ergothioneine-producing ability.
• Non-Patent Document 2 describes that microorganisms belonging to the genus Aureobasidium and Rhodotorula have the ability to produce ergothioneine.
• Non-Patent Document 3 describes that microorganisms belonging to the genus Pleurotus have the ability to produce ergothioneine.
• Patent Document 2 describes that microorganisms belonging to the genus Methylobactrium and Rhodotorula have the ability to produce ergothioneine.
• Patent Document 3 describes that microorganisms belonging to the genus Moniliella have the ability to produce ergothioneine.
• Patent Document 4 describes that microorganisms belonging to the genera Dirkmeia, Papiliotrema and Apiotrichum have the ability to produce ergothioneine.
• Ergothioneine is not biosynthesized in the human body, and is known to be biosynthesized by some microorganisms. Therefore, as described in the above-mentioned prior art documents, research and development such as searching for microorganisms that produce ergothioneine and modification of microorganisms for enhancing ergothioneine production are underway.
• the present invention has been made in view of the above problems, and aims to provide a novel microorganism that produces a high amount of ergothioneine.
• the present inventors discovered a novel microorganism with a high production of ergothioneine and completed the present invention.
• the microorganism according to one aspect of the present invention is a microorganism belonging to Rhodosporidiobolus azolicus (NITE BP-03572) or a microorganism belonging to Vanrija sp. 03573).
• a method for producing ergothioneine is a method for producing ergothioneine, which includes the step of culturing a microorganism belonging to the genus Rhodosporidiobolus or the genus Wanlya to obtain a culture containing ergothioneine.
• a microorganism with high ergothioneine production can be provided.
• a to B representing a numerical range intends "A or more (including A and greater than A) and B or less (including B and less than B)".
• a microorganism according to an aspect of the present invention is a microorganism belonging to Rhodosporidiobolus, which has the ability to produce ergothioneine, or a microorganism belonging to the genus Wanlya, which has the ability to produce ergothioneine.
• the microorganism according to one aspect of the present invention has a high production amount of ergothioneine.
• Ergothioneine is a kind of sulfur-containing amino acid and has excellent antioxidant action.
• the microorganism according to one aspect of the present invention since it has not been modified by genetic recombination technology or the like, it can also be used in the food industry.
• Rhodospolidiobolus azolicus EB152 Rhodosporidiobolus azolicus EB152 (hereinafter sometimes abbreviated as "yeast EB152”) is a microorganism that was first isolated from plant leaves as an isolation source.
• EB152 was assigned to Rhodosporidiobolus azolicus. In addition, it exhibits physiological and biochemical properties almost similar to those of Rhodosporidiobolus azolicus, except for the assimilation of maltose and water-soluble starch as carbon sources and the requirement for vitamins.
• Yeast EB152 was deposited at the National Institute of Technology and Evaluation (hereinafter abbreviated as "NITE") Patent Microorganism Depositary Center, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture ( NPMD) (original date of deposit: December 15, 2021, accession number: NITE BP-03572).
• NITE National Institute of Technology and Evaluation
• the method of culturing yeast EB152 may be carried out according to the general method of culturing microorganisms belonging to the genus Rhodosporidiobolus.
• the culture form is a batch culture using a liquid medium or a fed-batch culture in which a carbon source and/or an organic nitrogen source is continuously added to the culture system, and aeration and agitation are desirable.
• a medium a medium containing necessary nutrients such as a carbon source, a nitrogen source, or inorganic salts that can be assimilated by a microorganism belonging to the genus Rhodosporidiobolus may be used.
• the culture pH is preferably 3 to 8
• the culture temperature is preferably 20° C. to 30° C.
• the culture time is preferably 2 to 14 days.
• Vanrija sp. EB891 (hereinafter sometimes abbreviated as "yeast EB891") is a microorganism isolated for the first time from a basidiomycete fruiting body.
• Yeast EB891 was deposited at the National Institute of Technology and Evaluation (hereinafter abbreviated as "NITE") Patent Microorganism Depositary Center, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture ( NPMD) (original date of deposit: December 15, 2021, accession number: NITE BP-03573).
• NITE National Institute of Technology and Evaluation
• the method of culturing yeast EB891 may be carried out according to the general method of culturing microorganisms of the genus Wanrya.
• the culture form is a batch culture using a liquid medium or a fed-batch culture in which a carbon source and/or an organic nitrogen source is continuously added to the culture system, and aeration and agitation are desirable.
• a medium a medium containing necessary nutrients such as a carbon source, a nitrogen source, or inorganic salts that can be assimilated by microorganisms belonging to the genus Wanrya may be used.
• the culture pH is preferably 3 to 8
• the culture temperature is preferably 20° C. to 30° C.
• the culture time is preferably 2 to 14 days.
• a culture according to one aspect of the invention is a culture of yeast EB152 or yeast EB891.
• the culture according to one aspect of the present invention includes culture supernatants, culture sediments, culture media, cultured cells, cultured cells crushed products, cultured cells processed products such as freeze-dried cultured cells, and the like.
• a culture according to one aspect of the present invention contains ergothioneine.
• An extract according to one aspect of the invention is an extract of yeast EB152 or yeast EB891.
• microorganism extract refers to those obtained by subjecting microorganisms to extraction treatment, and those obtained by subjecting microorganism cultures to extraction treatment.
• an extract according to one aspect of the present invention is obtained, for example, by extracting yeast EB152 or yeast EB891 or extracting a culture of yeast EB152 or yeast EB891.
• An extract according to one aspect of the present invention contains ergothioneine.
• Extraction treatments include hot water extraction; solvent extraction with organic solvents, etc.; pressurized extraction; chemical extraction with enzymes and surfactants, etc.; extraction by freezing and thawing; extraction by liquid nitrogen; extraction by high-speed stirring;
• the extraction treatment is preferably hot water extraction in order to exhibit an excellent plant growth effect.
• One type of extraction processing may be performed, or two or more types of extraction processing may be performed.
• Hot water extraction is an extraction 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 water used for hot water extraction is preferably 40° C. or higher, more preferably 60° C. or higher.
• the extract according to one aspect of the present invention is a hot water extract, a solvent extract such as with an organic solvent; a pressurized extract; a chemical extract such as with enzymes and surfactants of the microorganism, yeast EB152 or yeast EB891.
• osmotic extraction; comminution extraction; grinding extraction; freeze-thaw extraction; liquid nitrogen extraction; may be
• Applications of the culture or extract according to one aspect of the present invention include a plant growth regulator containing the culture or extract as an active ingredient.
• a method for producing ergothioneine according to an aspect of the present invention includes a step of culturing a microorganism belonging to the genus Rhodosporidiobolus or the genus Wanliya to obtain a culture containing ergothioneine.
• the microorganism belonging to the genus Rhodosporidiobolus is preferably a microorganism belonging to Rhodosporidiobolus azolicus because of the high production of ergothioneine. It is more preferable to have In terms of high ergothioneine production, the microorganism belonging to the genus Wanrya is preferably a microorganism belonging to Wanrya sp., which is a species closely related to Wanrya Humicola, and more preferably yeast EB891.
• the recovery of ergothioneine from a culture containing ergothioneine may be performed, for example, by a method of recovering and purifying ergothioneine from a normal microbial culture.
• the culture is centrifuged to recover the cells.
• an extract containing ergothioneine is obtained by subjecting the collected cells to hot water extraction or the like.
• Ergothioneine can be recovered by purifying the extract.
• the amount of ergothioneine produced by a microorganism can be quantified, for example, by measuring the obtained extract with a high-performance liquid chromatography device and a mass spectrometer such as LCMS.
• the microorganism according to aspect 1 of the present invention is a microorganism belonging to Rhodosporidiobolus azolicus (NITE BP-03572) or a microorganism belonging to Wanrya sp. is.
• the culture according to aspect 2 of the present invention is the culture of the microorganism.
• the extract according to aspect 3 of the present invention is an extract of the microorganism.
• a method for producing ergothioneine according to aspect 4 of the present invention includes the step of culturing a microorganism belonging to the genus Rhodosporidiobolus or the genus Wanlya to obtain a culture containing ergothioneine.
• the microorganism belonging to the genus Rhodosporidiobolus may be Rhodosporidiobolus azolicus.
• the microorganism belonging to the genus Wanrya may be Wanrya sp.
• the microorganism belonging to the genus Rhodosporidiobolus is a microorganism belonging to Rhodosporidiobolus azolicus (NITE BP-03572) There may be.
• the microorganism belonging to the genus Wanrya belongs to the species closely related to Wanrya Humicola (NITE BP -03573).
• % represents % by mass.
• each of the collected samples 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.
• YM medium containing antibiotics was used as the screening medium. Specifically, a medium containing 1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, 0.01% streptomycin sulfate, and 0.005% chloramphenicol is used. bottom.
• the colonies grown on the agar medium of the selected 112 samples were visually observed for morphology and color, and 181 yeast-like colonies of different types (106 in the first run, 75 in the second run) were selected. .
• LCMS-2020 manufactured by Shimadzu Corporation was used.
• a mixture of 10 mM ammonium formate and acetonitrile (10 mM ammonium formate/acetonitrile 30/70 (v/v)) was used as the LC mobile phase.
• the flow rate was set to 0.1 mL/min, and the analysis was performed at 25°C.
• MS detection ionization was performed in the DUIS mode in which ESI ionization and APCI ionization were performed simultaneously.
• the culture solution was collected as appropriate on days 3 to 7 of culture. After centrifuging and washing the cells in the same manner as in (3) above, the extract was collected by hot water extraction.
• the resulting extract was analyzed by LCMS in the same manner as in (4) above, and two strains (EB152, EB891) with high ergothioneine production were selected.
• Table 1 shows the ergothioneine production amount and production rate of the two selected colonies.
• Table 2 shows the production amounts and production rates of known microorganisms.
• the unit of ergothioneine (EGT) production is mg/L
• the EGT production rate is mg/L/d (ergothioneine production per day).
• the EGT production amount in Table 1 indicates the ergothioneine production amount on the fifth day of culture.
• the extract was obtained by aerobically culturing the microorganism at 25°C for 5 days using a 5 L jar fermenter containing 2 L of YM medium. It was obtained by hot water extraction from dried cells. Then, the amount of EGT in the extract was measured by LCMS.
• the taxonomic groups belonging to the two selected strains were estimated by analyzing the base sequences of the D1/D2 region and the ITS region of the 26S rDNA of the ribosomal RNA gene.
• the EB152 strain was cultured on a YM agar plate medium at 25°C for 3 days, and colonies formed were observed.
• the shape of the rim of the colony was whole, and the raised state was cushion-shaped.
• the shape of the colony surface was smooth.
• the colonies were buttery and moist, and pink to light orange in color.
• strain EB152 was determined to be a novel microorganism belonging to Rhodosporidiobolus azolicus.
• the ITS-5.8 rDNA base sequence of strain EB891 was subjected to BLAST homology search against the international base sequence database. As a result, it showed 98.5 to 100% identity to multiple base sequences of Vanrija humicola, a kind of basidiomycete yeast.
• the EB891 strain is included in the phylogenetic group composed of the genus Wanrya, with multiple base sequences of Wanrya Humicola and a high bootstrap value of 99%. Formed a supported cluster, indicating the possibility of belonging to Wanrya Humicola.
• the EB891 strain was cultured on a YM agar plate medium at 25°C for 3 days, and colonies formed were observed.
• the shape of the periphery of the colony was whole, and the raised state was flat to cushion-like.
• the shape of the surface of the colony was smooth to slightly rough.
• the colonies were buttery, moist, and white to cream colored.
• the cell morphology was also observed.
• the vegetative cells were oval to clavate-shaped, and proliferation was observed to be by budding. No formation of sexual reproductive organs was observed on the plates after 6 weeks or more of culture.
• the EB891 strain was determined to be a novel microorganism belonging to Wanrya Humicola or a species closely related to it.
• the microorganism of the present invention has a high production of ergothioneine and can be used in fields such as health and beauty.

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• 2022
  • 2022-10-31 WO PCT/JP2022/040698 patent/WO2023132122A1/ja not_active Ceased
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