WO2023234307A1 - エルゴチオネイン生産性を高めるように改変された核酸、並びに遺伝子改変された微生物 - Google Patents

エルゴチオネイン生産性を高めるように改変された核酸、並びに遺伝子改変された微生物 Download PDF

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WO2023234307A1
WO2023234307A1 PCT/JP2023/020131 JP2023020131W WO2023234307A1 WO 2023234307 A1 WO2023234307 A1 WO 2023234307A1 JP 2023020131 W JP2023020131 W JP 2023020131W WO 2023234307 A1 WO2023234307 A1 WO 2023234307A1
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seq
positions
region
amino acid
ergothioneine
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French (fr)
Japanese (ja)
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遼子 田嶋
惠一 市川
考太郎 伊藤
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Kikkoman Corp
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Kikkoman Corp
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Priority to KR1020247039586A priority Critical patent/KR20250007610A/ko
Priority to CN202380043295.8A priority patent/CN119343451A/zh
Priority to JP2024524880A priority patent/JPWO2023234307A1/ja
Priority to EP23816064.2A priority patent/EP4534673A1/en
Publication of WO2023234307A1 publication Critical patent/WO2023234307A1/ja
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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

Definitions

  • the present disclosure relates to a nucleic acid modified to increase ergothioneine (hereinafter sometimes referred to as ERG) productivity, and a microorganism genetically modified to increase ERG productivity.
  • ERG ergothioneine
  • the present invention also relates to a method for producing ERG using genetically modified microorganisms.
  • Ergothioneine was discovered as a sulfur-containing amino acid isolated from Claviceps purpurea, and has also been found to exist in living organisms of plants and animals. However, plants and animals cannot synthesize ergothioneine, and ergothioneine in living organisms is thought to be derived from ergothioneine synthesized by microorganisms such as basidiomycetes. In particular, it is also contained in some edible mushrooms of the Basidiomycete family, such as oyster mushrooms, shiitake mushrooms, maitake mushrooms, and eryngii mushrooms, and is known to be particularly contained in large amounts in Tamogitake mushrooms.
  • Ergothioneine is known to have a biosynthetic pathway from histidine, and the sulfur atom is supplied from cysteine. Ergothioneine has high antioxidant properties, and has been reported to have elastase and tyrosinase inhibitory effects, and is attracting particular attention in the beauty and food fields such as skin whitening and wrinkle prevention. Furthermore, it has been found that ergothioneine is involved in the biological oxidative defense system, and attempts are being made to apply it in the medical field. Ergothioneine has high thermal stability and pH stability, and can maintain its antioxidant effect even at high temperatures.In addition to being added to foods for its physiological effects, it is also expected to be used in foods as an antioxidant. has been done.
  • Methods for producing ergothioneine include extraction from basidiomycetes such as Tamogitake, chemical synthesis, and fermentation using microorganisms. Extraction from basidiomycetes such as Tamogitake takes time to obtain raw materials and is not suitable for mass production. Although chemical synthesis is suitable for mass production, it requires the use of expensive synthetic reagents and also has the problem of high purification costs (Patent Document 1). Therefore, fermentation using bacteria or yeast capable of converting C1 compounds (Patent Document 2, Non-Patent Document 1), and fermentation using a microorganism overexpressing the ergothioneine biosynthetic gene (Patent Document 3) are methods of production. It is a promising method in that it can be used as a microbial fermentation product as it is, or as an extract obtained by a simple extraction procedure.
  • the purpose is to increase ERG productivity in ERG production by microbial culture.
  • the present inventors conducted intensive research with the aim of increasing the ERG productivity of Aspergillus aspergillus and found that ergothioneine productivity was enhanced by introducing a specific genetic mutation into a gene whose function is unknown. This led to the present invention.
  • the invention therefore relates to: [1] In the sequence corresponding to SEQ ID NO: 1, which includes the conserved region included in SEQ ID NO: 1, the function of the functional domain is added to the region included in the functional domain corresponding to the functional domain W404 to Q515 of SEQ ID NO: 1. or a nucleic acid encoding a sequence having at least 90% identity to said sequence, which enhances ergothioneine production when introduced into an ergothioneine-producing microorganism.
  • nucleic acid according to item 1 wherein the conserved region is the conserved region at positions 433 to 440 of SEQ ID NO: 1.
  • the mutation that modulates the function of the functional domain is a deletion of a region included in a functional domain corresponding to functional domains W404 to Q515.
  • the nucleic acid according to item 1, wherein the deleted region includes a position corresponding to position 428 of SEQ ID NO: 1.
  • the nucleic acid according to item 1, wherein the deleted region consists of a deletion of 1 to 120 amino acids.
  • the deleted region is at least one region selected from the group consisting of positions 427 to 429, positions 427 to 440, positions 427 to 444, and positions 419 to 444 of SEQ ID NO: 1.
  • the nucleic acid according to item 4 which is a corresponding region.
  • the deleted region corresponds to at least one region selected from the group consisting of positions 431 to 475, positions 419 to 475, and positions 404 to 515 of SEQ ID NO: 1.
  • the nucleic acid according to item 1 wherein the mutation that modulates the function of the functional domain has a mutation in G at the position corresponding to position 428 and/or C at the position corresponding to position 459 of SEQ ID NO: 1.
  • nucleic acid according to item 1 wherein the nucleic acid is derived from a microorganism selected from the group consisting of the genus Aspergillus, the genus Penicillium, the genus Rasamsonia, and the genus Talaromysis.
  • a vector comprising the nucleic acid according to any one of items 1 to 9.
  • a method for producing ergothioneine which comprises culturing the microorganism according to item 11.
  • a method for producing a microorganism with enhanced ergothioneine-producing ability which comprises the steps of W404 to Q515 of SEQ ID NO: 1 in the sequence corresponding to SEQ ID NO: 1, including the conserved region from positions 433 to 440 of SEQ ID NO: 1.
  • the method comprises introducing a mutation that modulates the function of the functional domain into a region included in the functional domain corresponding to the functional domain, and the microorganism is selected from the group consisting of the genus Aspergillus, the genus Penicillium, the genus Rasamsonia, and the genus Talaromysis.
  • the above-mentioned production method which is an ergothioneine-producing microorganism.
  • the production method according to item 13, wherein the mutation that modulates the function of the functional domain is a deletion of a region included in a functional domain corresponding to the functional domains W404 to Q515.
  • the conserved region is the conserved region at positions 433 to 440 of SEQ ID NO: 1.
  • the production method according to item 14, wherein the deleted region includes a position corresponding to position 428 of SEQ ID NO: 1.
  • the deleted region consists of 1 to 120 amino acids.
  • the deleted region is at least one selected from the group consisting of positions 428, 427 to 429, 427 to 440, 427 to 444, and 419 to 444 of SEQ ID NO: 1.
  • the manufacturing method according to item 16 wherein the region corresponds to the region.
  • the deleted region corresponds to at least one region selected from the group consisting of positions 431 to 475, positions 419 to 475, and positions 404 to 515 of SEQ ID NO: 1.
  • the manufacturing method according to item 14. [20]
  • the production method according to item 14, wherein the mutation that modulates the function of the functional domain has a mutation in G at the position corresponding to position 428 and/or C at the position corresponding to position 459 of SEQ ID NO: 1. .
  • a microorganism containing a mutation that modulates the function of the functional domain in the region included in the functional domain corresponding to the functional domains W404 to Q515 of SEQ ID NO: 1 produces ERG. can be promoted.
  • FIG. 1 shows the amino acid sequence of AO090005000664 (SEQ ID NO: 1) (A) and the base sequence of AO090005000664 (SEQ ID NO: 2) (B).
  • the conserved region from positions 411 to 467 is shown in color.
  • FIG. 2 shows a diagram comparing the conserved region (positions 411 to 479 of SEQ ID NO: 1:) of the amino acid sequence of AO090005000664 with orthologous genes of the genus Aspergillus, Penicillium, Rasamsonia, and Talaromysis.
  • FIG. 3 shows the amount of ERG produced in a mutant strain in which the G428S mutation and C459F mutation were introduced in Aspergillus oryzae AO090005000664.
  • FIG. 1 shows the amino acid sequence of AO090005000664 (SEQ ID NO: 1) (A) and the base sequence of AO090005000664 (SEQ ID NO: 2) (B).
  • FIG. 4 shows the ERG production amount in a mutant strain in which a mutation corresponding to G428S of SEQ ID NO: 1 was introduced in the AO090005000664 ortholog gene of Aspergillus sojae.
  • FIG. 5 shows the ERG production amount in a mutant strain in which a mutation (G442S) corresponding to G428S was introduced in the AO090005000664 ortholog gene (An16g07990) of Aspergillus niger.
  • FIG. 6 shows the results of BLAST analysis of the amino acid sequence of AO090005000664. Ranks 412 to 462 are further enlarged.
  • Figure 7 shows the region including position 428 (deletion from position 427 to 429 (3DEL), deletion from position 427 to 444 (18DEL), and deletion from position 419 to 444 (26DEL) in the amino acid sequence encoded by the AO090005000664 gene. )) ERG production amount in Aspergillus oryzae (A) deleted, and the region including position 428 (1DEL) and 427 in the amino acid sequence encoded by the ortholog gene that has 98% identity with the AO090005000664 gene.
  • FIG. 8 shows the predicted three-dimensional structure of the protein expressed from the AO090005000664 gene by homology modeling using SWISS-MODEL (https://swissmodel.expasy.org/) (A). An enlarged view of the supersecondary structure surrounded by the dotted line is shown in FIG. 8B (B).
  • the microorganism into which genetic mutations are introduced is a microorganism that has ergothioneine productivity.
  • Such microorganisms are preferably filamentous fungi, and more preferably any fungi belonging to the genus Aspergillus, Penicillium, Rasamsonia, and Talaromyces can be used.
  • filamentous fungi of the genus Aspergillus include Aspergillus oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus tamarii, Aspergillus niger, Examples include Aspergillus nidulans.
  • filamentous fungal strains examples include publicly available strains such as Aspergillus oryzae RIB40 strain and RIB326 strain, strains contained in seed koji commercially available from seed koji shops, and food and beverage sources such as sake and soy sauce breweries.
  • Bacterial strains isolated from product manufacturing environments can be used. A strain obtained by isolation can be used.
  • the gene to be mutated relates to the gene named AO090005000664 in Aspergillus oryzae RIB40 strain and its ortholog gene.
  • This gene has a translated region of unknown function and consists of a base sequence (SEQ ID NO: 2) that encodes a protein consisting of a 657-residue amino acid sequence (SEQ ID NO: 1).
  • the base sequence of the Aspergillus sojae ortholog gene of the AO090005000664 gene corresponds to REGION 19840...21877 of Accession No. BaCA02000013 (SEQ ID NO: 4), and its amino acid sequence is represented by SEQ ID NO: 3.
  • the ortholog genes of the AO090005000664 gene in Aspergillus tamari, Aspergillus luthuensis, Aspergillus niger, and Aspergillus nidulans are BDV40DRAFT_264902 (amino acid sequence: SEQ ID NO: 5, nucleotide sequence: SEQ ID NO: 6) and AAWM_07753 (amino acid sequence: SEQ ID NO: 7, base sequence: SEQ ID NO: 8), An16g07990 (amino acid sequence: SEQ ID NO: 9, base sequence: SEQ ID NO: 10), and ANIA_10201 (amino acid sequence: SEQ ID NO: 11, base sequence: SEQ ID NO: 12).
  • AO090005000664 has multiple conserved regions that are highly conserved with other species.
  • An example of such a conserved region is the region corresponding to positions 411 to 479 of SEQ ID NO: 1 (SEQ ID NO: 13).
  • the conserved region is preferably a region corresponding to positions 411 to 467 of SEQ ID NO: 1 (SEQ ID NO: 14), and a region corresponding to positions 411 to 462 (SEQ ID NO: 15) is preferable.
  • the region corresponding to positions 411 to 444 is even more preferred, the region corresponding to positions 427 to 444 (SEQ ID NO: 17) is even more preferred, and the region corresponding to positions 433 to 440 (SEQ ID NO: 18) is more preferred. Corresponding regions are even more preferred.
  • the region corresponding to positions 433 to 440 (SEQ ID NO: 18) is highly conserved among Aspergillus species, and more preferably can be completely identical.
  • the nucleic acids of the present invention may be derived from filamentous fungi, such as Aspergillus, Penicillium, Rasamsonia or Talaromysis.
  • the nucleic acid of the present invention is highly conserved in these filamentous fungi.
  • a nucleic acid derived from a filamentous fungus belonging to the genus Aspergillus encodes an amino acid sequence having 75-100% homology to SEQ ID NO:1.
  • a nucleic acid derived from a filamentous fungus belonging to the genus Penicillium encodes an amino acid sequence having 68-75% homology to SEQ ID NO:1.
  • a nucleic acid derived from a filamentous fungus belonging to the genus La Samsonia encodes an amino acid sequence with 70% homology to SEQ ID NO:1.
  • a nucleic acid derived from a filamentous fungus belonging to the genus Talaromysis encodes an amino acid sequence having 64-68% homology to SEQ ID NO:1.
  • the sequence corresponding to SEQ ID NO: 1 refers to the amino acid sequence of a gene that is an ortholog of AO090005000664, which is possessed by a specific filamentous fungus.
  • Orthologous genes can be selected from the protein database registered at NCBI by using NCBI's blastp program. Such orthologs typically have 40% or more, 50% or more, or 60% or more homology.
  • the sequence corresponding to SEQ ID NO: 1 is an amino acid sequence encoded by an orthologous gene of the genus Aspergillus, Penicillium, Rasamsonia, or Talaromysis.
  • the sequence corresponding to SEQ ID NO: 1 can be defined by including conserved regions. Therefore, the sequence corresponding to SEQ ID NO: 1 is an amino acid sequence encoding a gene that is an ortholog of AO090005000664, and is specified by a predetermined conserved region included in SEQ ID NO: 1. Specific examples of sequences corresponding to SEQ ID NO: 1 include amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11.
  • the conserved regions are, for example, positions 427 to 444 of SEQ ID NO: 1 (SEQ ID NO: 17), positions 411 to 444 (SEQ ID NO: 16), positions 411 to 462 (SEQ ID NO: 15), or positions 433 to 440 (SEQ ID NO: 15). Located at number 18).
  • the sequence corresponding to SEQ ID NO: 1 can be defined by including the conserved region of positions 433 to 440 (SEQ ID NO: 18) of SEQ ID NO: 1 from the viewpoint of matching in the genus Aspergillus.
  • the conserved region included in the sequence corresponding to SEQ ID NO: 1 is the following sequence instead of this conserved region: 427 444
  • X430 is at least one amino acid selected from the group consisting of Q, H, P, and D
  • X431 is at least one amino acid selected from the group consisting of Q and L
  • X432 is at least one amino acid selected from the group consisting of P, Q and G
  • X433 is at least one amino acid selected from the group consisting of D and E
  • X435 is at least one amino acid selected from the group consisting of R and W
  • X437 is at least one amino acid selected from the group consisting of L and I
  • X438 is at least one amino acid selected from the group consisting of V and M
  • X441 is at least one amino acid selected from the group consisting of V, I and L] It may be specified by
  • the conserved region is preferably a sequence that is conserved, particularly in the genus Aspergillus.
  • Such storage areas include: 427 444
  • X430 is at least one amino acid selected from the group consisting of Q, H, and P
  • X431 is at least one amino acid selected from the group consisting of Q and L
  • X432 is at least one amino acid selected from the group consisting of P and G
  • X441 is at least one amino acid selected from the group consisting of V and I] can be mentioned.
  • X412 is at least one amino acid selected from the group consisting of E and D;
  • X416 is at least one amino acid selected from the group consisting of Q, H and E;
  • X417 is at least one amino acid selected from the group consisting of N, H and D;
  • X418 is at least one amino acid selected from the group consisting of Y, F and C;
  • X422 is at least one amino acid selected from the group consisting of N and S;
  • X424 is at least one amino acid selected from the group consisting of W, C, and M;
  • X425 is at least one amino acid selected from the group consisting of Y, Q, S, A and G;
  • X426 is at least one amino acid selected from the group consisting of L, I, V and M;
  • X430 is at least one amino acid selected from the group consisting of E and D;
  • X417 is at least one amino acid selected from the group consisting of N, H and D;
  • X412 is at least one amino acid selected from the group consisting of E and D;
  • X416 is at least one amino acid selected from the group consisting of Q, H and E;
  • X417 is at least one amino acid selected from the group consisting of N, H and D;
  • X418 is at least one amino acid selected from the group consisting of Y, F and C;
  • X422 is at least one amino acid selected from the group consisting of N and S;
  • X424 is at least one amino acid selected from the group consisting of W, C, and M;
  • X425 is at least one amino acid selected from the group consisting of Y, Q, S, A and G; X426
  • amino acid sequence having such a conserved region and having at least 60% homology to the amino acid sequence of SEQ ID NO: 1 can be defined as a sequence corresponding to SEQ ID NO: 1.
  • the sequence corresponding to SEQ ID NO: 1 includes the above-mentioned conserved region and is at least 60%, at least 63%, at least 70%, at least 75%, at least 80%, at least 90%, at least 94%, with respect to SEQ ID NO: 1.
  • the present invention in the sequence corresponding to SEQ ID NO: 1, modulates the function of the functional domain in a region included in the functional domain corresponding to the functional domain W404 to Q515 of SEQ ID NO: 1.
  • the present invention relates to a nucleic acid encoding a sequence containing a mutation; or a sequence having at least 90% identity to the sequence, and which enhances ergothioneine production when introduced into an ergothioneine-producing microorganism.
  • the functional domain refers to the functional domain W404 to Q515 of SEQ ID NO: 1, or a functional domain corresponding to the functional domain in a protein encoded by the AO090005000664 gene or its ortholog gene. It has been shown that such a functional domain has a supersecondary structure composed of five ⁇ -helices (FIGS. 8A and B). While the specific function of this functional domain is unknown, there are deletions from positions 427 to 429 (3DEL), deletions from positions 427 to 444 (18DEL), and deletions from positions 419 to 444 (26DEL), which constitute the functional domain.
  • 3DEL deletions from positions 427 to 429
  • 18DEL deletions from positions 427 to 444
  • 26DEL deletions from positions 419 to 444
  • positions 428 and 459 of SEQ ID NO: 1 are located in the connecting region of five ⁇ -helices, and mutations at these positions are considered to have a high possibility of causing structural changes in the super-secondary structure.
  • a mutation that modulates the function of a functional domain may be any mutation that affects the function of the functional domain W404 to Q515 of SEQ ID NO: 1 or the functional domain corresponding to the functional domain. may be a deletion, addition or substitution of one or more amino acids in.
  • the corresponding functional domain refers to a functional domain in the ortholog gene of the gene named AO090005000664.
  • mutations that modulate the functional domain are deletions in the regions included in the functional domains corresponding to the functional domains W404 to Q515, or deletions of G and/or at the position corresponding to position 428. Mutations, especially substitutions, at position C corresponding to position 459.
  • the number of amino acids to be deleted is any number as long as the production of ERG is enhanced in the microorganism in which the deletion occurs.
  • the number of amino acids to be deleted may be in any range selected from the group consisting of 1 to 120, 1 to 112, 1 to 60, 1 to 57, and 1 to 47.
  • the number of amino acids deleted is preferably any number from 1 to 30, more preferably from 1 to 26. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, At least one number of amino acids selected from the group consisting of 25 and 26 is deleted.
  • amino acid at the position corresponding to position 428 may be deleted, or a plurality of amino acids including the amino acid at the position corresponding to position 428 may be deleted. In another embodiment, amino acids not including the amino acid at the position corresponding to position 428 may be deleted.
  • any sequence included in the conserved region from position 411 to position 462 (SEQ ID NO: 15) and including position 428 may be deleted.
  • it is selected from the group consisting of positions 428, 427 to 429, 427 to 440 (SEQ ID NO: 20), 427 to 444 (SEQ ID NO: 17), and 419 to 444 (SEQ ID NO: 21).
  • a region corresponding to at least one region may be deleted.
  • a region corresponding to at least one region selected from the group consisting of positions 431-475, 419-475, and 404-515 may be deleted.
  • amino acid positions are indicated based on the amino acid positions of SEQ ID NO: 1 encoded by the AO090005000664 gene, and represent the corresponding amino acid positions in the protein encoded by the corresponding gene, that is, the ortholog gene. Corresponding positions can be determined by aligning homologous sequences of the amino acid sequence of SEQ ID NO:1.
  • the identity to the sequence in which the region included in the functional domain corresponding to the functional domain of W404 to Q515 is deleted is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical.
  • a sequence that has such identity and that increases ergothioneine productivity when a region included in a functional domain corresponding to the functional domains W404 to Q515 in a filamentous fungus is deleted can be selected.
  • the function of contributing to ergothioneine production means that it plays some function in the ergothioneine production process.
  • Such functions may be functions related to transcriptional regulation of enzymes that contribute to the synthetic pathway or decomposition pathway of ergothioneine, enzymatic reactions that contribute to the synthetic pathway of ergothioneine, or functions of ergothioneine or its decomposition pathway. It may also be an enzymatic reaction that contributes to the decomposition pathway of the precursor.
  • the nucleic acid of the present invention which encodes a sequence corresponding to SEQ ID NO: 1 in which a region included in the functional domain corresponding to the functional domain W404 to Q515 of SEQ ID NO: 1 is deleted, can be used in an ergothioneine-producing filamentous fungus. When introduced, it has the function of enhancing ergothioneine production.
  • the present invention further relates to a method for producing a microorganism with enhanced ergothioneine-producing ability. More specifically, such a production method includes deleting, in the sequence corresponding to SEQ ID NO: 1, a region included in a functional domain corresponding to functional domains W404 to Q515 of SEQ ID NO: 1.
  • the region to be deleted is usually a region included in the functional domain corresponding to the functional domain W404 to Q515, but the adjacent region from positions 404 to 511 may also be deleted as long as it is possible to enhance the ergothioneine production ability. It's okay.
  • the deleted region may include a position corresponding to position 428 of SEQ ID NO: 1. However, it may not be included.
  • the region to be deleted includes a position corresponding to position 428 of SEQ ID NO: 1.
  • the region to be deleted corresponds to at least one region selected from the group consisting of positions 428, 427 to 429, 427 to 440, 427 to 444, and 419 to 444 of SEQ ID NO: 1. This is an area where The region to be deleted may be the entire conserved region from positions 411 to 462 (SEQ ID NO: 15), or even the entire functional domain from positions 404 to 515. good.
  • the scope of the deletion may be any deletion as long as the microorganism having such deletion can enhance ergothioneine production.
  • Contiguous amino acid sequences within the functional domain W404 to Q515, particularly within the conserved region from positions 411 to 462 (SEQ ID NO: 15), are deleted, but discontinuous amino acid sequences may also be deleted.
  • the microorganism is an ergothioneine-producing microorganism selected from the group consisting of Aspergillus, Penicillium, Rasamsonia, and Talaromysis.
  • the corresponding sequence of SEQ ID NO: 1 has a mutation in G at the position corresponding to position 428 of SEQ ID NO: 1 and/or C at the position corresponding to position 459 of SEQ ID NO: 1, and the ergothioneine-producing microorganism It may also relate to a nucleic acid that enhances ergothioneine production when introduced. Mutations at such positions may more specifically be G428S and/or C459F substitutions. By having a nucleic acid having a mutation at such a position, an ergothioneine-producing microorganism enhances ergothioneine production. More specifically, the corresponding sequence of SEQ ID NO: 1 has conserved regions as defined herein.
  • the present invention provides: 1) In the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, S is present at the position corresponding to position 428, and/or F is present at the position corresponding to position 459. or 2) has at least 60% identity to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, and 11, and at the position corresponding to position 428. It may also relate to a nucleic acid that encodes an amino acid sequence that has S and/or has F at the position corresponding to position 459, and that enhances ergothioneine production when introduced into an ergothioneine-producing filamentous fungus. By having the G428S and C459F mutations, ergothioneine production can be enhanced.
  • Identity to SEQ ID NO: 1, 3, 5, 7, 9, 11 includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% identity.
  • the orthologous genes of Aspergillus sojae and Aspergillus niger share 68% and 67% identity, respectively, while the mutation corresponding to G428S (G442S in Aspergillus niger) - Increased ERG production has been confirmed by introducing it into Niger ( Figures 4 and 5).
  • the present invention provides: Having at least 60% identity to the base sequences of SEQ ID NOs: 2, 4, 6, 8, 10, and 12, and corresponding to position 428 and/or position 459 of the amino acid sequence of SEQ ID NO: 1.
  • the base sequence that encodes the mutation at the position that It may also relate to a nucleic acid consisting of the following, which enhances ergothioneine production when introduced into an ergothioneine-producing microorganism. By having the G428S and C459F mutations, ergothioneine production can be enhanced.
  • Identity to SEQ ID NOs: 2, 4, 6, 8, 10, and 12 is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% identity has.
  • the orthologous genes of Aspergillus sojae and Aspergillus niger have 68% and 67% identity, respectively, by introducing the mutation corresponding to G428S (G442S in Aspergillus niger), Enhancement of ERG production has been confirmed ( Figures 4 and 5).
  • the nucleic acids of the present invention may be derived from filamentous fungi, such as Aspergillus, Penicillium, Rasamsonia or Talaromysis.
  • the nucleic acid of the present invention is highly conserved in these filamentous fungi.
  • the amino acid sequence encoded by a nucleic acid derived from a filamentous fungus belonging to the genus Aspergillus has 75 to 100% homology to SEQ ID NO:1.
  • the amino acid sequence encoded by a nucleic acid derived from a filamentous fungus belonging to the genus Penicillium has 68 to 75% homology to SEQ ID NO:1.
  • the amino acid sequence encoded by a nucleic acid derived from a filamentous fungus belonging to the genus La Samsonia has 70% homology to SEQ ID NO:1.
  • the amino acid sequence encoded by a nucleic acid derived from a filamentous fungus belonging to the genus Talaromysis has 64 to 68% homology to SEQ ID NO:1.
  • the function of contributing to ergothioneine production refers to being responsible for some enzymatic reaction in the ergothioneine production process. It may be an enzymatic reaction that contributes to the synthetic pathway of ergothioneine, or an enzymatic reaction that contributes to the decomposition pathway of ergothioneine or its precursor.
  • the protein encoded by the nucleic acid of the present invention may have an activity for synthesizing ergothioneine or its precursor.
  • the protein encoded by the nucleic acid of the present invention may have an activity to decompose ergothioneine or its precursor or metabolite.
  • the nucleic acid of the present invention encoding an amino acid sequence having S at the position corresponding to position 428 of SEQ ID NO: 1 and/or having F at the position corresponding to position 459 is introduced into an ergothioneine-producing filamentous fungus. Thus, it has the function of enhancing ergothioneine production.
  • the positions corresponding to positions 428 and 459 of SEQ ID NO: 1 can be determined by aligning homologous sequences of the amino acid sequence of SEQ ID NO: 1.
  • it can be determined from the neighboring sequences, and the position of G in LSGLQ, which is the neighboring sequence at position 428, corresponds to position 428.
  • Mutation of G at position 428 to S increases ergothioneine productivity.
  • the position C of APCED, which is a neighboring sequence to position 459, corresponds to position 459.
  • Mutation of C at position 459 to F increases ergothioneine productivity. Mutations may be introduced into the positions corresponding to position 428 and 459 separately, or at the same time.
  • the present invention further relates to a method for producing a microorganism with enhanced ergothioneine-producing ability. More specifically, such a production method involves introducing a mutation at a position corresponding to position 428 and/or position corresponding to position 459 of SEQ ID NO: 1 into the genome of a microorganism encoding the conserved region according to the present invention. including.
  • the mutation at the position corresponding to position 428 is G428S
  • the mutation at position 459 is C459F.
  • the microorganism is an ergothioneine-producing microorganism selected from the group consisting of Aspergillus, Penicillium, Rasamsonia, and Talaromysis.
  • X430 is at least one amino acid selected from the group consisting of Q, H, P, and D;
  • X431 is at least one amino acid selected from the group consisting of Q and L;
  • X432 is at least one amino acid selected from the group consisting of P, Q and G;
  • X433 is at least one amino acid selected from the group consisting of D and E;
  • X435 is at least one amino acid selected from the group consisting of R and W;
  • X437 is at least one amino acid selected from the group consisting of L and I;
  • X438 is at least one amino acid selected from the group consisting of V and M;
  • X441 is at least one amino acid selected from the group consisting of V, I and L] , the following: the microorganism is an er
  • Mutations may be introduced into the genome using any method. It may be introduced using a standard transformation system, it may be introduced as a result of introducing random mutations, or it may be introduced using genome editing technology.
  • the gene may be introduced using a gene cassette, site-specific gene introduction may be performed, or a vector or the like may be used.
  • Another aspect of the invention may relate to a vector into which the nucleic acid of the invention can be expressed.
  • Random mutation introduction methods include, for example, ultraviolet (UV) and X-ray irradiation, which physically damage DNA and introduce mutations, and N-methyl-N'-nitro irradiation, which chemically damages DNA and introduces mutations.
  • Examples include treatment with an alkylating reagent such as -N-nitrosoguanidine (NTG) and ethyl methanesulfonic acid (EMS).
  • NVG -N-nitrosoguanidine
  • EMS ethyl methanesulfonic acid
  • site-specific cleavage and homologous recombination using the CRISPR/Cas system can be used.
  • the invention also relates to microorganisms containing the nucleic acids according to the invention. These are microorganisms obtained as a result of the production method according to the present invention.
  • ergothioneine By culturing the microorganism according to the present invention, ergothioneine can be produced with high productivity.
  • the microorganism according to the present invention may be cultured in either solid culture or liquid culture.
  • ergothioneine can be produced with high productivity by culturing the microorganism according to the present invention.
  • the microorganism according to the present invention may be cultured in either solid culture or liquid culture.
  • rice malt with high ergothioneine content can be produced by culturing the microorganism according to the present invention in steamed rice. Using such koji, soy sauce, miso, salt koji, amazake, etc.
  • the invention may relate to a method for producing rice malt containing a high content of ergothioneine.
  • the produced ergothioneine can be purified from the culture based on a conventional method.
  • the culture can be ground and the water evaporated to obtain a dry powder.
  • aqueous ethanol By adding aqueous ethanol to the dried ergothioneine-containing raw material, ergothioneine is extracted into an ethanol solution. Insoluble substances may be removed and the ergothioneine in the ethanol solution may be purified by subjecting it to chromatography.
  • Ergothioneine is known to have various effects, including antioxidant effects, elastase inhibitory effects, tyrosinase inhibitory effects, and polyphenol oxidase (PPO) inhibitory effects. Therefore, the produced ergothioneine may be purified or directly blended into products such as foods, cosmetics, quasi-drugs, and pharmaceuticals, or may be prepared as a concentrate to be blended into products. When formulated as a food, it can also be a food with functional claims.
  • Such functional claims include ergothioneine's anti-wrinkle effect based on elastase inhibitory activity, skin beautification or whitening effect based on tyrosinase inhibitory activity, lifestyle-related disease preventive effect based on the production of lipid peroxide, and anti-dementia and Alzheimer's effect based on active oxygen removal. It can display functions such as disease prevention.
  • the produced ergothioneine may be a food with functional claims, a food with nutritional function claims, a food for specified health uses, or a nutritional supplement, which focuses on the physiological effects of ergothioneine.
  • Example 1 Analysis of ERG high-producing strains (1)
  • Eukaryotes of the genus Aspergillus were cultured using the following medium.
  • Czapek-DOX (Cz-DOX) medium (3.5% Czapek-DOX broth, manufactured by BD, pH 6.0)
  • PD medium 2% dextrin, 1% polypeptone, 0.5% KH 2 PO 4 , 0.1% NaNO 3 , 0.05% MgSO 4 , 0.1% Casamino Acid, pH 6.0
  • Pafmin medium 5% Pafmin SM, pH not adjusted).
  • Cz-DOX agar medium (3.5% zapek-DOX broth, manufactured by BD, supplemented with 0.1% trace element, 2% Agar) or Malt's agar medium (4.5% Malt Agar Nissui, Japan). (manufactured by Mizuyaku Co., Ltd., 0.1% trace element, 0.5% yeast extract) was used.
  • the homology between AO090005000664 and the orthologous genes of Aspergillus oryzae, Aspergillus sojae, Aspergillus tamari, Aspergillus lutuensis, Aspergillus niger, and Aspergillus nidulans is 100%, 98%, 96%, 67%, respectively. They were 67% and 62%.
  • Example 2 Preparation of mutation reproduction strain in Aspergillus oryzae RIB40 strain and confirmation of ERG productivity
  • G428S cassette G428S mutation is introduced into the AO090005000664 gene using Aspergillus oryzae RIB40_dKP strain ( ⁇ ku70, ⁇ pyrG) as a host.
  • Primers are shown in Table 1.
  • Gene fragments were amplified by PCR using the genome sequence of RIB40 strain as a template and primer sets of AoG428S_F1 and R1, AoG428S_F2 and R2, AoG428S_F3 and R3, and AopyrG F and R. PCR conditions followed the attached protocol.
  • the four purified PCR amplified fragments and Linerized pUC19 (Takara) were mixed with 2.5 ⁇ L of NEBuilder HiFi DNA Assembly Master Mix (NEB) and reacted at 60° C. for 1 hour. 4 ⁇ L of the reaction was added to ECOS Competent E.
  • the cells were introduced into E.coli JM109 (Nippon Gene) and transformed.
  • the plasmid extracted from this transformant was used as a template AoG428S_NF and a primer set of NR, and the gene fragment amplified by PCR was made into a G428S cassette.
  • 50 ⁇ L of the PCR product was subjected to ethanol precipitation and dissolved in 10 ⁇ L of sterile water.
  • the bacterial cells were collected with Miraclose and washed with KCl buffer (0.6 M KCl, 0.1 M NaH 2 PO 4 , pH 5.5).
  • the collected cells were suspended in 20 mL of cell wall lytic enzyme (0.5% Lysing enzyme (Sigma-Aldrich), 0.3% Yatalase (Takara), 1.5% Cellulase Onozuka (Yakult) /20 mL KCl buffer).
  • the filtrate was collected through a 70 ⁇ m cell strainer.
  • the obtained transformant was purified by plating three times on a Cz-DOX agar medium (3.5% Czapek-DOX borth, 0.1% trace element, 1.5% Agar).
  • the culture solution was extracted with hot water (90° C., 1 hour), and 0.5 mL of the supernatant was filtered using a Nanosep centrifugal filtration device (3K, manufactured by Nippon Pall Co., Ltd.), and diluted with ultrapure water as appropriate to prepare an analysis sample.
  • FIG. 3 shows the amount of ERG produced by each strain. When the ERG productivity of the mutant strain was compared, it was found that the ERG productivity was improved compared to the control strain and the wild strain. Although the C459F mutation-introduced strain produced less ERG than the G428S mutation-introduced strain, it became clear that the C459F mutation was also involved in improving ERG productivity.
  • Example 3 Preparation of a mutation-reproducing strain in Aspergillus sojae and confirmation of ERG productivity Aspergillus oryzae RIB40 strain AO090005000664 G428S in Example 1
  • Aspergillus sojae Using the NBRC4239_dKP strain ( ⁇ ku70, ⁇ pyrG) as a host, mutation introduction, transformation, and ERG production were confirmed.
  • the ortholog of the AO090005000664 gene in Aspergillus sojae has 98% amino acid identity and 100% amino acid homology.
  • Six of the 19 transformants that reproduced the G428S mutation contained the G428S mutation.
  • Figure 4 shows the amount of ERG produced by each strain. When the ERG productivity of the mutant strain was compared, it was found that the ERG productivity was improved compared to the control strain and the wild strain. It has also been revealed that this gene mutation is involved in improving ERG productivity in Aspergillus sojae.
  • Example 4 Preparation of a mutation-reproducing strain in Aspergillus niger and confirmation of ERG productivity Same method as described in the section of Aspergillus oryzae RIB40 strain AO090005000664 G428S mutation-reproducing strain of Example 1.
  • Aspergillus niger strain A1179 also known as MA70.15, Genotyping: kusA::amdS, cspA1 (mutation resulting in short conidiophores), pyrG -
  • the amino acid identity of the ortholog of the AO090005000664 gene in Aspergillus niger is 67%, and the amino acid homology is 79%.
  • the mutation points of the Aspergillus oryzae strain that showed high ERG production are also conserved in Aspergillus niger (G442 and C473). Therefore, a mutant strain was created in which glycine at position 442, which corresponds to G428S, whose mutational effect was confirmed in Aspergillus oryzae RIB40 strain, was replaced with serine.
  • Figure 5 shows the amount of ERG produced by each strain. When the ERG productivity of the mutant strain was compared, it was found that the ERG productivity was improved compared to the control strain and the wild strain. It has also been revealed that this gene mutation is involved in improving ERG productivity in Aspergillus niger.
  • Example 5 Analysis of ERG high-producing strains (1)
  • Medium Aspergillus eukaryotes were cultured using the following medium.
  • Czapek-DOX (Cz-DOX) medium (3.5% Czapek-DOX broth, manufactured by BD, pH 6.0)
  • PD medium 2% dextrin, 1% polypeptone, 0.5% KH 2 PO 4 , 0.1% NaNO 3 , 0.05% MgSO 4 , 0.1% Casamino Acid, pH 6.0
  • Pafmin medium 5% Pafmin SM, pH not adjusted).
  • Cz-DOX agar medium (3.5% zapek-DOX broth, manufactured by BD, supplemented with 0.1% trace element, 2% Agar) or Malt's agar medium (4.5% Malt Agar Nissui, Japan). (manufactured by Mizuyaku Co., Ltd., 0.1% trace element, 0.5% yeast extract) was used.
  • AO090005000664 A phasic search was performed on the amino acid sequence of AO090005000664 using the default settings of BLAST (Basic Logical Alignment Search Tool) at NCBI (National Center for Biotechnology Information), and it was found that it is highly conserved in the Aspergillus genus, and that SEQ ID NO. A highly conserved conserved region was found at positions 411-467 ( Figure 6).
  • the identity of AO090005000664 to the orthologous genes of Aspergillus oryzae, Aspergillus sojae, Aspergillus tamari, Aspergillus lutuensis, Aspergillus niger, and Aspergillus nidulans is 100%, 98%, 96%, 67%, respectively. They were 67% and 62%.
  • Example 6 Preparation of amino acid-deficient strain of Aspergillus oryzae RIB40 strain (1) Preparation of amino acid deletion cassette and acquisition of transformed strain of Aspergillus oryzae RIB40_dKP Using RIB40_dKP strain ( ⁇ ku70, ⁇ pyrG) as a host, a gene for deleting amino acids A cassette was made. The amino acids to be deleted were highly conserved amino acids centered around G428 ( Figure 6). Specifically, positions 428, 427-429, 427-440, 427-444, and 419-444 were deleted, respectively. Table 1 shows the primers used.
  • a gene fragment was amplified by PCR using primer sets of primers 1 and 2, primers 3 and 4, and primers 5 and 6. PCR conditions followed the attached protocol.
  • the three purified PCR amplified fragments and Linerized pUC19 (Takara) (0.5 ⁇ L each) were mixed with 2.5 ⁇ L of NEBuilder HiFi DNA Assembly Master Mix (NEB) and reacted at 60° C. for 1 hour. 4 ⁇ L of the reaction was added to ECOS Competent E.
  • the cells were introduced into E.coli JM109 (Nippon Gene) and transformed.
  • a gene fragment was extracted by PCR using the sets of primers 7 and 8 (Ao3DEL), primers 9 and 8 (Ao18DEL), and primers 9 and 10 (Ao26DEL). Amplified. PCR conditions followed the attached protocol. DpnI was added to each PCR reaction solution and reacted at 37°C for 1 hour. Next, 2 ⁇ l of each DpnI-treated PCR product was added to Ligation high Ver. 2 (Toyobo), 1 ⁇ l of T4 Polynucleotide Kinase, and 7 ⁇ l of sterile water, and reacted at 16° C. for 1 hour.
  • Example 7 Preparation of an amino acid-deficient strain of Aspergillus sojae NBRC4239 (1) Preparation of an amino acid-deficient cassette and acquisition of a transformed strain of Aspergillus sojae NBRC4239_dKP Using the NBRC4239_dKP strain ( ⁇ ku70, ⁇ pyrG) as a host, the above-mentioned Aspergillus oryzae A gene cassette with amino acid deletion was created in the same way as for the RIB40 strain. Using the genome sequence of NBRC4239 strain as a template, a gene fragment was amplified by PCR with primer sets of primers 13 and 14, primers 3 and 15, and primers 5 and 6. PCR conditions followed the attached protocol.
  • the three purified PCR amplified fragments and Linerized pUC19 (Takara) (0.5 ⁇ L each) were mixed with 2.5 ⁇ L of NEBuilder HiFi DNA Assembly Master Mix (NEB) and reacted at 60° C. for 1 hour. 4 ⁇ L of the reaction was added to ECOS Competent E. The cells were introduced into E.coli JM109 (Nippon Gene) and transformed.
  • primers 16 and 17 (As1DEL), primers 7 and 8 (As3DEL), primers 18 and 8 (As14DEL), primers 9 and 8 (As18DEL), and The gene fragment was amplified by PCR using a set of primers 19 and 10 (As26DEL).
  • As26DEL a set of primers 19 and 10
  • the AsXDEL cassette was introduced to transform the NBRC4239_dKP strain.
  • the transformed strain was purified by spot planting on Cz-DOX agar medium three times.
  • Example 8 Preparation of an amino acid-deficient strain of Aspergillus oryzae RIB40 strain (1) Preparation of an amino acid deletion cassette and acquisition of a transformed strain of Aspergillus oryzae RIB40_dKP Using the RIB40_dKP strain ( ⁇ ku70, ⁇ pyrG) as a host, a gene for deleting an amino acid A cassette was made. Based on the predicted structure of Example 10, amino acids were selected to delete part or all of the domain. Specifically, positions 431-475, 419-475, and 404-515 were deleted. Table 1 shows the primers used.
  • a gene fragment was amplified by PCR with a set of primers 22 and 23 (Ao45DEL), primers 22 and 10 (Ao57DEL), and primers 24 and 25 (Ao112DEL).
  • PCR conditions followed the attached protocol.
  • DpnI was added to each PCR reaction solution and reacted at 37°C for 1 hour.
  • 2 ⁇ l of each DpnI-treated PCR product was added to Ligation high Ver. 2 (Toyobo), 1 ⁇ l of T4 Polynucleotide Kinase, and 7 ⁇ l of sterile water, and reacted at 16° C. for 1 hour.
  • Example 9 Preparation of amino acid-deficient strain of Aspergillus niger FGSC A1179 strain
  • Preparation of amino acid deletion cassette and acquisition of transformed strain of Aspergillus niger FGSC A1179 The above-mentioned procedure was carried out using FGSC A1179 strain ( ⁇ kusA, pyrG-) as a host.
  • a gene cassette with amino acid deletion was prepared in the same manner as in Aspergillus oryzae RIB40 strain.
  • a gene fragment was amplified by PCR using primer sets of primers 26 and 27 and primers 28 and 29. PCR conditions followed the attached protocol.
  • Linearized pUC19 (Takara) (0.5 ⁇ L each) of the two purified PCR amplified fragments and the gene fragments of primers 5 and 6 purified in Example 2 was mixed with 2.5 ⁇ L of NEBuilder HiFi DNA Assembly Master Mix (NEB), The reaction was carried out at 60°C for 1 hour. 4 ⁇ L of the reaction was added to ECOS Competent E. The cells were introduced into E.coli JM109 (Nippon Gene) and transformed.
  • primers 30 and 31 (Ang3DEL), primers 32 and 31 (Ang18DEL), primers 32 and 33 (Ang26DEL), primers 34 and 33 (Ang57DEL), and The gene fragment was amplified by PCR using a set of primers 35 and 36 (Ang112DEL).
  • Ang112DEL a set of primers 35 and 36
  • RNA sample 50 ⁇ L of the PCR product was subjected to ethanol precipitation and dissolved in 10 ⁇ L of sterile water.
  • the AngXDEL cassette was introduced to transform FGSC A1179 strain.
  • the transformed strain was purified by spot planting on Cz-DOX agar medium three times.
  • Example 10 Structural analysis of protein expressed from AO090005000664 gene Using the homology modeling function of SWISS-MODEL (https://swissmodel.expasy.org/), the predicted three-dimensional structure of PAAG_04735 was used as a template for the AO090005000664 gene. The three-dimensional structure was predicted ( Figure 8). Regarding the 3D structure of the template, we searched for genes with high homology to AO090005000664 whose 3D structure has been clarified or whose predicted 3D structure has been published, and found that the identity was 43% and was derived from Paracoccidioides lutzii. The predicted three-dimensional structure of PAAG_04735 was selected.
  • deletion at position 428 (1DEL), deletion at positions 427-429 (3DEL), and deletion at positions 427-440 (14DEL) from the amino acid sequence encoded by an orthologous gene with 98% identity with the AO090005000664 gene.
  • a deletion from positions 427 to 444 (18DEL) was shown that ergothioneine production was enhanced in S. sojae (FIG. 7B).
  • a deletion of positions 441 to 443 (3DEL), a deletion of positions 441 to 458 (18DEL), and a deletion of positions 433 to 458 were detected.
  • the function of the super-secondary structure domain contained in W404 to Q515 including position G428 is still unknown, but by at least partially inhibiting the function of the super-secondary structure domain, , it was shown that ergothioneine productivity can be increased. Note that the position in the AO090005000664 gene is referred to, and those skilled in the art can fully understand that the corresponding position differs depending on the gene.

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