Nucleic acids altered in manner to improve ergothioneine productivity, and genetically altered microorganisms

Abstract

The purpose of the present invention is to improve the productivity of ergothioneine in the production of ergothioneine by culturing an ergothioneine-producing filamentous bacterium. In an ergothioneine-producing filamentous bacterium, a genetic variation that increases the production of ergothioneine is identified. Provided are: a nucleic acid in which a region contained in a functional domain corresponding to a functional domain of W404-Q515 in SEQ ID NO: 1 is deleted in a sequence corresponding to SEQ ID NO: 1, said nucleic acid containing a conserved region at positions 433-440 in SEQ ID NO: 1; and a filamentous bacterium containing the nucleic acid.

Description

[Technical field]

[0001] The present disclosure relates to a nucleic acid modified in a manner to increase the productivity of ergothioneine (hereinafter, sometimes referred to as ERG), and a microorganism genetically modified in a manner to increase the productivity of ERG. Furthermore, it relates to a method for producing ERG using a genetically modified microorganism. [Background technology]

[0002] Ergothioneine is found as a sulfur-containing amino acid isolated from Claviceps purpurea, and is found to be present in the organism of plants or animals. However, plants or animals believe that ergothioneine cannot be synthesized, and the ergothioneine in the organism is derived from the ergothioneine synthesized by the microorganisms of basidiomycetes, etc. It is particularly known that it is also contained in the mushrooms of a part of basidiomycetes, for example, edible mushrooms such as oyster mushrooms, shiitake mushrooms, maitake mushrooms, and king oyster mushrooms, and is particularly contained in elm wood. Ergothioneine is known from the biosynthetic pathway of histidine, and sulfur atoms are supplied from cysteine. Ergothioneine has high antioxidant properties, and in addition, elastase inhibition and tyrosinase inhibition have been reported, and it is particularly concerned in the beauty food field such as whitening or wrinkle prevention. In addition, it is confirmed that ergothioneine is related to the oxidative defense system of the organism, and the application in the medical field is also tried. Ergothioneine has high thermal stability and pH stability, and can maintain its antioxidant effect even at high temperatures. It is added to foods in anticipation of physiological effects, and is also expected to be used in foods as an antioxidant.

[0003] As the manufacture method of thioneine, use the extraction, chemosynthesis, and fermentation of microorganisms from the basidiomycetes of elm wood velvet etc. Extraction from the basidiomycetes of elm wood velvet etc. consumes time in the acquisition of raw materials, and is not suitable for mass production. Suitable chemosynthesis is used in mass production, but the necessity of using expensive synthetic reagents is arranged, and in addition, there is the problem (patent documentation 1) that purification cost becomes high. Thereby, using the fermentation (patent documentation 2, non-patent documentation 1) of the bacterium or yeast of C1 compound usefulness, moreover, using the fermentation (patent documentation 3) of the microorganism of overexpression thioneine biosynthesis gene as manufacture method is powerful, can directly use microbial fermentation product, or use the point of extract obtained by simple extraction operation to be powerful.

[0004] [Prior art literature]

[0005] [Patent Literature]

[0006] Patent Document 1: Japanese Patent Application Publication No. 2006-160748

[0007] Patent Document 2: International Publication No. 2016 / 104437

[0008] Patent Document 3: International Publication No. 2017 / 150304

[0009]

Non-patent literature

[0010] Non-patent literature 1: PLOS One 2014vol.9(5)e97774

[0011] [Summary of the Invention]

[0012] [Problems to be solved by the invention]

[0013] In the production of ERG by microbial culture, the goal is to improve ERG productivity.

[0014]

Methods for solving the problem

[0015] The present inventors conducted intensive studies with the purpose of improving the ERG productivity of Aspergillus niger and found that the productivity of ergothioneine is enhanced by introducing a specific gene mutation into a gene whose function is unknown, thereby completing the present invention.

[0016] Therefore, the present invention is next concerned with:

[0017] [1] Nucleic acid,

[0018] A sequence encoding a conserved region contained in SEQ ID NO: 1, wherein the region contained in the functional domain corresponding to the functional domain of W404 to Q515 of SEQ ID NO: 1 in the sequence corresponding to SEQ ID NO: 1 contains a variation that changes the function of the functional domain; or a sequence having at least 90% identity with the sequence,

[0019] When introduced into a microorganism that produces ergothioneine, the production of ergothioneine is enhanced.

[0020] [2] The nucleic acid according to item 1, wherein the conserved region is the conserved region from position 433 to position 440 of SEQ ID NO: 1.

[0021] [3] The nucleic acid according to item 1, wherein the mutation that alters the function of the functional domain is a deletion of a region contained in the functional domain corresponding to the functional domain of W404 to Q515.

[0022] [4] The nucleic acid according to item 1, wherein the deleted region includes a position corresponding to position 428 of SEQ ID NO: 1.

[0023] [5] The nucleic acid according to item 1, wherein the deleted region consists of a deletion of 1 to 120 amino acids.

[0024] [6] The nucleic acid according to item 4, wherein the deleted region corresponds to 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.

[0025] [7] The nucleic acid according to item 3, wherein 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.

[0026] [8] The nucleic acid according to item 1, wherein the mutation that changes the function of the functional domain has a mutation in the G at position corresponding to position 428 and / or the C at position corresponding to position 459 of SEQ ID NO: 1.

[0027] [9] The nucleic acid according to item 1, wherein the nucleic acid is derived from a microorganism selected from the genera Aspergillus, Penicillium, Rasamsonia, and Talaromyces.

[0028]

[10] A vector comprising the nucleic acid according to any one of items 1 to 9.

[0029]

[11] A microorganism comprising the nucleic acid according to any one of items 1 to 9.

[0030]

[12] A method for producing ergothioneine, comprising culturing the microorganism described in item 11.

[0031]

[13] A method for producing a microorganism that enhances the function of ergothioneine production, comprising introducing a mutation that changes the function of a functional domain into a region contained in a functional domain corresponding to the functional domains W404 to Q515 of SEQ ID NO: 1 in a sequence corresponding to SEQ ID NO: 1, which contains a conserved region from positions 433 to 440 of SEQ ID NO: 1, wherein the microorganism is an ergothioneine-producing microorganism selected from the genera Aspergillus, Penicillium, Rasamsonia and Talaromyces.

[0032]

[14] The production method according to item 13, wherein the mutation that changes the function of the functional domain is a deletion of a region contained in the functional domain corresponding to the functional domain of W404 to Q515.

[0033]

[15] The production method according to item 13, wherein the above-mentioned conserved region is the conserved region from position 433 to position 440 of SEQ ID NO: 1.

[0034]

[16] The manufacturing method described in item 14, wherein the deleted region includes the position corresponding to position 428 of SEQ ID NO: 1.

[0035]

[17] The production method according to item 14, wherein the deleted region consists of 1 to 120 amino acids.

[0036]

[18] The method of claim 16, wherein the deleted region 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.

[0037]

[19] The production method according to item 14, wherein the deleted region corresponds to at least one region selected from positions 431 to 475, positions 419 to 475, and positions 404 to 515 of SEQ ID NO: 1.

[0038]

[20] The manufacturing method described in item 14, wherein the mutation that changes the function of the above-mentioned functional domain has a mutation in the G at the position corresponding to position 428 and / or the C at the position corresponding to position 459 of SEQ ID NO: 1.

[0039] [Effects of the invention]

[0040] According to the present invention, a microorganism containing a mutation that changes the function of a functional domain in a region corresponding to the functional domain of W404 to Q515 of SEQ ID NO: 1 in the gene corresponding to AO090005000664 can enhance ERG production.

[0041] [Brief description of the attached drawings]

[0042]

Figure 1

[0043]

Figure 2

[0044]

Figure 3

[0045]

Figure 4

[0046]

Figure 5

[0047]

Figure 6

[0048]

Figure 7A-B

[0049]

Fig. 7C-D

[0050]

Figure 8

[0051] In the present invention, as the microorganism of introducing gene mutation, it is the microorganism with ergothioneine productivity.As such microorganism, preferably filamentous fungi, more preferably, can use any bacterium belonging to Aspergillus (Aspergillus) genus, Penicillium (Penicillium) genus, Rasamsonia (Rasamsonia) genus, Talaromyces (Talaromyces) genus.As an example of the filamentous fungi of Aspergillus (Aspergillus) genus, Aspergillus oryzae (Aspergillus oryzae), Aspergillus sojae (Aspergillus sojae), Ryukyu Aspergillus (Aspergillus luchuensis), Aspergillus lustamarii (Aspergillus lustamarii), Aspergillus niger (Aspergillus nidulans) can be enumerated. As the strain of filamentous fungi, for example, publicly available strains such as Aspergillus oryzae RIB40 and RIB326, strains contained in commercially available koji from koji-brewing houses, or strains isolated from food production environments such as sake and soy sauce breweries can be used. Isolated strains can be used.

[0052] In the present invention, the gene to be introduced into the mutation is a gene named AO090005000664 in the RIB40 strain of Aspergillus oryzae, and its orthologous genes. This gene has a translation region of unknown function, and is composed of a base sequence (SEQ ID NO: 2) encoding a protein composed of an amino acid sequence of 657 residues (SEQ ID NO: 1). The base sequence of the orthologous gene of the AO090005000664 gene in Aspergillus sojae 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 orthologous genes of the AO090005000664 gene in Aspergillus lus tamarii, Aspergillus luchuensis, Aspergillus lus niger, and Aspergillus nidulans are: BDV40DRAFT_264902 (amino acid sequence: SEQ ID NO: 5, base sequence: SEQ ID NO: 6), 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). It is highly conserved among the genus Aspergillus, with 98% amino acid sequence identity in Aspergillus sojae, 96% amino acid sequence identity in Aspergillus tamarii, 67% amino acid sequence identity in Aspergillus luchuensis, 67% amino acid sequence identity in Aspergillus niger, and 62% amino acid sequence identity in Aspergillus nidulans.These genes have 100% amino acid sequence identity in Aspergillus sojae, 98% amino acid sequence identity in Aspergillus tamarii, 79% amino acid sequence identity in Aspergillus luchuensis, 79% amino acid sequence identity in Aspergillus niger, and 75% amino acid sequence identity in Aspergillus nidulans.

[0053] In AO090005000664, there are a plurality of conserved regions that are highly conserved with other species. As such a conserved region, in particular, there is a region corresponding to positions 411 to 479 of SEQ ID NO: 1 (SEQ ID NO: 13). From the viewpoint of a more highly conserved region, the conserved region is preferably a region corresponding to positions 411 to 467 of SEQ ID NO: 1 (SEQ ID NO: 14), more preferably a region corresponding to positions 411 to 462 (SEQ ID NO: 15), more preferably a region corresponding to positions 411 to 444 (SEQ ID NO: 16), more preferably a region corresponding to positions 427 to 444 (SEQ ID NO: 17), and more preferably a region corresponding to positions 433 to 440 (SEQ ID NO: 18). The region corresponding to positions 433 to 440 (SEQ ID NO: 18) may be extremely highly conserved in the genus Aspergillus, and more preferably completely identical.

[0054] The nucleic acid of the present invention may also be derived from filamentous fungi, such as Aspergillus, Penicillium, Rasamsonia or Talaromyces. The nucleic acid of the present invention is highly conserved in these filamentous fungi. The nucleic acid derived from the filamentous fungi belonging to the genus Aspergillus encodes an amino acid sequence having 75 to 100% identity with SEQ ID NO: 1. The nucleic acid derived from the filamentous fungi belonging to the genus Penicillium encodes an amino acid sequence having 68 to 75% identity with SEQ ID NO: 1. The nucleic acid derived from the filamentous fungi belonging to the genus Rasamsonia encodes an amino acid sequence having 70% identity with SEQ ID NO: 1. The nucleic acid derived from the filamentous fungi belonging to the genus Talaromyces encodes an amino acid sequence having 64 to 68% identity with SEQ ID NO: 1.

[0055] In the present invention, the sequence corresponding to SEQ ID NO: 1 refers to the amino acid sequence of a gene that is an ortholog of AO090005000664 possessed by a specific filamentous fungus. The gene that is an ortholog can be selected from the protein database logged into NCBI by using the blastp program of NCBI. Such orthologs usually have an identity of more than 40%, more than 50%, or more than 60%. More preferably, such a sequence corresponding to SEQ ID NO: 1 is an amino acid sequence encoded by an ortholog gene of the genus Aspergillus, the genus Penicillium, the genus Rasamsonia or the genus Talaromyces. More preferably, it is an amino acid sequence of a gene of an ortholog of Aspergillus lusoryzae, Aspergillus lus sojae, Aspergillus lus tamarii, Aspergillus luchuensis, Aspergillus niger, or Aspergillus lusnidulans. In one embodiment of the present invention, the sequence corresponding to SEQ ID NO: 1 can be defined by containing a conserved region. Thus, the sequence corresponding to SEQ ID NO: 1 is an amino acid sequence of a gene encoding an ortholog of AO090005000664, and is determined by the specified conserved region contained in SEQ ID NO: 1. As specific examples of the sequence corresponding to SEQ ID NO: 1, there can be mentioned an amino acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, and 11.

[0056] As an example, the conserved region is located at positions 427 to 444 (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: 18) of SEQ ID NO: 1. Specifically, from the viewpoint of consistency among Aspergillus species, the sequence corresponding to SEQ ID NO: 1 can be defined by the conserved region including positions 433 to 440 (SEQ ID NO: 18) of SEQ ID NO: 1. The conserved region included in the sequence corresponding to SEQ ID NO: 1 can also be defined by the following sequence instead of the conserved region:

[0057]

[0058] [In the sequence,

[0059] X430 is at least one amino acid selected from Q, H, P, and D;

[0060] X431 is at least one amino acid selected from Q, and L;

[0061] X432 is at least one amino acid selected from P, Q and G;

[0062] X433 is at least one amino acid selected from D, and E;

[0063] X435 is at least one amino acid selected from R, and W;

[0064] X437 is at least one amino acid selected from L and I;

[0065] X438 is at least one amino acid selected from V, and M;

[0066] X441 is at least one amino acid selected from V, I and L].

[0067] Furthermore, as another example, the conserved region is particularly preferably a sequence conserved in the genus Aspergillus. As such a conserved region, the following sequences can be cited:

[0068]

[0069] [In the sequence,

[0070] X430 is at least one amino acid selected from Q, H, and P;

[0071] X431 is at least one amino acid selected from Q, and L;

[0072] X432 is at least one amino acid selected from P, and G;

[0073] X441 is at least one amino acid selected from V and I].

[0074] As a further example, the following sequences can be cited as conserved regions:

[0075]

[0076] [In the sequence,

[0077] X412 is at least one amino acid selected from E and D;

[0078] X416 is at least one amino acid selected from Q, H and E;

[0079] X417 is at least one amino acid selected from N, H and D;

[0080] X418 is at least one amino acid selected from Y, F and C;

[0081] X422 is at least one amino acid selected from N and S;

[0082] X424 is at least one amino acid selected from W, C, and M;

[0083] X425 is at least one amino acid selected from Y, Q, S, A and G;

[0084] X426 is at least one amino acid selected from L, I, V and M;

[0085] X430 is at least one amino acid selected from Q, H, and P;

[0086] X431 is at least one amino acid selected from Q, and L;

[0087] X432 is at least one amino acid selected from P, and G;

[0088] X441 is at least one amino acid selected from V and I].

[0089] As a further example, the following sequences can be cited as conserved regions:

[0090]

[0091] [In the sequence,

[0092] X412 is at least one amino acid selected from E and D;

[0093] X416 is at least one amino acid selected from Q, H and E;

[0094] X417 is at least one amino acid selected from N, H and D;

[0095] X418 is at least one amino acid selected from Y, F and C;

[0096] X422 is at least one amino acid selected from N and S;

[0097] X424 is at least one amino acid selected from W, C, and M;

[0098] X425 is at least one amino acid selected from Y, Q, S, A and G;

[0099] X426 is at least one amino acid selected from L, I, V and M;

[0100] X430 is at least one amino acid selected from Q, H, and P;

[0101] X431 is at least one amino acid selected from Q, and L;

[0102] X432 is at least one amino acid selected from P, and G;

[0103] X441 is at least one amino acid selected from V and I;

[0104] X445 is at least one amino acid selected from Y, and F;

[0105] X446 is at least one amino acid selected from Q, and K;

[0106] X447 is at least one amino acid selected from V, and I;

[0107] X448 is at least one amino acid selected from F, V, I, Y, C, and L;

[0108] X450 is at least one amino acid selected from N, G, D, H, E, Q, and S;

[0109] X452 is at least one amino acid selected from S, G, D, H, A, and E;

[0110] X453 is at least one amino acid selected from F, Y, D, C, and H;

[0111] X454 is at least one amino acid selected from D, E, N, K, and Q;

[0112] X456 is at least one amino acid selected from P, H, T, S, and N;

[0113] X461 is at least one amino acid selected from D, N, and S;

[0114] X464 is at least one amino acid selected from I, L, and V;

[0115] X465 is at least one amino acid selected from N, D, S, C, Q, R, H, and G;

[0116] X466 is at least one amino acid selected from H, and R].

[0117] An amino acid sequence having the conserved region and having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 can be defined as a sequence corresponding to SEQ ID NO: 1. A sequence corresponding to SEQ ID NO: 1 can be determined by having at least 60%, at least 63%, at least 70%, at least 75%, at least 80%, at least 90%, at least 94%, at least 95%, at least 97%, at least 98%, or at least 99% identity or homology with SEQ ID NO: 1 while containing the conserved region.

[0118] In one embodiment of the present invention, the present invention relates to a sequence encoding a region containing a variation that changes the function of the functional domain in a sequence corresponding to SEQ ID NO: 1, which corresponds to the functional domain of W404 to Q515 of SEQ ID NO: 1; or a sequence having at least 90% identity with the sequence, which, when introduced into a microorganism that produces ergothioneine, enhances the production of ergothioneine.

[0119] In the present invention, the functional domain refers to the functional domain of W404 to Q515 of SEQ ID NO: 1 in the protein encoded by the AO090005000664 gene or its orthologous gene, or a functional domain corresponding to the functional domain. The functional domain involved shows that there is a domain with a super secondary structure composed of five α helices ( Figure 8 A and B). The specific function of this functional domain is unknown. In addition, since the production of ergothioneine is enhanced when the 427-429 position deletion (3DEL), the 427-444 position deletion (18DEL), and the 419-444 position deletion (26DEL), the 431-475 position (45DEL), the 419-475 position (57DEL), and the 404-515 position (112DEL) constituting the functional domain are deleted, the functional change of the functional domain may contribute to the production of ergothioneine. In particular, from the viewpoint that the production of ergothioneine is increased by the deletion of the amino acids constituting the functional domain, the increase in the production of ergothioneine may hinder the function in the necessary functional domain. In addition, the 428th and 459th positions of SEQ ID NO: 1 are located in the connection region of 5 α helices, and the variation at the above position is considered to have a high possibility of causing structural changes in the super secondary structure.

[0120] The variation that changes the function of the functional domain is only required to affect the functional domain of W404 to Q515 of SEQ ID NO: 1, or the variation of the function of the functional domain corresponding to the functional domain, and the variation may be the deletion, addition or substitution of one or more amino acids in the functional domain. The corresponding functional domain refers to the functional domain in the orthologous gene of the gene named AO090005000664. By affecting the function of the functional domain, the productivity of ergothioneine can be enhanced in the microorganism producing ergothioneine having the AO090005000664 gene or its orthologous gene. From the viewpoint of enhancing the production of ergothioneine, the variation that changes the functional domain is the deletion of the region contained in the functional domain corresponding to the functional domain of W404 to Q515, or the variation, especially substitution, of G at the position corresponding to position 428 and / or C at the position corresponding to position 459.

[0121] The number of amino acids deleted is an arbitrary number that the production of ERG is enhanced in the microorganism in which the deletion occurs. As an example, the number of amino acids deleted can be deleted in a number selected from 1 to 120, 1 to 112, 1 to 60, 1 to 57 and 1 to 47. The number of amino acids deleted is preferably 1 to 30, and more preferably 1 to 26. As an example, at least one number of amino acids selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 is deleted. More specifically, the amino acid corresponding to position 428 can be deleted, or multiple amino acids containing the amino acid corresponding to position 428 can be deleted. In another embodiment, the amino acid not containing the amino acid corresponding to position 428 can also be deleted.

[0122] As an example, the conserved region included in positions 411 to 462 (SEQ ID NO: 15) and any sequence including position 428 may be deleted. As an example, the region corresponding to at least one region selected from 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) may be deleted. Furthermore, as another example, the region corresponding to at least one region selected from positions 431 to 475, 419 to 475, and 404 to 515 may be deleted.

[0123] In the present invention, the amino acid position is shown based on the amino acid position of SEQ ID NO: 1 encoded by the AO090005000664 gene, indicating the corresponding amino acid position in the protein encoded by the corresponding gene, i.e., the orthologous gene. The corresponding position can be determined by comparing the same sequence of the amino acid sequence of SEQ ID NO: 1.

[0124] The sequence of the region deleted relative to the functional domain corresponding to the functional domain of W404~Q515 has 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% identity. The sequence having the identity and improving the productivity of ergothioneine when the region contained in the functional domain corresponding to the functional domain of W404~Q515 is deleted in the filamentous fungus can be selected.

[0125] The function contributing to the generation of ergothioneine refers to some functions in the generation process of ergothioneine. As such a function, it can be a function of transcriptional regulation of an enzyme contributing to the synthesis pathway or decomposition pathway of ergothioneine, it can also be an enzyme reaction contributing to the synthesis pathway of ergothioneine, it can also be an enzyme reaction contributing to the decomposition pathway of ergothioneine or its precursor. The nucleic acid of the present invention encoding the sequence of the region contained in the functional domain corresponding to the functional domain of W404 to Q515 of SEQ ID NO: 1 is missing in the sequence corresponding to SEQ ID NO: 1 has the function of promoting the production of ergothioneine by being introduced into the filamentous fungi of ergothioneine production.

[0126] Furthermore, the present invention relates to a method for producing a microorganism that makes ergothioneine produce hyperfunction. More specifically, the method comprises deleting the region contained in the functional domain corresponding to the functional domain of W404 to Q515 of SEQ ID NO:1 in the sequence corresponding to SEQ ID NO:1. The deleted region is usually the region contained in the functional domain corresponding to the functional domain of W404 to Q515, and as long as ergothioneine can be made to produce hyperfunction, the adjacent region of 404 to 511 may also be deleted. When deleting the region contained in the functional domain corresponding to the functional domain of 404 to 511 of SEQ ID NO:1, the deleted region may contain the position corresponding to 428 of SEQ ID NO:1, or may not contain it. In the deleted region, it is preferably contained the position corresponding to 428 of SEQ ID NO:1. The deleted region is a region corresponding to at least one region selected from positions 428, 427 to 429, 427 to 440, 427 to 444, and 419 to 444 of SEQ ID NO: 1. The deleted region may be the entirety of the conserved region of positions 411 to 462 (SEQ ID NO: 15), or the entirety of the functional domain of positions 404 to 515. The scope of the deletion may be any deletion that can enhance the production of ergothioneine in the microorganism having the deletion. The continuous amino acid sequence in the functional domain of W404 to Q515, particularly the conserved region of positions 411 to 462 (SEQ ID NO: 15), may also be deleted in a discontinuous manner. The microorganism is an ergothioneine-producing microorganism selected from the genera Aspergillus, Penicillium, Rasamsonia and Talaromyces.

[0127] In another embodiment of the present invention, it may also be related to the corresponding sequence of SEQ ID NO: 1, the G at the position corresponding to position 428 of SEQ ID NO: 1 and / or the C at the position corresponding to position 459 have a variation, and when introduced into the microorganism producing ergothioneine, ergothioneine is produced hyperactive nucleic acid. The variation at the position, more specifically, may also be a substitution of G428S and / or C459F. By having the nucleic acid having a variation at the position, the microorganism producing ergothioneine produces hyperactive ergothioneine. More specifically, the corresponding sequence of SEQ ID NO: 1 has a conserved region defined by this specification.

[0128] Furthermore, the present invention may also relate to the following nucleic acids:

[0129] (1) an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, and 11, having S at the position corresponding to position 428 and / or having F at the position corresponding to position 459, or

[0130] (2) relative to the amino acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, having at least 60% identity, and having S at the position corresponding to position 428, and / or having F at the position corresponding to position 459, encoding an amino acid sequence, when introduced into a filamentous fungus that produces ergothioneine, the production of ergothioneine is enhanced. The production of ergothioneine can be enhanced by having G428S and C459F mutations.

[0131] The gene has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11. In particular, the orthologous gene of Aspergillus sojae and the orthologous gene of Aspergillus niger have 68% and 67% identity, respectively, and by introducing a mutation corresponding to G428S into Aspergillus sojae and Aspergillus niger (G442S in Aspergillus niger), enhancement of ERG production was confirmed ( Figure 4 and 5 ).

[0132] Furthermore, the present invention may also relate to the following nucleic acid: having at least 60% identity with the base sequence of SEQ ID NO: 2, 4, 6, 8, 10 and 12, being encoded at the position corresponding to position 428 and / or the position corresponding to position 459 of the amino acid sequence of SEQ ID NO: 1, and constituting a base sequence having a mutated amino acid sequence, and when introduced into a microorganism producing ergothioneine, ergothioneine is produced hyperactively. Ergothioneine can be produced hyperactively by having G428S and C459F mutations.

[0133] The homology with respect to SEQ ID NO: 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%. In particular, the homology with respect to Aspergillus sojae and Aspergillus niger is 68% and 67% respectively, and by introducing a mutation corresponding to G428S (G442S in Aspergillus niger), it was confirmed that the production of ERG was enhanced ( Figure 4 and 5 ).

[0134] The nucleic acid of the present invention may also be derived from filamentous fungi, such as Aspergillus, Penicillium, Rasamsonia or Talaromyces. The nucleic acid of the present invention is highly conserved in these filamentous fungi. The amino acid sequence encoded by the nucleic acid derived from the filamentous fungi belonging to the genus Aspergillus has 75-100% identity with SEQ ID NO: 1. The amino acid sequence encoded by the nucleic acid derived from the filamentous fungi belonging to the genus Penicillium has 68-75% identity with SEQ ID NO: 1. The amino acid sequence encoded by the nucleic acid derived from the filamentous fungi belonging to the genus Rasamsonia has 70% identity with SEQ ID NO: 1. The amino acid sequence encoded by the nucleic acid derived from the filamentous fungi belonging to the genus Talaromyces has 64-68% identity with SEQ ID NO: 1.

[0135] The function of contributing to the generation of ergothioneine refers to some enzyme reactions in the generation process of ergothioneine. It can be an enzyme reaction that contributes to the synthesis pathway of ergothioneine, or it can be an enzyme reaction that contributes to the decomposition pathway of ergothioneine or its precursor. When the enzyme reaction that contributes to the synthesis pathway is undertaken, the protein encoded by the nucleic acid of the present invention may have the synthesis activity of ergothioneine or its precursor. When the enzyme reaction that contributes to the decomposition pathway is undertaken, the protein encoded by the nucleic acid of the present invention may have the decomposition activity of ergothioneine or its precursor or metabolite. The nucleic acid of the present invention encoding the 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 has the function of promoting the production of ergothioneine by being introduced into the filamentous fungi that produce ergothioneine.

[0136] The position corresponding to position 428 of SEQ ID NO: 1 and the position corresponding to position 459 can be determined by aligning the same sequence of the amino acid sequence of SEQ ID NO: 1. In one example, it can be determined from the nearby sequence, and the position of G of LSGLQ, which is a nearby sequence of position 428, corresponds to position 428. The productivity of ergothioneine is enhanced by the mutation of G at position 428 to S. The position of C of APCED, which is a nearby sequence of position 459, corresponds to position 459. The productivity of ergothioneine is enhanced by the mutation of C at position 459 to F. The position corresponding to position 428 and the position corresponding to position 459 can be introduced with mutations individually or simultaneously.

[0137] Furthermore, the present invention relates to a method for producing a microorganism that makes ergothioneine produce hyperfunction. More specifically, the method comprises introducing a mutation to the position corresponding to position 428 and / or the position corresponding to position 459 of SEQ ID NO:1 relative to the genome of the microorganism encoding the conservative region to which the present invention relates. The mutation at the position corresponding to position 428 is G428S, and the mutation at position 459 is preferably C459F. The above-mentioned microorganism is an ergothioneine-producing microorganism selected from the genus Aspergillus, the genus Penicillium, the genus Rasamsonia and the genus Talaromyces. As an example, it relates to the above-mentioned method, which comprises introducing a mutation to the following conservative region corresponding to the partial sequence (positions 427 to 444) of SEQ ID NO:1:

[0138]

[0139] [In the sequence,

[0140] X430 is at least one amino acid selected from Q, H, P, and D;

[0141] X431 is at least one amino acid selected from Q, and L;

[0142] X432 is at least one amino acid selected from P, Q and G;

[0143] X433 is at least one amino acid selected from D, and E;

[0144] X435 is at least one amino acid selected from R, and W;

[0145] X437 is at least one amino acid selected from L and I;

[0146] X438 is at least one amino acid selected from V, and M;

[0147] X441 is at least one amino acid selected from V, I and L],

[0148] With respect to a genome having a conserved region encoding a mutation at position 428 and / or position 459, a mutation is introduced at a position corresponding to position 428 and / or a position corresponding to position 459,

[0149] The microorganism is an ergothioneine-producing microorganism selected from the genera Aspergillus, Penicillium, Rasamsonia and Talaromyces.

[0150] The introduction of genomic mutations can also be introduced using any method. It can be introduced using a fixed transformation system, or it can be introduced as a result of random mutations, or it can be introduced using genome editing technology. As an example, in the transformation system, a gene cassette can be used for introduction, a site-specific gene introduction can be performed, or it can be introduced by a vector, etc. In another embodiment of the present invention, it may also involve integrating a vector capable of expressing the nucleic acid of the present invention. As a method for introducing random mutations, for example, ultraviolet (UV) or X-ray irradiation that physically damages DNA to introduce mutations, treatment with alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonic acid (EMS) that chemically damages DNA to introduce mutations, etc. can be cited. As a genome editing technology, as an example, site-specific cutting and homologous recombination using the CRISPR / Cas system can be used. The present invention also relates to a microorganism containing the nucleic acid of the present invention. The present invention relates to a microorganism obtained as a result of the manufacturing method described.

[0151] By culturing the microorganisms to which the present invention relates, ergothioneine can be manufactured with high productivity. The microorganisms to which the present invention relates can also be cultured with either solid culture or liquid culture. As an example, by culturing the microorganisms to which the present invention relates, ergothioneine can be manufactured with high productivity. The microorganisms to which the present invention relates can also be cultured with either solid culture or liquid culture. As an example, by culturing the microorganisms to which the present invention relates in steamed rice, rice koji with high ergothioneine content can be manufactured. The koji can be used to manufacture soy sauce, sauce, salt koji, sweet sake, etc. through a known process in the art. In addition, aspergillus can also be collected to obtain mycoprotein. Thus, in one embodiment of the present invention, the present invention may also relate to a method for manufacturing rice koji containing high content of ergothioneine. The generated ergothioneine can be purified from the culture based on a certain method. As an example, the culture can be crushed to evaporate water to obtain a dry powder. By adding aqueous ethanol relative to the dried raw material containing ergothioneine, ergothioneine is extracted into an ethanol solution. It is also possible to purify ergothioneine by removing insoluble substances and subjecting the ergothioneine in the ethanol solution to chromatography.

[0152] Ergothioneine is known to have various effects such as elastase inhibition, tyrosinase inhibition, polyphenol oxidase (PPO) inhibition, etc., headed by antioxidation. Thus, the ergothioneine manufactured can be purified or directly coordinated in the products of food, cosmetics, quasi-drugs, and medicines, and can also be used as the concentrate modulation coordinated in the products. When coordinated as food, it can also be used as functional representation food. As the functional representation involved, the anti-wrinkle effect based on the elastase inhibition of ergothioneine, the beauty of skin or whitening effect based on tyrosinase inhibition activity, the preventive effect of lifestyle-related diseases based on the generation of lipid peroxides, the functions of the preventive effect of dementia or Alzheimer's disease based on the removal of active oxygen, etc. can be represented. The ergothioneine manufactured can also be the functional representation food, nutritional functional food, specific health food, or nutritional supplement (supplement) that pay special attention to the physiological effect of ergothioneine.

[0153] All documents mentioned in this disclosure are incorporated herein by reference in their entirety. Homogeneity was determined using the blastp program of NCBI using the protein database logged into NCBI. In this disclosure, except for homogeneity and identity, % refers to mass % unless otherwise specified.

[0154] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the patent claims. The present invention may be modified without departing from the purpose of the present invention, for example, the addition, deletion and replacement of the constituent elements of the present invention.

[0155] [Example]

[0156] [Example 1: Analysis of ERG high-producing strains]

[0157] (1) Culture medium

[0158] Eukaryotic organisms of the genus Aspergillus are cultured using the following culture media. In liquid culture, Czapek-DOX (Cz-DOX) medium (3.5% Czapek-DOX broth, manufactured by BD, pH 6.0), PD medium (2% dextrin, 1% polypeptone, 0.5% KH2PO4, 0.1% NaNO3, 0.05% MgSO4, 0.1% casamino acid, pH 6.0), Pafmin medium (5% PafminSM, pH not adjusted) are used. In plate culture, Cz-DOX agar medium (3.5% zapek-DOX broth, manufactured by BD, 0.1% trace element, 2% Agar added) or Malt agar medium (4.5% Malt Agar Nissui, manufactured by Nissui Pharmaceutical Co., Ltd., 0.1% trace element, 0.5% yeast extract) is mainly used.

[0159] (2) Genomic analysis of ERG high-producing strains

[0160] The whole genome of the ERG high-producing strain was analyzed and it was found that the mutation G428S or C459F in the gene called AO090005000664, whose function is unknown, contributes to the hyperproduction of ERG. The amino acid sequence (SEQ ID NO: 1) and base sequence (SEQ ID NO: 2) of AO090005000664 are as follows: Figure 1 .

[0161] When the amino acid sequence of AO090005000664 was searched using the default setting of BLAST (Basic Logical Alignment Search Tool) at NCBI (National Center for Biotechnology Information), it was found that the amino acid sequence was highly conserved in the genus Aspergillus, and a highly conserved region was found at positions 411 to 467 of SEQ ID NO: 1 ( Figure 2). The gene identities of AO090005000664 and the orthologous genes of Aspergillus oryzae, Aspergillus sojae, Aspergillus tamarii, Aspergillus luchuensis, Aspergillus niger, and Aspergillus nidulans are 100%, 98%, 96%, 67%, 67%, and 62%, respectively.

[0162] [Example 2: Preparation of mutant reproducing strains in Aspergillus oryzae RIB40 strain and confirmation of ERG productivity]

[0163] (1) Preparation of G428S box

[0164] Aspergillus oryzae RIB40_dKP strain (Δku70, ΔpyrG) was used as a host to prepare a gene cassette for introducing the G428S mutation into the AO090005000664 gene. Primers are shown in Table 1. Using the genomic sequence of the RIB40 strain as a template, the gene fragment was amplified by PCR using primer sets of AoG428S_F1 and R1, AoG428S_F2 and R2, AoG428S_F3 and R3, AopyrG F and R. PCR conditions were based on the attached process. The 4 purified PCR amplification fragments and linearized pUC19 (Takara) (0.5 μL each) were mixed with 2.5 μL of NEBuilder HiFi DNA Assembly Master Mix (NEB) and reacted at 60°C for 1 hour. 4 μL of the reaction product was introduced into ECOS Competent E.coli JM109 (Japan Gene) for transformation. Next, the plasmid extracted from the transformant was used as a template, and the primer set of AoG428S_NF and NR was used to amplify the gene fragment by PCR as the G428S cassette. 50 μL of the PCR product was ethanol precipitated and dissolved in 10 μL of sterile water.

[0165] [Table 1: G428S mutant reproducing strain of Aspergillus oryzae RIB40 strain]

[0166]

[0167] (2) Preparation of C459F box

[0168] A C459F cassette was prepared in the same manner as in (1) above, except that primers AoG428S_R2 were replaced with AoC459F_R2, and primers AoG428S_F3 were replaced with AoC459F_F3.

[0169] (3) Transformation of Aspergillus oryzae RIB40_dKP

[0170] The RIB40_dKP strain was spread on Cz-DOX_UraUri medium containing 10mM uridine and 10mM uracil and cultured at 30°C for 5 days. The meristems grown on the plate were picked with a microloop and inoculated into 40mL of PD medium containing 10mM uridine and 10mM uracil in a 150mL triangular flask. The culture was vortexed at 30°C and 180rpm for 20 hours and the cells were recovered.

[0171] The cultured cells were collected with Miraclose and washed with KCl buffer (0.6M KCl, 0.1M NaH2PO4, pH 5.5). The recovered cells were suspended in 20 mL of cell wall lytic enzyme (0.5% Lysing enzyme (Sigma-aldrich), 0.3% Yatalase (Takara), 1.5% Cellurase Onozuka (Yakult) / 20 mL KCl buffer), shaken at 30°C for 3 hours, and the filtrate was recovered through a 70 μm cell filter.

[0172] After the enzyme treatment, the protoplasts were precipitated by centrifugation (4°C, 3000 rpm, 10 min) and the supernatant was removed. Next, 30 mL of SolB buffer (1.2 M Sorbitol, 0.1 M KCl, 10 mM Tris-HCl; pH 7.5) was added to resuspend the protoplasts and transferred to an Eppendorf tube, and the protoplasts were recovered by centrifugation. Furthermore, 30 mL of SolB buffer was added and the same washing operation was repeated again. The recovered protoplasts were suspended in 0.4-0.8 mL of Sol B. To 100 μL of the protoplast suspension, 5-10 μg of the G428S cassette or the C459F cassette and 12.5 μL of SolC (50% PEG3350, 50 mM CaCl2, 10 mM Tris-HCl; pH 7.5) were added and mixed slowly, and then incubated on ice for 30 minutes. Then, add 1 mL of SolC, mix by pipetting, and let stand at room temperature for 10 minutes. Then, add 10 mL of SolB, precipitate protoplasts by centrifugation, and remove the supernatant. Load the entire amount on agar medium for regeneration (3.5% Czapek-DOX broth, 1.2M sorbitol, 0.1% trace element, 2% Agar), cover with 50°C Top Agar (3.5% Cz-DOX borth, 1.2M sorbitol, 0.1% trace element, 0.5% Agar), and culture at 30°C for about 1 week.

[0173] The obtained transformant was purified by plating three times on Cz-DOX agar medium (3.5% Czapek-DOX borth, 0.1% traceelement, 1.5% Agar).

[0174] (4) Confirmation of ERG production by mutant reproducing strains

[0175] The ERG production levels of the transformants obtained above and the RIB40 wild-type strain as a control were compared.

[0176] (4-1) Culture / Extraction

[0177] Prepare 10 mL of Pafmin medium (5% PafminSM, pH not adjusted) in a 50 mL triangular flask and sterilize by autoclaving at 120°C for 30 minutes. Add 100 μL of the meristem suspension (1.0×10 7 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 filter device (3K manufactured by Pall Corporation, Japan), and diluted with appropriate ultrapure water to prepare the analysis sample.

[0178] (4-2) Analysis conditions

[0179] The amount of ERG in the analysis sample was measured under the following analysis conditions.

[0180]

LC / MS analysis conditions

[0181] Analytical device: UPLC CQ micro; Waters UPLC

[0182] Column: 2.5HILIC 3.0mml.D.×150mm

[0183] Solvent A: 0.1% formic acid / acetonitrile

[0184] Solvent B: 0.1% formic acid / H2O

[0185] Flow rate: 0.5ml / min 80%A

[0186] inject:3μl

[0187] Mass spectrometer

[0188] ESI: ES+

[0189] Cone V: 21V

[0190] Capillary V: 4.5kV

[0191] Source temperature: 120℃

[0192] Desolvation temperature: 400℃

[0193] MS Scan Mode

[0194] [Analysis conditions of ERG by LC / MS / MS]

[0195] Analytical device: UPLC CQ micro; Waters UPLC

[0196] Column: 2.5HILIC 3.0mml.D.×150mm

[0197] Solvent A: 0.1% formic acid / acetonitrile

[0198] Solvent B: 0.1% formic acid / H2O

[0199] Flow rate: 0.5ml / min 80%A

[0200] Wash the column with 100% B for each sample.

[0201] inject:3μL

[0202] Mass spectrometer

[0203] ESI: ES+

[0204] Cone V: 21V

[0205] Capillary V: 4.5kV

[0206] Source temperature: 120℃

[0207] Desolvation temperature: 400℃

[0208] Collision energy: 11V

[0209] Trace: m / z 230.1>186.1

[0210] Collision energy: 20V

[0211] Trace: m / z 230.1>127.0

[0212] (5) Sequence confirmation of transformants

[0213] To 19.2 μL of KOD one (Toyobo) PCR reaction solution, add 0.8 μL of the meristem suspension of the bacteria cultured in Cz-DOX agar medium for 4 days with claw poplar branches suspended in 100 μL of TE at a degree of 2 picks, and perform colony PCR. The PCR product was purified with QIAquick PCR purification kit (QIAGEN). Elution Buffer (QIAGEN) was used in the elution from the column, and the elution amount was eluted with 50 μL of the same amount as the PCR system. The purified PCR product was entrusted to Fasmac to confirm the sequence.

[0214] G428S mutation was introduced into 2 of 48 transformants in which G428S mutation was reproduced. C459F mutation was introduced into 1 of 40 transformants in which C459F mutation was reproduced. The ERG production amount of each strain is shown in Figure 3 When the ERG productivity of the mutant strains was compared, the ERG productivity was improved compared with the control strain and the wild-type strain. The ERG production of the C459F mutant strain was less than that of the G428S mutant strain, indicating that the C459F mutation is also involved in the improvement of ERG productivity.

[0215] [Example 3: Preparation of mutant reproducing strains in Aspergillus sojae and confirmation of ERG productivity]

[0216] The mutation introduction, transformation, and confirmation of ERG production were performed using Aspergillus sojae NBRC4239_dKP strain (Δku70, ΔpyrG) as a host in the same manner as described in the section of the preparation of the G428S mutant reproducing strain of Aspergillus oryzae RIB40 strain in Example 1. The amino acid identity of the orthologous gene of the AO090005000664 gene in Aspergillus sojae was 98%, and the amino acid identity was 100%.

[0217] [Table 2: Primer list]

[0218] Primer name Sequence (5′-3′) SEQ ID NO: AsG428S_F1 CGGTACCCGG GGATCcaccg cagccaatcc ccagtt SEQ ID NO:34 AsG428S_R1 tagcaataag cccaagacat cgggaattgg gtcatccgg SEQ ID NO:35 AsG428S_F2 tttcttctga gggtgcctcgc acagccatgc tgtgcc SEQ ID NO:51 AsG428S_R2 GCTGTTGCAG GCtCGAGAGA TACC SEQ ID NO:52 AsgG428S_F3 GGTATCTCTC GaGCCTGCAA CAGC SEQ ID NO:53 AsG428S_R3 CGACTCTAGA GGATCgacca ccttggaaga aagctgacct ga SEQ ID NO:36 AopyrG_F ttgggctttat tgctatgtcc ctgaaagg SEQ ID NO:54 AspyrG_R gcacctcaga agaaaaggat gatcaatacc SEQ ID NO:55 AsG428S_NF gccgtgggca taggcacgat tg SEQ ID NO:41 AsG428S_NR gctagcatca catctcacga cacgtcc SEQ ID NO:42

[0219] Among the 19 transformants, 6 strains reproduced the G428S mutation. The ERG production amount of each strain is shown in Figure 4 When the ERG productivity of the mutant strains was compared, the ERG productivity was improved compared with the control strain and the wild-type strain. It was found that the present gene mutation also improved the ERG productivity in Aspergillus lus sojae.

[0220] [Example 4: Preparation of mutant reproducing strains in Aspergillus niger and confirmation of ERG productivity]

[0221] In the same manner as described in the section of the preparation of the G428S mutant reproducing strain of Aspergillus oryzae RIB40 strain AO090005000664 in Example 1, Aspergillus niger A1179 strain (also known as MA70.15, Genotyping: kusA::amdS, cspA1 (a mutation resulting in short conidiophores), pyrG - ) was used as a host, mutations were introduced and transformed, and the number of culture days was changed to 6 days to confirm the ERG production amount.

[0222] [Table 3: Primers used for mutation introduction and gene disruption strain preparation of Aspergillus niger A1179 strain]

[0223]

[0224] The amino acid identity of the orthologous gene of AO090005000664 in Aspergillus niger is 67%, and the amino acid identity is 79%. The mutation points (G428S and C459F) of the Aspergillus oryzae strain with high ERG productivity are also conserved in Aspergillus niger (G442 and C473). Thus, a mutant strain was prepared in which the 442nd glycine equivalent to G428S, which was confirmed to have a mutation effect in the Aspergillus oryzae RIB40 strain, was replaced with serine. The ERG production amount of each strain is shown in Figure 5 When the ERG productivity of the mutant strains was compared, the ERG productivity was improved compared with the control strain and the wild-type strain. It was found that the present gene mutation also improved the ERG productivity in Aspergillus niger.

[0225] [Example 5: Analysis of ERG high-producing strains]

[0226] (1) Culture medium

[0227] Eukaryotic organisms of the genus Aspergillus are cultured using the following culture media. For liquid culture, Czapek-DOX (Cz-DOX) medium (3.5% Czapek-DOX broth, manufactured by BD, pH 6.0), PD medium (2% dextrin, 1% polypeptone, 0.5% KH2PO4, 0.1% NaNO3, 0.05% MgSO4, 0.1% casamino acid, pH 6.0), and Pafmin medium (5% PafminSM, pH not adjusted) are used. For plate culture, Cz-DOX agar medium (3.5% zapek-DOX broth, manufactured by BD, 0.1% trace element, 2% Agar added) or Malt agar medium (4.5% Malt Agar Nissui, manufactured by Nissui Pharmaceutical Co., Ltd., 0.1% trace element, 0.5% yeast extract) is mainly used.

[0228] (2) Genomic analysis of ERG high-producing strains

[0229] The whole genome of the ERG high-producing strain was analyzed and it was found that the amino acid deficiency in a gene called AO090005000664, whose function is unknown, contributes to the hyperproduction of ERG. The amino acid sequence (SEQ ID NO: 1) and base sequence (SEQ ID NO: 2) of AO090005000664 are as follows: Figure 1 .

[0230] When the amino acid sequence of AO090005000664 was searched using the default setting of BLAST (Basic Logical Alignment Search Tool) at NCBI (National Center for Biotechnology Information), it was found that the amino acid sequence was highly conserved in the genus Aspergillus, and a highly conserved region was found at positions 411 to 467 of SEQ ID NO: 1 ( Figure 6 ). The gene identities of AO090005000664 and the orthologous genes of Aspergillus oryzae, Aspergillus sojae, Aspergillus tamarii, Aspergillus luchuensis, Aspergillus niger, and Aspergillus nidulans are 100%, 98%, 96%, 67%, 67%, and 62%, respectively.

[0231] [Example 6: Preparation of amino acid-deficient strain of Aspergillus oryzae RIB40 strain]

[0232] (1) Preparation of amino acid deficiency cassette and acquisition of transformant strain of Aspergillus oryzae RIB40_dKP

[0233] The RIB40_dKP strain (Δku70, ΔpyrG) was used as a host to produce a gene cassette with amino acid deletions. The amino acids to be deleted were highly conserved amino acids with G428 as the center ( Figure 6). Specifically, positions 428, 427-429, 427-440, 427-444, and 419-444 were deleted respectively. The primers used are shown in Table 1. Using the genome sequence of the RIB40 strain as a template, the gene fragment was amplified by PCR using primer sets of primers 1 and 2, primers 3 and 4, and primers 5 and 6. The PCR conditions were according to the attached process. The three purified PCR amplification fragments and the linearized pUC19 (Takara) (0.5 μL each) were mixed with 2.5 μL of NEBuilder HiFi DNA Assembly Master Mix (NEB) and reacted at 60°C for 1 hour. 4 μL of the reaction product was introduced into ECOS Competent E. coli JM109 (Japan Gene) for transformation. Next, the plasmid extracted from the transformant was used as a template, and the gene fragment was amplified by PCR using the set of primers 7 and 8 (Ao3DEL), primers 9 and 8 (Ao18DEL), and primers 9 and 10 (Ao26DEL). The PCR conditions were based on the attached process. DpnI was added to each PCR reaction solution and reacted at 37°C for 1 hour. Next, 2μl of each PCR product treated with DpnI was mixed with 5μl of Ligation high Ver.2 (Toyobo), 1μl of T4 Polynucleotide Kinase, and 7μl of sterile water, and reacted at 16°C for 1 hour. 5μL of each reaction product was introduced into ECOSCompetent E.coli JM109 (Japan Gene) for transformation. Next, using each plasmid extracted from the transformant as a template, using the set of primers 11 and 12, the gene fragment amplified by PCR was used as the respective amino acid deficiency box AoXDEL box (X=3, 18, 26). 50 μL of the PCR product was subjected to ethanol precipitation and dissolved in 10 μL of sterile water. The AoXDEL cassette was introduced to transform the RIB40_dKP strain. The transformant was purified by spot-plating three times in Cz-DOX agar medium.

[0234] (2) Meristem PCR

[0235] The cassette introduction position was confirmed from the size of the band using primers 1 and 4. In addition, the PCR product was purified and sequenced to confirm the amino acid deletion.

[0236] [Table 4: Primers used in the preparation of amino acid-deficient strains]

[0237]

[0238]

[0239] (3) Confirmation of ERG production in amino acid-deficient strains

[0240] The transformants obtained above and the control were cultured in 50 ml triangular flasks with 10 ml of 5% PafminSM medium at 26°C for 7 days. After the culture, the cells were heated at 90°C for 1 hour in the dissolution and insulation mode of the autoclave, and the supernatant was filtered with a Nanosep centrifugal filter device (3K manufactured by Pall Corporation, Japan) and quantified by LC / MS / MS ( FIG. 7A ).

[0241] (3-2) Analysis conditions

[0242] The amount of ERG in the analysis sample was measured under the following analysis conditions.

[0243]

LC / MS analysis conditions

[0244] Analytical device: UPLC CQ micro; Waters UPLC

[0245] Column: 2.5HILIC 3.0mml.D.×150mm

[0246] Solvent A: 0.1% formic acid / acetonitrile

[0247] Solvent B: 0.1% formic acid / H2O

[0248] Flow rate: 0.5ml / min 80%A

[0249] inject:3μl

[0250] Mass spectrometer

[0251] ESI: ES+

[0252] Cone V: 21V

[0253] Capillary V: 4.5kV

[0254] Source temperature: 120℃

[0255] Desolvation temperature: 400℃

[0256] MS Scan Mode

[0257] [Analysis conditions of ERG by LC / MS / MS]

[0258] Analytical device: UPLC CQ micro; Waters UPLC

[0259] Column: 2.5HILIC 3.0mml.D.×150mm

[0260] Solvent A: 0.1% formic acid / acetonitrile

[0261] Solvent B: 0.1% formic acid / H2O

[0262] Flow rate: 0.5ml / min 80%A

[0263] Wash the column with 100% B for each sample.

[0264] inject:3μL

[0265] Mass spectrometer

[0266] ESI: ES+

[0267] Cone V: 21V

[0268] Capillary V: 4.5kV

[0269] Source temperature: 120℃

[0270] Desolvation temperature: 400℃

[0271] Collision energy: 11V

[0272] Trace: m / z 230.1>186.1

[0273] Collision energy: 20V

[0274] Trace: m / z 230.1>127.0

[0275] [Example 7: Preparation of amino acid-deficient strain of Aspergillus lus sojae NBRC4239]

[0276] (1) Preparation of amino acid deficiency cassette and acquisition of transformant strain of Aspergillus sojae NBRC4239_dKP

[0277] Using NBRC4239_dKP strain (Δku70, ΔpyrG) as the host, a gene cassette with amino acid deficiency was prepared in the same manner as the above-mentioned Aspergillus oryzae RIB40 strain. Using the genomic sequence of the NBRC4239 strain as a template, the gene fragment was amplified by PCR using a primer set of primers 13 and 14, primers 3 and 15, and primers 5 and 6. The PCR conditions were based on the attached process. The three purified PCR amplification fragments and the linearized pUC19 (Takara) (0.5 μL each) were mixed with 2.5 μL of NEBuilder HiFi DNA Assembly Master Mix (NEB) and reacted at 60°C for 1 hour. 4 μL of the reaction product was introduced into ECOS Competent E. coli JM109 (Japan Gene) for transformation. Next, the gene fragment was amplified by PCR using the plasmid extracted from the transformant as a template, using the set of primers 16 and 17 (As1DEL), primers 7 and 8 (As3DEL), primers 18 and 8 (As14DEL), primers 9 and 8 (As18DEL), and primers 19 and 10 (As26DEL). Using each plasmid obtained in the same manner as the above-mentioned Aspergillus oryzae RIB40 strain as a template, using the set of primers 20 and 21, the gene fragment amplified by PCR was used as each amino acid deficiency box AsXDEL box (X=1, 3, 14, 18, 26). 50 μL of the PCR product was ethanol precipitated and dissolved in 10 μL of sterile water. The AsXDEL box was introduced to transform the NBRC4239_dKP strain. The transformant was purified by spot planting three times in Cz-DOX agar medium.

[0278] (2) Meristem PCR

[0279] The cassette introduction position was confirmed from the size of the band using primers 13 and 15. In addition, the PCR product was purified and sequenced to confirm the amino acid deletion.

[0280] (3) Confirmation of ERG production in amino acid-deficient strains

[0281] The same procedure as that for the Aspergillus oryzae RIB40 strain described in Example 2 (3) was carried out to confirm the ERG production amount of the amino acid-deficient strain ( FIG. 7B ).

[0282] [Example 8: Preparation of amino acid-deficient strain of Aspergillus oryzae RIB40 strain]

[0283] (1) Preparation of amino acid deficiency cassette and acquisition of transformant strain of Aspergillus oryzae RIB40_dKP

[0284] Using RIB40_dKP strain (Δku70, ΔpyrG) as a host, a gene cassette that causes amino acid deficiency was prepared. Based on the predicted structure of Example 10, the amino acid was used to cause a part or the entirety of the domain to be deleted. Specifically, positions 431 to 475, 419 to 475, and 404 to 515 were each deleted. The primers used are shown in Table 1. The plasmid prepared in Example 2 was used as a template, and the gene fragment was amplified by PCR using a group of primers 22 and 23 (Ao45DEL), primers 22 and 10 (Ao57DEL), and primers 24 and 25 (Ao112DEL). The PCR conditions were based on the attached process. DpnI was added to each PCR reaction solution and reacted at 37°C for 1 hour. Next, 2 μl of each PCR product treated with DpnI was mixed with 5 μl of Ligation high Ver.2 (Toyobo), 1 μl of T4 Polynucleotide Kinase, and 7 μl of sterile water, and reacted at 16°C for 1 hour. 5 μL of each reaction product was introduced into ECOS Competent E. coli JM109 (Japan Gene) for transformation. Next, each plasmid extracted from this transformant was used as a template, and a set of primers 11 and 12 was used, and the gene fragment amplified by PCR was used as each amino acid deficiency box AoXDEL box (X=45, 57, 112). 50 μL of the PCR product was ethanol precipitated and dissolved in 10 μL of sterile water. The AoXDEL box was introduced to transform the RIB40_dKP strain. The transformant was purified by spot planting three times in Cz-DOX agar medium.

[0285] (2) Meristem PCR

[0286] The cassette introduction position was confirmed from the size of the band using primers 1 and 4. In addition, the PCR product was purified and sequenced to confirm the amino acid deletion.

[0287] (3) Confirmation of ERG production in amino acid-deficient strains

[0288] The same procedure as that for the Aspergillus oryzae RIB40 strain described in Example 2 (3) was carried out to confirm the ERG production amount of the amino acid-deficient strain ( FIG. 7C ).

[0289] [Example 9: Preparation of amino acid-deficient strain of Aspergillus niger FGSC A1179 strain]

[0290] (1) Preparation of amino acid deficiency cassette and acquisition of transformant strain of Aspergillus niger FGSC A1179

[0291] Using FGSC A1179 strain (ΔkusA, pyrG-) as the host, a gene cassette with the same amino acid deficiency as the above-mentioned Aspergillus oryzae RIB40 strain was prepared. Using the genomic sequence of Aspergillus niger IAM2533 strain as a template, the gene fragment was amplified by PCR using primer sets of primers 26 and 27, primers 28 and 29. The PCR conditions were based on the attached process. pUC19 (Takara) (0.5 μL each) linearized with the two purified PCR amplification fragments and the gene fragments of primers 5 and 6 purified in Example 2 was mixed with 2.5 μL of NEBuilder HiFi DNA Assembly Master Mix (NEB) and reacted at 60°C for 1 hour. 4 μL of the reaction product was introduced into ECOS Competent E.coli JM109 (Japan Gene) for transformation. Next, the gene fragment was amplified by PCR using the plasmid extracted from the transformant as a template, using the set of primers 30 and 31 (Ang3DEL), primers 32 and 31 (Ang18DEL), primers 32 and 33 (Ang26DEL), primers 34 and 33 (Ang57DEL), and primers 35 and 36 (Ang112DEL). Using each plasmid obtained in the same manner as the above-mentioned Aspergillus oryzae RIB40 strain as a template, the set of primers 37 and 38 was used, and the gene fragment amplified by PCR was used as each amino acid deficiency box AsXDEL box (X=3, 18, 26, 57, 112). 50 μL of the PCR product was ethanol precipitated and dissolved in 10 μL of sterile water. The AngXDEL box was introduced to transform the FGSC A1179 strain. The transformant was purified by spot planting three times in Cz-DOX agar medium.

[0292] (2) Meristem PCR

[0293] The position of the cassette introduction was confirmed from the size of the band using primers 26 and 29. In addition, the PCR product was purified and sequenced to confirm the amino acid deletion.

[0294] (3) Confirmation of ERG production in amino acid-deficient strains

[0295] The same procedure as for the Aspergillus oryzae RIB40 strain described in Example 2 (3) was carried out to confirm the ERG production amount of the amino acid-deficient strain ( FIG. 7D ).

[0296] [Example 10: Structural analysis of the protein expressed from AO090005000664 gene]

[0297] Using the homology modeling function of SWISS-MODEL (https: / / swissmodel.expasy.org / ), the predicted three-dimensional structure of the AO090005000664 gene was used as a template to predict the three-dimensional structure ( Figure 8 ). As the three-dimensional structure of the template, among the genes with high homology to AO090005000664, the three-dimensional structure is known, or the predicted three-dimensional structure is searched and disclosed, and the predicted three-dimensional structure of PAAG_04735 from Paracoccidioides lutzii with 43% identity is selected. In addition, the predicted three-dimensional structure of PAAG_04735 is predicted using αFold2. Currently, the predicted structure of OAory_01008280, which has 100% identity with the AO090005000664 gene, is disclosed in the α-fold protein structure database (https: / / alphafold.com / ), and the predicted structure of homology modeling is similar to this. In addition, at the time of homology modeling, the predicted structure of OAory_01008280 was not disclosed.

[0298] According to the research of the present inventors, in the amino acid sequence encoded by the AO090005000664 gene, positions 427 to 429 are deleted (3DEL), positions 427 to 444 are deleted (18DEL), positions 419 to 444 (26DEL), positions 431 to 475 (45DEL), positions 419 to 475 (57DEL), and positions 404 to 515 (112DEL) are deleted (Figures 7A and C). Furthermore, positions 431 to 475 correspond to the second α helix and the third helix in the super secondary structure domain composed of 5 α helices. Positions 404 to 515 correspond to the entire super secondary structure domain. In addition, in Aspergillus sojae (A. sojae), which has a gene modified in such a way that the amino acid sequence encoded by the orthologous gene having 98% identity with the AO090005000664 gene has a deletion at position 428 (1DEL), a deletion at positions 427 to 429 (3DEL), a deletion at positions 427 to 440 (14DEL), a deletion at positions 427 to 444 (18DEL), and a deletion at positions 419 to 444 (26DEL), ergothioneine production was enhanced ( Figure 7B ). Furthermore, in Aspergillus niger (A. niger), which has a gene modified in such a way that the An16g07990 gene, which has only 67% identity with the AO090005000664 gene, has a deletion at positions 441 to 443 (3DEL), a deletion at positions 441 to 458 (18DEL), a deletion at positions 433 to 458 (26DEL), a deletion at positions 433 to 489 (57DEL), and a deletion at positions 418 to 529 (112DEL), ergothioneine production was enhanced ( FIG. 7D ).

[0299] For AO090005000664 gene and its corresponding gene, the function of the super secondary structure domain contained in W404~Q515 containing G428 position is still unclear, and it can be shown that the function of the super secondary structure domain is suppressed at least in part, and thioneine productivity is improved. Furthermore, referring to the position in AO090005000664 gene, the position corresponding to the gene is different, and as long as it is a person skilled in the art, it can be fully understood.

Claims

1. A nucleic acid encoding: A region containing a conserved region contained in SEQ ID NO: 1, in which a region contained in a functional domain corresponding to the functional domain of W404 to Q515 of SEQ ID NO: 1 in a sequence corresponding to SEQ ID NO: 1 contains a variant sequence that changes the function of the functional domain; or a sequence that is at least 90% identical to said sequence, and When introduced into a microorganism that produces ergothioneine, the production of ergothioneine is enhanced. 2 . The nucleic acid according to claim 1 , wherein the conserved region is the conserved region from position 433 to position 440 of SEQ ID NO:

1. 3 . The nucleic acid according to claim 1 , wherein the mutation that changes the function of the functional domain is a deletion of a region contained in the functional domain corresponding to the functional domain of W404 to Q515.

4. The nucleic acid of claim 1, wherein the deleted region comprises a position corresponding to position 428 of SEQ ID NO:

1. The nucleic acid according to claim 1 , wherein the deleted region consists of a deletion of 1 to 120 amino acids. 6 . The nucleic acid of claim 4 , wherein the deleted region corresponds to 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. 7 . The nucleic acid of claim 3 , wherein the deleted region is a region corresponding 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.

8. The nucleic acid of claim 1, wherein the variation that changes the function of the functional domain has a variation in G at the position corresponding to position 428 and / or C at the position corresponding to position 459 of SEQ ID NO:

1.

9. The nucleic acid of claim 1, wherein the nucleic acid is of microbial origin selected from the group consisting of Aspergillus, Penicillium, Rasamsonia, and Talaromyces.

10. A vector comprising the nucleic acid according to any one of claims 1 to 9.

11. A microorganism comprising the nucleic acid according to any one of claims 1 to 9.

12. A method for producing ergothioneine, comprising culturing the microorganism according to claim 11.

13. A method for producing a microorganism that produces hyperfunction of ergothioneine, comprising: Introducing a mutation that changes the function of the functional domain into a region contained in a functional domain corresponding to the functional domains W404 to Q515 of SEQ ID NO: 1 in a sequence corresponding to SEQ ID NO: 1, including a conserved region at positions 433 to 440 of SEQ ID NO: 1, The microorganism is an ergothioneine-producing microorganism selected from the genera Aspergillus, Penicillium, Rasamsonia and Talaromyces. 14 . The production method according to claim 13 , wherein the mutation that changes the function of the functional domain is a deletion of a region contained in the functional domain corresponding to the functional domain of W404 to Q515. The production method according to claim 13 , wherein the conserved region is a conserved region from position 433 to position 440 of SEQ ID NO:

1. The manufacturing method according to claim 14 , wherein the deleted region includes a position corresponding to position 428 of SEQ ID NO:

1.

17. The production method according to claim 14, wherein the deleted region consists of 1 to 120 amino acids. 18 . The production method of claim 16 , wherein the deleted region is a region corresponding to at least one region selected from the group consisting of position 428, positions 427 to 429, positions 427 to 440, positions 427 to 444, and positions 419 to 444 of SEQ ID NO:

1. 19 . The production method according to claim 14 , wherein the deleted region is a region corresponding 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. 20 . The production method of claim 14 , wherein the variation that changes the function of the functional domain has a variation in G at the position corresponding to position 428 and / or C at the position corresponding to position 459 of SEQ ID NO: 1.

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