Method for synthesizing cerocyanine II, method for synthesizing betaxanthin, amyloid beta polymerization inhibitor or Alzheimer's treatment or prevention agent, amyloid peptide aggregation inhibitor, and HIV-1 protease activity inhibitor

Abstract

The present invention provides: a method for synthesizing celosianin II; a method for synthesizing betaxanthin; an amyloid-β polymerization inhibitor, or a therapeutic or prophylactic agent for Alzheimer's disease; an amyloid peptide aggregation inhibitor; and an HIV-1 protease activity inhibitor.　A celosianin II synthesis method according to the present invention was realized through the isolation, from quinua, of a gene having a capability of synthesizing celosianin II. Furthermore, it was confirmed that celosianin II and the like can serve as an active ingredient of: an amyloid-β polymerization inhibitor, or a therapeutic or prophylactic agent for Alzheimer's disease; an amyloid peptide aggregation inhibitor; and an HIV-1 protease activity inhibitor.

Description

[Technical Field]

[0001] The present invention relates to a method for synthesizing cerocyanine II, a method for synthesizing betaxanthin, an inhibitor of amyloid β polymerization or an agent for treating or preventing Alzheimer's disease, an inhibitor of amyloid peptide aggregation, and an inhibitor of HIV-1 protease activity. This application claims priority from Japanese Patent Application No. 2020-119563 and Japanese Patent Application No. 2019-229647, which are incorporated herein by reference. [Background technology]

[0002] (betalain) Betalains, a type of plant pigment, are produced by the Caryophyllaceae and some fungi. Betalain pigments are broadly divided into two types: red to purple betacyanins and yellow to orange betaxanthins. They are known to be involved in the resistance of plants to environmental stress (Non-Patent Documents 1 and 2). Due to their vivid colors, betalain pigments are used as food additives. Furthermore, because betalain pigments have high antioxidant activity, they are expected to be used as pharmaceuticals and supplements. Previous studies have reported that betalain pigments have physiological effects such as anti-inflammatory and anti-cancer effects, suppression of LDL cholesterol oxidation, and inhibition of HIV-1 protease activity (Non-Patent Documents 3-9, Figure 1). Betalain pigments have various physiological effects, but their production is difficult because they are mainly extracted from betalain-producing plants. Recently, it has been reported that betalains have been successfully produced by introducing betalain biosynthesis genes into non-betalain-producing plants (Non-Patent Document 2). The present inventors have also succeeded in synthesizing amaranthine, a betalain pigment (Non-Patent Document 4). However, of the many types of betalain pigments that exist, only a limited number can be artificially synthesized, and the physiological activities of only a few individual betalain pigments have been clarified.

[0003] (Quinoa) Quinoa (Chenopodium quinoa) is a plant belonging to the genus Chenopodium in the subfamily Chenopodiaceae of the family Amaranthaceae, and is rich in nutrients and has excellent environmental stress tolerance. It is known that three types of betalains (betanin, amaranthin, and cerocyanin II) are synthesized and accumulated in the hypocotyl of quinoa (Figures 1 and 2). The present inventors have succeeded in synthesizing betanin and amaranthin in tobacco BYII cells (Non-Patent Document 4).

[0004] The synthetic gene for cerocyanin II has not been identified, and no artificial production system has been established in non-betalain-producing plants. Furthermore, the properties and physiological activities of cerocyanin II have not been reported and are unknown (Figure 1).

[0005] Patent Document 1 discloses "a method for producing betacyanins, comprising a step (conversion step) of converting a raw material into betacyanins in an aqueous medium in the presence of a microorganism or a processed product thereof, which has the enzyme activity of hydroxylating the 3-position of the phenolic ring of tyrosine, DOPA 4,5-dioxygenase activity, L-DOPA oxidase activity, and enzyme activity of adding sugar to a phenolic hydroxyl group." However, the present invention does not disclose or suggest the cerocyanine II synthase gene or the genes required for betaxanthin synthesis. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2016-182044 [Non-patent literature]

[0007] [Non-Patent Document 1] Jain , G. , Schwinn , KE and Gould , KS(2015) Betalain induction byl-DOPA application confers photoprotection of tosaline-exposed leaves of Dysphymaaustrale . New Phytol. Rev. 207, 1075–1

Outdoor Tool2

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Outdoor Tools 4

Direct Environment 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

[0008] An objective of the present invention is to provide a method for synthesizing cerocyanine II, a method for synthesizing betaxanthin, an amyloid β polymerization inhibitor or an agent for treating or preventing Alzheimer's disease, an amyloid peptide aggregation inhibitor, and an inhibitor of HIV-1 protease activity. [Means for solving the problem]

[0009] In order to achieve the above object, the present inventors isolated a gene capable of synthesizing cerocyanine II from quinoa and constructed a method for synthesizing cerocyanine II of the present invention. Furthermore, in order to solve the above problems, the present inventors isolated the gene required for betaxanthin synthesis from quinoa and constructed the method for synthesizing betaxanthin of the present invention. In addition, it has been confirmed that cerocyanine II and the like are active ingredients in amyloid beta polymerization inhibitors or therapeutic or preventive agents for Alzheimer's disease, amyloid peptide aggregation inhibitors, and HIV-1 protease activity inhibitors.

[0010] That is, the present invention is as follows. 1. A method for synthesizing cerocyanine II, comprising: A host into which the following genes have been introduced, which are a gene encoding cerocyanine II synthase, a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine, a gene encoding an enzyme having L-DOPA oxidase activity and / or a gene encoding an enzyme having DOPA 4,5-dioxygenase activity, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid, and which has the ability to produce tyrosine or 3-hydroxy-L-tyrosine (L-DOPA), is cultured, and cerocyanine II is extracted from the cultured host. Or, a host into which a gene encoding the following cerocyanine II synthase has been introduced, and which has an enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine, an L-DOPA oxidase activity and / or a DOPA 4,5-dioxygenase activity, an enzyme activity of adding a sugar to a phenolic hydroxyl group, an enzyme activity of adding glucuronic acid, and an ability to produce tyrosine or 3-hydroxy-L-tyrosine; and extracting cerocyanine II from the cultured host; wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (3) a gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, (5) A gene comprising a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and encodes a polypeptide having the ability to synthesize cerocyanine II; (6) A gene consisting of DNA in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added in DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15. (7) A gene consisting of DNA having 90% or more homology to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, A method for producing cerocyanine II, characterized by: 2. A method for synthesizing cerocyanine II, comprising the steps of: a host into which a gene encoding the following cerocyanine II synthase has been introduced and which has the ability to produce amaranthin is cultured, and cerocyanine II is extracted from the cultured host; wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (3) a gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, (5) A gene comprising a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and encodes a polypeptide having the ability to synthesize cerocyanine II; (6) A gene consisting of DNA in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added in DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15. (7) A gene consisting of a DNA having 90% or more homology to a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, A method for producing cerocyanine II, characterized by: 3. A method for synthesizing cerocyanine II, comprising the steps of: a host having the ability to produce betanin, into which a gene encoding the cerocyanine II synthase and a gene encoding an enzyme having the activity of adding glucuronic acid are introduced, and cerocyanine II is extracted from the host after the culture; Or, A host into which a gene encoding the following cerocyanine II synthase has been introduced and which has an enzyme activity of adding glucuronic acid and an ability to produce betanin is cultured, and cerocyanine II is extracted from the cultured host; or wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (3) a gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, (5) A gene comprising a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and encodes a polypeptide having the ability to synthesize cerocyanine II; (6) A gene consisting of DNA in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added in DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15. (7) A gene consisting of a DNA having 90% or more homology to a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, A method for producing cerocyanine II, characterized by: 4. A method for synthesizing cerocyanine II, comprising the steps of: a host having the ability to produce betanidin, into which a gene encoding cerocyanine II synthase, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid are introduced, and cerocyanine II is extracted from the host after the culture; Or, a host into which a gene encoding the following cerocyanine II synthase has been introduced, and which has an enzyme activity of adding sugar to a phenolic hydroxyl group, an enzyme activity of adding glucuronic acid, and an ability to produce betanidin, and then extracting cerocyanine II from the cultured host; wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (3) a gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, (5) A gene comprising a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and encodes a polypeptide having the ability to synthesize cerocyanine II; (6) A gene consisting of DNA in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added in DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15. (7) A gene consisting of DNA having 90% or more homology to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, A method for producing cerocyanine II, characterized by: 5. A method for synthesizing cerocyanine II according to any one of items 1 to 4 above, wherein the gene encoding the cerocyanine II synthase is selected from one or more genes consisting of DNA having the base sequence set forth in SEQ ID NOs: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 and 15. 6. A cerocyanine II synthetic composition comprising any one of the following genes or a vector carrying said gene: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (3) a gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, (5) A gene comprising a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and encodes a polypeptide having the ability to synthesize cerocyanine II; (6) A gene consisting of DNA in which 1 to 50 base sequences have been substituted, deleted, inserted and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, and (7) a gene consisting of DNA having 90% or more homology to DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15. 7. A synthetic composition of cerocyanine II having a peptide represented by any one of the following amino acid sequences: (1) an amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) an amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and which forms a polypeptide having the ability to synthesize cerocyanine II of substantially the same quality as the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; and (3) An amino acid sequence that has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and forms a polypeptide having the ability to synthesize cerocyanine II of substantially the same quality as the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. 8. A host producing cerocyanine II into which the synthetic composition according to the preceding paragraph 6 or 7 has been introduced. 9. A method for synthesizing cerocyanine II according to the following (1) or (2): (1) contacting amaranthin with the cerocyanine II synthetic composition described in the preceding item 7; (2)(a) contacting betanin with an enzyme having glucuronic acid-adding activity; (b) A step of contacting the amaranthin obtained in (a) with the cerocyanine II synthetic composition described in the above item 7. 10. A host producing cerocyanine II, The host is The following genes have been introduced: a gene encoding cerocyanine II synthase; a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine; a gene encoding an enzyme having L-DOPA oxidase activity and / or a gene encoding an enzyme having DOPA 4,5-dioxygenase activity; a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group; and a gene encoding an enzyme having the activity of adding glucuronic acid, and the gene has the ability to produce tyrosine or L-DOPA. Or, A gene encoding the following cerocyanine II synthase has been introduced, and the enzyme has the activity of hydroxylating the 3-position of the phenol ring of tyrosine, L-DOPA oxidase activity and / or DOPA 4,5-dioxygenase activity, the activity of adding sugar to a phenolic hydroxyl group, the activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine. wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, or 12; (3) a gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, (5) A gene comprising a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a DNA comprising the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and encodes a polypeptide having the ability to synthesize cerocyanine II; (6) A gene consisting of DNA in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added in DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15. (7) A gene consisting of DNA having 90% or more homology to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, and (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, A host producing cerocyanine II. 11. A method for synthesizing betaxanthin, comprising: a host into which a gene encoding an enzyme having an enzyme activity of hydroxylating the phenolic ring of tyrosine but not having L-DOPA oxidase activity and a gene encoding an enzyme having DOPA 4,5-dioxygenase activity have been introduced, and which is capable of producing tyrosine or 3-hydroxy-L-tyrosine, is cultured with the addition of an amine, and betaxanthin is extracted from the cultured host; Or, a host into which a gene encoding an enzyme having an enzyme activity of hydroxylating the phenolic ring of tyrosine but not having L-DOPA oxidase activity has been introduced, and which has an enzyme activity of DOPA 4,5-dioxygenase activity and the ability to produce tyrosine or 3-hydroxy-L-tyrosine, is cultured with the addition of an amine, and betaxanthin is extracted from the cultured host; Or, a host having the ability to produce tyrosine or 3-hydroxy-L-tyrosine, into which a gene encoding an enzyme having the enzyme activity of hydroxylating the phenolic ring of tyrosine but not having L-DOPA oxidase activity, a gene encoding an enzyme having DOPA 4,5-dioxygenase activity, and an amine synthesis gene have been introduced, and the host is then cultured, and betaxanthin is extracted from the cultured host; Or, a host into which a gene encoding an enzyme having an enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine but not having L-DOPA oxidase activity has been introduced, and which has an enzyme activity having DOPA 4,5-dioxygenase activity, an enzyme activity having amine synthesis activity, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine; and extracting betaxanthin from the host after cultivation. Here, the gene encoding an enzyme having an enzyme activity that hydroxylates the 3-position of the phenol ring of tyrosine but not having L-DOPA oxidase activity is selected from any one or more of the following: (1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, which is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (3) A gene encoding a polypeptide that has 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 50, 52, or 54. (5) A gene consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54, and encodes a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (6) A gene consisting of DNA in which 1 to 50 bases have been substituted, deleted, inserted, and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54. (7) A gene consisting of DNA having 90% or more homology to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 50, 52, or 54, and (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 50, 52, or 54. A method for producing betaxanthin, comprising: 12. The method for synthesizing betaxanthin according to the preceding paragraph 11, wherein the gene encoding an enzyme having the enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine but not having L-DOPA oxidase activity is selected from any one or more of SEQ ID NOs: 50, 52, and 54. 13. A betalamic acid synthesis composition for betaxanthin synthesis, comprising any one of the following genes or a vector carrying said gene: (1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, which is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (3) A gene encoding a polypeptide that has 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 50, 52, or 54. (5) A gene consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54, and encodes a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (6) A gene consisting of DNA in which 1 to 50 bases have been substituted, deleted, inserted, and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54. (7) A gene consisting of DNA having 90% or more homology to the DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54, and (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 50, 52, or 53. 14. A betalamic acid synthesis composition for betaxanthin synthesis, comprising a peptide represented by any one of the following amino acid sequences: (1) an amino acid sequence set forth in SEQ ID NO: 51, 53, or 55; (2) an amino acid sequence having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and forming a polypeptide having the ability to hydroxylate the 3-position of the phenol ring of tyrosine, which is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, but does not have L-DOPA oxidase activity; (3) An amino acid sequence that has 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and forms a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. 15. A betaxanthin-producing host into which the synthetic composition according to the preceding paragraph 13 or 14 has been introduced. 16. A betaxanthin-producing host, The host is A gene encoding an enzyme that has the enzyme activity of hydroxylating the phenolic ring of tyrosine at the 3-position but does not have L-DOPA oxidase activity, and a gene encoding an enzyme that has DOPA 4,5-dioxygenase activity have been introduced, and the gene has the ability to produce tyrosine or 3-hydroxy-L-tyrosine. Or, A gene encoding an enzyme that has the enzyme activity of hydroxylating the phenolic ring of tyrosine at the 3-position but does not have L-DOPA oxidase activity has been introduced, and the enzyme activity has DOPA 4,5-dioxygenase activity and the ability to produce tyrosine or 3-hydroxy-L-tyrosine. Here, the gene encoding an enzyme having an enzyme activity that hydroxylates the 3-position of the phenol ring of tyrosine but not having L-DOPA oxidase activity is selected from any one or more of the following: (1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55; (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, which is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and which has the ability to hydroxylate the 3-position of the phenol ring of tyrosine but does not have L-DOPA oxidase activity. (3) A gene encoding a polypeptide that has 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 50, 52, or 54. (5) A gene consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54, and encodes a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (6) A gene consisting of DNA in which 1 to 50 bases have been substituted, deleted, inserted, and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54. (7) A gene consisting of DNA having 90% or more homology to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 50, 52, or 54, and (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 50, 52, or 54. A betaxanthin-producing host characterized by: 17. An amyloid beta polymerization inhibitor or an agent for treating or preventing Alzheimer's disease, comprising any one of the following: (1) Celocyanine II (2) Celocyanine II obtained by the method for synthesizing Celocyanine II according to any one of the above items 1 to 5 and 9. (3) Celocyanine II obtained from the Celocyanine II-producing host according to the above item 8 or 10. (4) Amaranthine (5) Betanin (6) Ferulic acid (7) Betaxanthin (8) Betaxanthin obtained by the method for synthesizing betaxanthin according to the preceding paragraph 11 or 12. (9) Betaxanthin obtained from the betaxanthin-producing host described in the preceding paragraph 16. 18. An amyloid peptide aggregation inhibitor, including any one of the following: (1) Celocyanine II (2) Celocyanine II obtained by the method for synthesizing Celocyanine II according to any one of the above items 1 to 5 and 9. (3) Celocyanine II obtained from the Celocyanine II-producing host according to the above item 8 or 10. (4) Amaranthine (5) Betanin (6) Ferulic acid (7) Betaxanthin 19. A preventive and / or therapeutic agent for neurodegenerative diseases, Alzheimer's disease, systemic amyloidosis, Parkinson's disease, Huntington's disease, prion disease, multiple sclerosis, or amyotrophic lateral sclerosis, comprising the amyloid peptide aggregation inhibitor described in the preceding item 18 as an active ingredient. 20. HIV-1 protease inhibitors, including any one of the following: (1) Celocyanine II (2) Celocyanine II obtained by the method for synthesizing Celocyanine II according to any one of the above items 1 to 5 and 9. (3) Celocyanine II obtained from the Celocyanine II-producing host according to the above item 8 or 10. [Effects of the Invention]

[0011] The present invention can provide a method for synthesizing cerocyanine II, a method for synthesizing betaxanthin, an amyloid β polymerization inhibitor or an agent for treating or preventing Alzheimer's disease, an amyloid peptide aggregation inhibitor, and an inhibitor of HIV-1 protease activity. [Brief explanation of the drawings]

[0012] [Figure 1] Structure of betalain pigments. [Figure 2] Accumulation of betalain pigments in hypocotyls. [Figure 3] Evaluation results of the stability of betalain pigments. [Figure 4] Evaluation results of inhibitory activity against HIV-1 protease activity. [Figure 5] Evaluation results of the polymerization inhibitory activity of betalain dyes against amyloid beta. [Figure 6] Summary of the search results for the cerocyanine II synthase gene. [Figure 7] Schematic diagram of the betalain pigment biosynthetic pathway. [Figure 8] Schematic diagram of cerocyanine II biosynthesis. [Figure 9] Schematic diagram of the betaxanthin biosynthetic pathway. [Figure 10] Activity evaluation in tobacco BY2 cells. [Figure 11] Evaluation results of betaxanthin's inhibitory activity against amyloid beta polymerization. [Figure 12] Electron microscopy of amyloid peptide aggregation inhibition. A and B: 25 μM Aβ40 + water, C and D: 25 μM Aβ40 + 50 μM betanin, E: 25 μM Aβ40 + 50 μM amaranthin, F: 25 μM Aβ40 + 50 μM cerocyanine II, G: 25 μM Aβ40 + 50 μM ferulic acid, H: 25 μM Aβ40 + 50 μM betaxanthin. Scale bar: 200 μm. [Figure 13] A: The percentage of non-paralyzed nematodes in the control group where nematodes were fed dextrin, B: The percentage of non-paralyzed nematodes in the control group where nematodes were fed betanin. Vertical axis: The percentage of non-paralyzed nematodes, horizontal axis: The number of days until adulthood. C: Nematodes raised on a diet mixed with dextrin, D: Normal nematodes. [Figure 14]Results of amaranth plants expressing XM_021915966 in assessing cerocyanine II synthesis activity. The gene XM_021915966 was transiently expressed, and the pigment was analyzed by HPLC. Infected plants: Amaranth (which does not accumulate cerocyanine II). Accumulation of cerocyanine II was newly observed in amaranth plants expressing XM_021915966. [Figure 15] Results of quinoa transiently expressing XM_021915966 in assessing cerocyanine II synthesis activity. The candidate gene was transiently expressed and the pigment was analyzed by HPLC. Infected plant: quinoa. In quinoa transiently expressing XM_021915966, cerocyanine II accumulation increased approximately two-fold. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to a method for synthesizing cerocyanine II, a composition for synthesizing cerocyanine II, and a host for producing cerocyanine II. The present invention relates to a method for synthesizing betaxanthin, a betalamic acid synthesis composition for synthesizing betaxanthin, and a betaxanthin-producing host. The present invention will be described in detail below.

[0014] (betalain pigments) The betalain pigments referred to in the present invention are classified into betaxanthins and betacyanins based on their structural characteristics. Note that betaxanthins produce a yellow color, while betacyanins produce a reddish-purple color, and so they have traditionally been used as natural colorants. Note that betacyanins are a collective term for a group of compounds in which sugars are glycosidicly bonded to the phenolic hydroxyl group of betanidin.

[0015] (Cerocyanine II synthetic system) The outline of the betalain pigment biosynthetic pathway in the present invention is shown in FIGS. As is clear from Figures 7 and 8, a host capable of producing tyrosine (L-tyrosine) or 3-hydroxy-L-tyrosine (L-DOPA), as well as amino acids and amines (e.g., putrescine, spermidine, spermine) (including hosts capable of taking in these substances from the outside) can synthesize cerocyanine II if it has the following enzymatic activity: Enzyme activity that hydroxylates the 3-position of the phenol ring of tyrosine (e.g., CqCYP76AD1), L-DOPA oxidase (e.g., CqCYP76AD1) activity and / or DOPA 4,5-dioxygenase (e.g., CqDODA-1) activity, enzyme activity that adds sugar to a phenolic hydroxyl group (e.g., enzyme activity having betanidin 5-O-glucosyltransferase (5GT) activity (Cyclo-DOPA 5-O-glucosyltransferase, CqCDOPA5GT, etc.), enzyme activity that adds glucuronic acid (CqAmaSyl1, etc.), and celosianin II synthetase activity of the present invention. CqCYP76AD1 has both the enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine and the L-DOPA oxidase activity. The results of the examples below show that a host plant into which a group of genes, including a gene encoding an enzyme (CqCYP76AD1-1) that has the activity of hydroxylating the 3-position of the phenolic ring of tyrosine, a gene encoding an enzyme with L-DOPA oxidase activity and / or a gene encoding an enzyme with DOPA 4,5-dioxygenase activity (CqDODA-1), a gene encoding an enzyme (CqCDOPA5GT) that has the activity of adding sugar to a phenolic hydroxyl group, a gene encoding an enzyme (CqAmaSy1) that has the activity of adding glucuronic acid, and a gene encoding the cerocyanine II synthase of the present invention, has the ability to synthesize cerocyanine II, a betalain pigment.

[0016] As is clear from the description of FIGS. 7 and 8, the present invention also covers the following method for synthesizing cerocyanine II. (1) Amaranthin is contacted with the cerocyanine II synthase of the present invention, and if necessary, a metal ion is added. (2) Amaranthin is obtained by contacting betanin with an enzyme having the activity of adding glucuronic acid, and then the resulting product is contacted with the cerocyanine II synthase of the present invention, and if necessary, metal ions are added. (3) Betadinin is contacted with an enzyme capable of adding sugar to a phenolic hydroxyl group to obtain betanin, which is then contacted with an enzyme capable of adding glucuronic acid to obtain amaranthin, which is then contacted with the cerocyanine II synthase of the present invention, and if necessary, metal ions are added.

[0017] (An enzyme that hydroxylates the 3-position of the phenol ring of tyrosine) In the present invention, the enzyme that hydroxylates the 3-position of the phenol ring of tyrosine has the activity of adding a hydroxyl group to the 3-position of the phenol ring of tyrosine, and may be derived from any species as long as it has this activity. Enzymes that hydroxylate the 3-position of the phenol ring of tyrosine include, for example, tyrosinase, cytochrome P450 (particularly, CYP76AD1, CYP76AD2, CYP76AD3, etc.), catechol oxidase, etc., but preferably CqCYP76AD1 (base sequence: SEQ ID NO: 17, amino acid sequence: SEQ ID NO: 18, accession number XP_021769302).

[0018] (Enzyme with L-DOPA oxidase activity) In the present invention, the enzyme having L-DOPA oxidase activity has the activity of converting L-DOPA to cyclo-DOPA, and may be derived from any species as long as it has this activity. Examples of enzymes having L-DOPA oxidase activity include tyrosinase and cytochrome P450 subfamily alpha of the CYP76AD group, such as CYP76AD1, CYP76AD2, and CYP76AD3, but preferably CqCYP76AD1 (base sequence: SEQ ID NO: 17, amino acid sequence: SEQ ID NO: 18).

[0019] (An enzyme that can add sugars to phenolic hydroxyl groups) In the present invention, the enzyme having the activity of adding sugars to phenolic hydroxyl groups refers to an enzyme having the activity of adding sugars to phenolic hydroxyl groups, i.e., the ability to synthesize betanin by adding sugars to the phenolic hydroxyl groups present at the 5th and 6th positions of the cyclo-DOPA skeleton, and can be derived from any species as long as it has this activity. Enzymes that have the activity of adding sugars to phenolic hydroxyl groups include the enzyme that synthesizes betanin from betanidin. Examples of enzymes that have the activity of adding sugars to phenolic hydroxyl groups include cyclo-DOPA 5-O-glucosyltransferase (CDOPA5GT), betanidin 5-O-glucosyltransferase (B5GT), and betanidin 6-O-glucosyltransferase, with CqCDOPA5GT (nucleotide sequence: SEQ ID NO: 19, amino acid sequence: SEQ ID NO: 20, accession number XP_021748306) being preferred.

[0020] (synthetic enzyme of gomufrenin I from betanidin) The enzyme used in the present invention to synthesize gomphrenin I from betanidin is not particularly limited as long as it is capable of synthesizing gomphrenin I from betanidin. Examples include betanidin 6-O-glucosyltransferase (6GT (B6GT): SEQ ID NOs: 21 and 22) and cyclodopa 6-O-glucosyltransferase (CDOPA6GT) (see: Substrate specificity and sequence analysis define a polyphyletic origin of betanidin 5- and 6-O-glucosyltransferase from Dorotheanthus bellidiformis. Planta 214, 492-495).

[0021] (Enzyme with glucuronic acid-adding activity) The enzyme having the activity of adding glucuronic acid in the present invention has the ability to synthesize amaranthin by the enzymatic activity of adding glucuronic acid, and may be derived from any species as long as it has this activity. Enzymes with the activity of adding glucuronic acid include enzymes that synthesize amaranthin from betanin, and enzymes that have the activity of synthesizing amaranthin by linking glucuronic acid to betanin (e.g., the activity of synthesizing UDP-glucoronate (betanin beta-D-glucuronosyltransferase)). Examples of genes encoding enzymes having the activity of adding glucuronic acid (amaranthin synthase genes) include the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32, or 34. (2) A gene encoding a polypeptide in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34, and which has the ability to synthesize amaranthin that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34. (3) A gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34 and having the ability to synthesize amaranthin substantially identical to the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34. (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 23, 25, 27, 29, 31 or 33. (5) A gene consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence set forth in SEQ ID NO: 23, 25, 27, 29, 31 or 33 and encodes a polypeptide having the ability to synthesize amaranthin. (6) A gene consisting of DNA having the nucleotide sequence set forth in SEQ ID NO: 23, 25, 27, 29, 31, or 33, in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added. (7) A gene consisting of DNA having 90% or more homology to the DNA consisting of the base sequence set forth in SEQ ID NO: 23, 25, 27, 29, 31 or 33. (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 23, 25, 27, 29, 31 or 33.

[0022] The gene (2) above is a gene encoding a polypeptide into which a mutation has been introduced to the extent that the ability to synthesize amaranthin is not lost. Such mutations include not only mutations that occur in nature but also artificial mutations. Methods for generating artificial mutations include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982). The number of mutated amino acids is usually 20 or less, preferably 10 or less, more preferably 5 or less, and most preferably 3. Whether a mutated polypeptide retains the ability to synthesize amaranthin can be determined, for example, by introducing a gene encoding the mutated polypeptide into a plant or the like and confirming the ability of the plant to synthesize amaranthin. With respect to the gene (3) above, the "ability to synthesize amaranthin substantially identical to the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32, or 34" means that the degree of activity may be stronger or weaker than the ability to synthesize amaranthin substantially identical to the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32, or 34. Examples of the ability to synthesize amaranthin include about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, and about 150% compared to the ability to synthesize amaranthin of the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32, or 34. Alternatively, identity can be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, using default or initial setting parameters). The gene (5) above is a gene obtained by DNA hybridization. The term "stringent conditions" in this context refers to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. Such conditions typically include hybridization at 37°C in a buffer containing 5×SSC and 1% SDS and washing at 37°C in a buffer containing 1×SSC and 0.1% SDS. Preferred conditions include hybridization at 42°C in a buffer containing 5×SSC and 1% SDS and washing at 42°C in a buffer containing 0.5×SSC and 0.1% SDS. More preferred conditions include hybridization at 65°C in a buffer containing 5×SSC and 1% SDS and washing at 65°C in a buffer containing 0.2×SSC and 0.1% SDS. Whether or not the DNA obtained by hybridization encodes a polypeptide having activity can be determined, for example, by introducing the DNA into a plant or the like and confirming the plant's ability to synthesize amaranthin. The DNA obtained by hybridization usually has a high identity to the gene (SEQ ID NO: 23, 25, 27, 29, 31, or 33) described in (4) above. A high identity refers to an identity of 90% or more, preferably 95% or more, and more preferably 98% or more. The gene (6) above is a gene consisting of DNA comprising the base sequence set forth in SEQ ID NO: 23, 25, 27, 29, 31, or 33, in which 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, and even more preferably 1 to 5 bases have been substituted, deleted, inserted, and / or added. The gene (7) above is a gene consisting of DNA having 90% or more, preferably 93% or more, more preferably 95% or more, and most preferably 98% or more identity to DNA consisting of the base sequence set forth in SEQ ID NO: 23, 25, 27, 29, 31 or 33.

[0023] The enzyme of the present invention having an enzymatic activity of adding glucuronic acid has an amino acid sequence selected from any one or more of the following: (1) The amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34. (2) An amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34, and which forms a polypeptide having the ability to synthesize amaranthin of substantially the same quality as the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16. (3) An amino acid sequence that has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34 and forms a polypeptide that has the ability to synthesize amaranthin that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32 or 34. In addition, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations into the peptide, it is easily conceivable to substitute, for example, mutually homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) with each other.

[0024] (Enzyme with DOPA 4,5-dioxygenase activity) The enzyme having DOPA 4,5-dioxygenase activity of the present invention catalyzes the extradiol cleavage of L-3,4-dihydroxyphenylalanine, thereby converting L-DOPA to 4,5-seco-DOPA, which is then converted to betalaminic acid through a spontaneous reaction.

[0025] In the present invention, the gene encoding an enzyme having DOPA 4,5-dioxygenase activity is selected from one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 35 (accession number XP_021769303), 37 (accession number XP_021769301), 39, 41, 43, 45, or 47. (2) A gene encoding a polypeptide having a substitution, deletion, insertion, and / or addition of 1 to 20 amino acids in the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45, or 47, and having the ability to convert L-DOPA to 4,5-seco-DOPA, which is substantially identical to the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, or 45. (3) A gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45, or 47 and having the ability to convert L-DOPA to 4,5-seco-DOPA, which is substantially identical to the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45, or 47. (4) A gene consisting of DNA having the nucleotide sequence set forth in SEQ ID NO: 36, 38, 40, 42, 44, 46 or 48. (5) A gene comprising DNA that hybridizes under stringent conditions with DNA comprising a base sequence complementary to DNA comprising the base sequence set forth in SEQ ID NO: 36, 38, 40, 42, 44, 46, or 48, and encodes a polypeptide having the ability to convert L-DOPA to 4,5-seco-DOPA. (6) A gene consisting of DNA having the nucleotide sequence set forth in SEQ ID NO: 36, 38, 40, 42, 44, 46, or 48, in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added. (7) A gene consisting of DNA having 90% or more homology to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 36, 38, 40, 42, 44, 46 or 48. (8) One or more genes according to the above (1) to (7), including a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 33. (9) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 36, 38, 40, 42, 44, 46 or 48. The gene (2) encodes a polypeptide into which a mutation has been introduced that does not impair the ability to convert L-DOPA to 4,5-seco-DOPA. Such a mutation can be introduced by the method described above. With respect to the gene (3) above, the term "ability to convert L-DOPA to 4,5-seco-DOPA substantially identical to the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45, or 47" may refer to a level of activity that is stronger or weaker than the ability of the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45, or 47 to convert L-DOPA to 4,5-seco-DOPA. For example, the level of activity can be about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, or about 150% of the ability of the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45, or 47 to convert L-DOPA to 4,5-seco-DOPA. The gene (5) above is a gene obtained by hybridization between DNAs. The DNA obtained by hybridization usually has a high identity with the gene (4) above (SEQ ID NO: 18, 20, 22, 24, 26, 28, or 48). A high identity refers to an identity of 90% or more, preferably 95% or more, and more preferably 98% or more. The gene (6) above is a gene consisting of DNA comprising the base sequence set forth in SEQ ID NO: 36, 38, 40, 42, 44, 46, or 48, in which 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, and even more preferably 1 to 5 base sequences have been substituted, deleted, inserted, and / or added. The gene (7) above is a gene consisting of DNA having 90% or more, preferably 93% or more, more preferably 95% or more, and most preferably 98% or more identity to DNA consisting of the base sequence set forth in SEQ ID NO: 36, 38, 40, 42, 44, 46 or 48.

[0026] The enzyme having DOPA 4,5-dioxygenase activity of the present invention is selected from any one or more of the following: (1) The amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45 or 47. (2) An amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45, or 47, and which has the ability to convert L-DOPA to 4,5-seco-DOPA that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45, or 47. (3) An amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 35, 37, 39, 41, 43, 45 or 47, and having the ability to convert L-DOPA to 4,5-seco-DOPA that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, or 2. (4) One or more of the amino acid sequences (1) to (3) above, including the amino acid sequence set forth in SEQ ID NO: 33. In addition, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations into the peptide, it is easily conceivable to substitute, for example, mutually homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) with each other. In particular, the amino acid sequence set forth in SEQ ID NO: 49 contains the sequence of the active site, and since introduction of a mutation into this sequence is likely to result in the loss of the ability to convert L-DOPA to 4,5-seco-DOPA, it is preferable to introduce a mutation into an amino acid outside of this domain.

[0027] (cerocyanine II synthase) The cerocyanine II synthase of the present invention is not particularly limited as long as it has the ability to synthesize cerocyanine II through the activity of synthesizing cerocyanine II from amaranthin. In addition to synthesizing cerocyanine II by adding ferulic acid to amaranthin, cerocyanine II synthase also has the activity of adding aromatic carboxylic acids other than ferulic acid (e.g., caffeic acid, coumaric acid) to amaranthin. Furthermore, it also has the activity of adding aromatic carboxylic acids to betacyanins other than amaranthin (e.g., betanin, gomufrenin). Examples of the gene encoding cerocyanine II synthase (cerocyanine II synthase gene) include the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. (2) A gene encoding a polypeptide in which 1 to 20 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and which has the ability to synthesize cerocyanine II that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. (3) A gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15. (5) A gene consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, and encodes a polypeptide having the ability to synthesize cerocyanine II. (6) A gene consisting of DNA having the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added. (7) A gene consisting of DNA having 90% or more homology to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15. (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15.

[0028] The gene (2) above is a gene encoding a polypeptide into which a mutation has been introduced to the extent that the ability to synthesize cerocyanine II is not lost. Such mutations include not only naturally occurring mutations but also artificial mutations. Methods for generating artificial mutations include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982). The number of mutated amino acids is usually 20 or less, preferably 10 or less, more preferably 5 or less, and most preferably 3. Whether a mutated polypeptide retains the ability to synthesize cerocyanine II can be determined, for example, by introducing a gene encoding the mutated polypeptide into a plant or the like and confirming the ability of the plant to synthesize cerocyanine II. With respect to the gene (3) above, the "ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16" means that the degree of the activity may be stronger or weaker than the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. For example, examples of the ability to synthesize cerocyanine II include about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, and about 150% compared to the ability of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. Alternatively, identity can be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, using default or initial setting parameters). The gene (5) above is a gene obtained by DNA hybridization. The term "stringent conditions" in this context refers to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. Such conditions typically include hybridization at 37°C in a buffer containing 5×SSC and 1% SDS and washing at 37°C in a buffer containing 1×SSC and 0.1% SDS. Preferred conditions include hybridization at 42°C in a buffer containing 5×SSC and 1% SDS and washing at 42°C in a buffer containing 0.5×SSC and 0.1% SDS. More preferred conditions include hybridization at 65°C in a buffer containing 5×SSC and 1% SDS and washing at 65°C in a buffer containing 0.2×SSC and 0.1% SDS. Whether or not the DNA obtained by hybridization encodes a polypeptide having activity can be determined, for example, by introducing the DNA into a plant or the like and confirming the plant's ability to synthesize cerocyanin II. The DNA obtained by hybridization typically has a high identity to the gene (4) above (SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15). A high identity refers to an identity of 90% or more, preferably 95% or more, and more preferably 98% or more. The gene (6) above is a gene consisting of DNA comprising the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, in which 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, and even more preferably 1 to 5 bases have been substituted, deleted, inserted, and / or added. The gene (7) above is a gene consisting of DNA having 90% or more, preferably 93% or more, more preferably 95% or more, and most preferably 98% or more identity to DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15.

[0029] The enzyme having cerocyanine II synthase activity in the present invention has an amino acid sequence selected from one or more of the following: (1) The amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. (2) An amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and which forms a polypeptide having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16. (3) An amino acid sequence that has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and forms a polypeptide having the ability to synthesize cerocyanine II of substantially the same quality as the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. In addition, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations into the peptide, it is easily conceivable to substitute, for example, mutually homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) with each other.

[0030] (Method for synthesizing cerocyanine II) The method for synthesizing cerocyanine II of the present invention can be exemplified as follows. (1) A host having the ability to produce tyrosine or L-DOPA is cultured, into which a gene encoding cerocyanine II synthase, a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine, a gene encoding an enzyme having L-DOPA oxidase activity and / or a gene encoding an enzyme having DOPA 4,5-dioxygenase activity, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid are introduced, and cerocyanine II is extracted from the cultured host. (2) A host into which a gene encoding cerocyanine II synthase has been introduced and which has the enzyme activity of hydroxylating the 3-position of the phenolic ring of tyrosine, L-DOPA oxidase activity and / or DOPA 4,5-dioxygenase activity, the enzyme activity of adding sugar to the phenolic hydroxyl group, the enzyme activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine is cultured, and cerocyanine II is extracted from the cultured host.

[0031] (host) The host used in the method for synthesizing cerocyanine II or betaxanthin of the present invention is not particularly limited as long as it has the ability to produce tyrosine or 3-hydroxy-L-tyrosine. For example, known recombinant Escherichia coli protein synthesis systems, insect protein synthesis systems, yeast protein synthesis systems, plant cell protein synthesis systems, cell-free protein synthesis systems, plant protein synthesis systems, cultured animal cells, etc. can be used. The method for introducing each gene into a host used in the method for synthesizing cerocyanine II or betaxanthin of the present invention can be a method known per se, for example, by using a vector carrying the gene to introduce the gene into the host. As the vector, known viral vectors, particularly plant viral vectors (eg, vectors derived from viruses belonging to the genus Tobamovirus, tobacco mosaic virus vectors, tomato mosaic virus vectors) can be used. Alternatively, the Agrobacterium method using a Ti plasmid can be used.

[0032] (Method of introducing each gene into the host) In the method for synthesizing cerocyanine II or betaxanthin of the present invention, the genes can be introduced into a host by a method known per se. For example, the genes can be introduced into a host by applying a solution containing a plant virus vector carrying the genes to the leaves, stems, roots, panicles, etc. of the plant. Other examples of such methods include the particle gun method and the Agrobacterium method.

[0033] (Method of introducing each protein into the host) In the method for synthesizing cerocyanin II or betaxanthin of the present invention, the protein can be introduced into a host (especially a plant) by a method known per se. For example, the protein can be introduced into a host by applying a solution containing the protein to the leaves, stems, roots, ears, etc. of the plant. Other examples of such methods include the particle gun method and the Agrobacterium method.

[0034] (Cerocyanine II producing host) The host producing cerocyanine II of the present invention has, for example, one or more of the following constitutions and characteristics: (1) A gene encoding cerocyanine II synthase, a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine, a gene encoding an enzyme having L-DOPA oxidase activity and / or a gene encoding an enzyme having DOPA 4,5-dioxygenase activity, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid are introduced, and the strain has the ability to produce tyrosine or 3-hydroxy-L-tyrosine. (2) A gene encoding cerocyanine II synthase has been introduced, and the enzyme has the enzyme activity of hydroxylating the 3-position of the phenolic ring of tyrosine, L-DOPA oxidase activity and / or DOPA 4,5-dioxygenase activity, the enzyme activity of adding sugar to the phenolic hydroxyl group, the enzyme activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine. (3) The gene encoding cerocyanine II synthase has been introduced, and the strain has the ability to produce amaranthin. (4) A gene encoding cerocyanine II synthase and a gene encoding an enzyme having glucuronic acid-adding activity have been introduced, and the strain has the ability to produce betanin. (5) A gene encoding cerocyanine II synthase has been introduced, and the strain has an enzyme activity that adds glucuronic acid and the ability to produce betanin. (6) A gene encoding cerocyanine II synthase, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid have been introduced, and the strain has the ability to produce betanidin. (7) A gene encoding cerocyanine II synthase has been introduced, and the strain has an enzyme activity of adding sugar to a phenolic hydroxyl group, an enzyme activity of adding glucuronic acid, and the ability to produce betanidin. (8) Celocyanine II synthetic composition has been introduced.

[0035] (Cerocyanine II Synthetic Composition) The cerocyanine II synthesis composition (cerocyanine II synthesis agent, cerocyanine II synthesizing enzyme agent) of the present invention contains any one of the following genes or a vector carrying said gene: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. (2) A gene encoding a polypeptide in which 1 to 20 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16, and which has the ability to synthesize cerocyanine II that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. (3) A gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, (5) A gene consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, and encodes a polypeptide having the ability to synthesize cerocyanine II. (6) A gene consisting of DNA having the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, in which 1 to 50 nucleotides have been substituted, deleted, inserted, and / or added. (7) A gene consisting of DNA having 90% or more homology to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15. (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15.

[0036] Alternatively, the cerocyanine II synthetic composition of the present invention comprises a peptide represented by any one of the following amino acid sequences (1) to (3): (1) The amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16. (2) An amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and which forms a polypeptide having the ability to synthesize cerocyanine II substantially identical to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16. (3) An amino acid sequence that has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and forms a polypeptide having the ability to synthesize cerocyanine II of substantially the same quality as the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16.

[0037] (Amyloid beta polymerization inhibitor or Alzheimer's treatment or prevention agent) The amyloid β polymerization inhibitor or the agent for treating or preventing Alzheimer's disease of the present invention comprises one or more of the following: (1) Celocyanine II (2) Celocyanine II obtained by the method for synthesizing Celocyanine II described in this specification (3) Celocyanin II obtained from the Celocyanin II-producing host described herein (4) Amaranthine (5) Betanin (6) Ferulic acid (7) Betaxanthin (8) Betaxanthin obtained by the method for synthesizing betaxanthin described in this specification (9) Betaxanthin obtained from the betaxanthin-producing host described herein.

[0038] (HIV-1 protease inhibitor) The inhibitor of HIV-1 protease activity of the present invention comprises one or more of the following: (1) Celocyanine II (2) Celocyanine II obtained by the method for synthesizing Celocyanine II described in this specification (3) Celocyanin II obtained from the Celocyanin II-producing host described herein

[0039] (Amyloid peptide aggregation inhibitor) The amyloid peptide aggregation inhibitor of the present invention comprises any one or more of the following: (1) Celocyanine II (2) Celocyanine II obtained by the method for synthesizing Celocyanine II described in this specification (3) Celocyanin II obtained from the Celocyanin II-producing host described herein (4) Amaranthine (5) Betanin (6) Ferulic acid (7) Betaxanthin

[0040] It has been confirmed in the examples described below that the amyloid peptide aggregation inhibitor of the present invention inhibits the formation of amyloid peptide aggregates and is therefore effective in the prevention and / or treatment of neurodegenerative diseases, Alzheimer's disease, systemic amyloidosis, Parkinson's disease, Huntington's disease, prion disease, multiple sclerosis, and amyotrophic lateral sclerosis.

[0041] (Betaxanthin synthesis system) The outline of the betaxanthin biosynthetic pathway in the present invention is shown in FIGS. As is clear from the descriptions in Figures 7 and 9, a host capable of producing tyrosine or 3-hydroxy-L-tyrosine, as well as amino acids and amines (including hosts capable of taking in these from the outside) can synthesize betaxanthin if it has the following enzymatic activity: Enzyme activity that hydroxylates the 3-position of the phenol ring of tyrosine but does not have L-DOPA oxidase activity (e.g., CqCYP76AD5, CqCYP76AD6) and DOPA 4,5-dioxygenase (e.g., CqDODA-1) activity. The results of the examples below show that host plants into which a group of genes encoding enzymes (CqCYP76AD5, CqCYP76AD6) that have the enzymatic activity of hydroxylating the 3-position of the phenolic ring of tyrosine but do not have L-DOPA oxidase activity and a gene encoding an enzyme (CqDODA-1) with DOPA 4,5-dioxygenase activity have been introduced have the ability to synthesize betaxanthin. Alternatively, betaxanthin can be synthesized by spontaneously reacting betalaminic acid synthesized by a host that has the above-mentioned enzyme activity and is capable of producing tyrosine or 3-hydroxy-L-tyrosine but not amines with amino acids or amines as needed.

[0042] As is clear from the description of FIG. 9, the present invention also covers the following method for synthesizing betaxanthin. (1) Tyrosine or 3-hydroxy-L-tyrosine is contacted with an enzyme that has the enzymatic activity of hydroxylating the 3-position of the phenol ring of tyrosine but does not have L-DOPA oxidase activity to obtain L-DOPA, which is then contacted with an enzyme that has DOPA 4,5-dioxygenase activity to obtain betalamic acid, which is then contacted with an amine. (2) A host into which a gene group has been introduced, including a gene encoding an enzyme that has the enzymatic activity of hydroxylating the 3-position of the phenolic ring of tyrosine but does not have L-DOPA oxidase activity and a gene encoding an enzyme that has DOPA 4,5-dioxygenase activity, and which has the ability to produce tyrosine or 3-hydroxy-L-tyrosine and amino acids and / or amines, is cultured, and betaxanthin is extracted from the cultured host. (3) A host having the ability to produce tyrosine or 3-hydroxy-L-tyrosine is cultured, into which a gene cluster has been introduced, including a gene encoding an enzyme that has the enzymatic activity of hydroxylating the 3-position of the phenolic ring of tyrosine but does not have L-DOPA oxidase activity and a gene encoding an enzyme that has DOPA 4,5-dioxygenase activity. Betalamic acid is extracted from the cultured host and contacted with an amine. In the "culturing" in the above betaxanthin synthesis methods (2) and (3), the host may be cultured, for example, in the presence of an exogenous amine corresponding to the target betaxanthin. In addition, the betaxanthin synthesis methods (2) and (3) above may further include a step of purifying the target betaxanthin from the extracted pigment, or a step of separating the target betaxanthin from secondary endogenous amines and / or betaxanthins derived from amino acids from the extracted pigment.

[0043] (amine) The amine of the present invention may be a natural or non-natural amine, such as putrescine, spermidine, spermine, methylamine, ethanolamine, piperidine, histamine, dopamine, phenethylamine, tyramine, 3-methoxytyramine, γ-aminobutyric acid, noradrenaline, serotonin, muscimol, ethylamine, piperazine, morpholine, ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexamethylenediamine, 1,3-butadien-1-amine, 1,3,5-hexatrien-1-amine, aniline, 4-biphenylamine, amantadine, para-aminobenzoic acid (PABA), anthranilic acid (Ant.), 6AI (6-aminoindole), o-dianisidine (oDA), and the like.

[0044] (betaxanthin) The betaxanthin of the present invention is not particularly limited, but examples thereof include indicaxanthin, vulgaxanthin I, dopaxanthin, betaxanthin obtained by a condensation reaction of threonine and betalaminic acid, putrescine-betaxanthin, spermidine-betaxanthin, spermine-betaxanthin, methylamine-betaxanthin, ethanolamine-betaxanthin, piperidine-betaxanthin, histamine-betaxanthin, dopamine-betaxanthin, phenethylamine-betaxanthin, tyramine-betaxanthin, 3-methoxytyramine-betaxanthin, γ-aminobutyric acid-betaxanthin, noradrenaline-betaxanthin, serotonin, and the like. Examples of such betaxanthin include 1,3-butadiene-1-amine-betaxanthin, 1,3,5-hexatriene-1-amine-betaxanthin, aniline-betaxanthin, 4-biphenylamine-betaxanthin, and amantadine-betaxanthin.

[0045] (An enzyme that has the enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine but does not have L-DOPA oxidase activity) In the present invention, the enzyme having the enzymatic activity of hydroxylating the 3-position of the phenolic ring of tyrosine but not having L-DOPA oxidase activity may be derived from any species, as long as it has the activity to add a hydroxyl group to the 3-position of the phenolic ring of tyrosine but does not have L-DOPA oxidase activity. Examples of enzymes that have the enzymatic activity of hydroxylating the 3-position of the phenolic ring of tyrosine but do not have L-DOPA oxidase activity include cytochrome P450 (particularly subfamily β of the CYP76AD group, such as CYP76AD5 and CYP76AD6), with CqCYP76AD5 being preferred (CqCYP76AD5a has the base sequence of SEQ ID NO: 50 (accession number XM_021920495) and the amino acid sequence of SEQ ID NO: 51 (accession number XP_021776187); and CqCYP76AD5b has the base sequence of SEQ ID NO: 52 (accession number XM_021861483) and the amino acid sequence of SEQ ID NO: 53 (accession number XP_021717175).} or CqCYP76AD6 {nucleotide sequence: SEQ ID NO: 54 (accession number XM_021861500), amino acid sequence: SEQ ID NO: 55 (accession number XP_021717192)}.

[0046] Examples of genes encoding enzymes that have the enzymatic activity of hydroxylating the 3-position of the phenol ring of tyrosine but do not have L-DOPA oxidase activity include the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55. (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and having the ability to hydroxylate the 3-position of the phenol ring of tyrosine, which is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, but does not have L-DOPA oxidase activity. (3) A gene encoding a polypeptide that has 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 50, 52, or 54. (5) A gene consisting of DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54, and encodes a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (6) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 50, 52, or 54, in which 1 to 50 bases have been substituted, deleted, inserted, and / or added. (7) A gene consisting of DNA having 90% or more homology with the DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54. (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 50, 52, or 54.

[0047] The gene (2) above is a gene encoding a polypeptide into which a mutation has been introduced to the extent that it does not impair the ability to hydroxylate the 3-position of the phenol ring of tyrosine. Such mutations include not only naturally occurring mutations but also artificial mutations. Methods for generating artificial mutations include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982). The number of mutated amino acids is usually 20 or less, preferably 10 or less, more preferably 5 or less, and most preferably 3. Whether a mutated polypeptide retains the ability to synthesize amaranthin can be determined, for example, by introducing a gene encoding the mutated polypeptide into a plant or the like and confirming the ability of the plant to synthesize amaranthin. With respect to the gene (3) above, the "ability to hydroxylate the 3-position of the phenol ring of tyrosine substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55" means that the degree of activity may be stronger or weaker than the ability to synthesize amaranthin substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55. Examples of the ability to synthesize amaranthin include about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, and about 150% compared to the ability of the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55. Alternatively, identity can be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, using default or initial setting parameters). The gene (5) above is a gene obtained by DNA hybridization. The term "stringent conditions" in this context refers to conditions under which only specific hybridization occurs and nonspecific hybridization does not occur. Such conditions typically include hybridization at 37°C in a buffer containing 5×SSC and 1% SDS and washing at 37°C in a buffer containing 1×SSC and 0.1% SDS. Preferred conditions include hybridization at 42°C in a buffer containing 5×SSC and 1% SDS and washing at 42°C in a buffer containing 0.5×SSC and 0.1% SDS. More preferred conditions include hybridization at 65°C in a buffer containing 5×SSC and 1% SDS and washing at 65°C in a buffer containing 0.2×SSC and 0.1% SDS. Whether or not the DNA obtained by hybridization encodes a polypeptide having activity can be determined, for example, by introducing the DNA into a plant or the like and confirming the plant's ability to synthesize amaranthin. The DNA obtained by hybridization usually has a high identity with the gene (SEQ ID NO: 50, 52, or 54) described in (4) above. A high identity refers to an identity of 90% or more, preferably 95% or more, and more preferably 98% or more. The gene (6) above is a gene consisting of DNA comprising the base sequence set forth in SEQ ID NO: 50, 52 or 54, in which 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, and even more preferably 1 to 5 base sequences have been substituted, deleted, inserted and / or added. The gene (7) above is a gene consisting of DNA having an identity of 90% or more, preferably 93% or more, more preferably 95% or more, and most preferably 98% or more to DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54.

[0048] The enzyme of the present invention that has the enzymatic activity of hydroxylating the 3-position of the phenol ring of tyrosine but does not have L-DOPA oxidase activity has an amino acid sequence selected from any one or more of the following: (1) The amino acid sequence set forth in SEQ ID NO: 51, 53, or 55. (2) An amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and which forms a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (3) An amino acid sequence that has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and forms a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. In addition, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations into the peptide, it is easily conceivable to substitute, for example, mutually homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) with each other.

[0049] (Method for synthesizing betaxanthin) The method for synthesizing betaxanthin of the present invention can be exemplified as follows. (1) A host into which a gene group has been introduced, including a gene encoding an enzyme that has the enzymatic activity of hydroxylating the 3-position of the phenolic ring of tyrosine but does not have L-DOPA oxidase activity and a gene encoding an enzyme that has DOPA 4,5-dioxygenase activity, and which has the ability to produce tyrosine or 3-hydroxy-L-tyrosine and amino acids and / or amines, is cultured, and betaxanthin is extracted from the cultured host. (2) A host having the ability to produce tyrosine or 3-hydroxy-L-tyrosine is cultured, into which a gene group has been introduced, including a gene encoding an enzyme that has the enzymatic activity of hydroxylating the 3-position of the phenol ring of tyrosine but does not have L-DOPA oxidase activity and a gene encoding an enzyme that has DOPA 4,5-dioxygenase activity. Betalamic acid is extracted from the cultured host and contacted with an amine.

[0050] (betaxanthin-producing host) The betaxanthin-producing host of the present invention has, for example, one or more of the following configurations and characteristics: (1) A gene encoding an enzyme that has the enzymatic activity of hydroxylating the 3-position of the phenol ring of tyrosine but does not have L-DOPA oxidase activity and a gene encoding an enzyme that has DOPA 4,5-dioxygenase activity have been introduced, and the strain has the ability to produce tyrosine or 3-hydroxy-L-tyrosine and amino acids and / or amines. (2) A betalamic acid synthesis composition having the ability to produce amino acids and / or amines and for synthesizing betaxanthin is introduced.

[0051] (Betalamic acid producing host) The betalamic acid-producing host of the present invention has, for example, one or more of the following configurations and characteristics: A gene encoding an enzyme that has the enzymatic activity of hydroxylating the 3-position of the phenol ring of tyrosine but does not have L-DOPA oxidase activity, and a gene encoding an enzyme that has DOPA 4,5-dioxygenase activity have been introduced, and the strain has the ability to produce tyrosine or 3-hydroxy-L-tyrosine. (2) A betalamic acid synthesis composition for the synthesis of betaxanthin has been introduced.

[0052] (Betalamic acid synthesis composition for betaxanthin synthesis) The betalamic acid synthesis composition for betaxanthin synthesis (betalamic acid synthesis agent for betaxanthin synthesis, betalamic acid synthase agent for betaxanthin synthesis) of the present invention contains any one of the following genes or a vector carrying the gene: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55. (2) A gene encoding a polypeptide having 1 to 20 amino acids substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and having the ability to hydroxylate the 3-position of the phenol ring of tyrosine, which is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, but does not have L-DOPA oxidase activity. (3) A gene encoding a polypeptide that has 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 50, 52, or 54. (5) A gene comprising DNA that hybridizes under stringent conditions with DNA comprising a base sequence complementary to DNA comprising the base sequence set forth in SEQ ID NO: 50, 52, or 54, and encodes a polypeptide that has the ability to hydroxylate the 3-position of the phenol ring of tyrosine and does not have L-DOPA oxidase activity. (6) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 50, 52, or 54, in which 1 to 50 bases have been substituted, deleted, inserted, and / or added. (7) A gene consisting of DNA having 90% or more homology with the DNA consisting of the base sequence set forth in SEQ ID NO: 50, 52, or 54. (8) A gene consisting of DNA comprising a degenerate isomer of the nucleotide sequence set forth in SEQ ID NO: 50, 52, or 54.

[0053] Alternatively, the betalamic acid synthesis composition for betaxanthin synthesis of the present invention comprises a peptide represented by any one of the following amino acid sequences (1) to (3): (1) The amino acid sequence set forth in SEQ ID NO: 51, 53, or 55. (2) An amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and which forms a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. (3) An amino acid sequence that has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, and forms a polypeptide that is substantially identical to the amino acid sequence set forth in SEQ ID NO: 51, 53, or 55, has the ability to hydroxylate the 3-position of the phenol ring of tyrosine, and does not have L-DOPA oxidase activity. [Example]

[0054] The present invention will be described in detail below with reference to specific examples, but the present invention is not limited to these examples. [Example]

[0055] <Stability of betalain pigments> The stability of aqueous solutions of cerocyanine II, amaranthin, and betanin at 37°C was evaluated.

[0056] [Betalain pigment extraction and HPLC analysis] For the extraction of amaranthin and betanin, we used amaranthin-synthesizing tobacco BYII cells and betanin-synthesizing tobacco BYII cells prepared according to a previously reported method (Non-Patent Document 4). For the extraction of cerocyanin II, wild-type quinoa plants were used. Plant samples in which cerocyanin II, amaranthin, or betanin had accumulated were crushed, water was added, and the mixture was centrifuged (20,000 g, 10 and 15 minutes). The supernatant was collected, an equal volume of acetonitrile was added, and the mixture was centrifuged (20,000 g, 10 and 15 minutes). The supernatant obtained by the centrifugation was concentrated using a concentrating centrifuge (CC-105, Tomy Seiko). The resulting concentrate was used as a pigment extract and subjected to HPLC analysis. HPLC analysis was performed using a reverse-phase column (Shim-pack GWSC18 column, 5 μm; 200 × 4.6 mm id; Shimadzu GLC). Solution A was water with 0.05% TFA added, and solution B was acetonitrile with 0.05% TFA added. The flow rate was 0.5 ml / min. The analytical program was set so that the acetonitrile concentration increased linearly from 0% at 0 minutes to 45% at 45 minutes. The analytical wavelength was set to 536 nm, which allows detection of betacyanin. The purified betalain pigments were measured using a UV-2450 spectrophotometer (Shimadzu) and the molar extinction coefficients of amaranthin and betanin (ε = 54,000 M -1 cm -1 The solution concentrations were calculated using a chromatographic method (at 536 nm) (Gandia-Herrero, F., Escribano, J., and Garcia-Carmona, F. (2010) Structural implications on color, fluorescence, and antiradical activity in betalains. Planta 232, 449-460.; Schwartz, SJ, and VonElbe, JH (1980) Quantitative determination of individual beta-lactam pigments by high-performance liquid chromatography. J. Agric. Food Chem. 28, 540-543.).

[0057] [Stability of betalain pigments] Aqueous solutions of 1 mM betanin, amaranthin, and cerocyanine II were prepared and placed in an incubator at 37°C. The absorbance at 536 nm was measured on days 0, 1, 2, 3, and 4, and the relative amounts of the obtained values ​​to the absorbance on day 0 were calculated.

[0058] [result] As a result of measuring the residual amount of betalain pigments using the absorbance at 536 nm as an index, cyanidin was present in a larger amount compared to amarantin and betanin. Furthermore, in the samples treated at 37°C for 4 days, the red color of betanin and amarantin had disappeared, but cyanidin II still exhibited a red color. From the above, it became clear that cyanidin II is more stable than amarantin and betanin among the pigments under the condition of 37°C (Figure 3).

Example

[0059] <Effect of Cyanidin II on HIV-1 Protease Activity> It was verified whether cyanidin II inhibits the activity of HIV-1 protease.

[0060] [Evaluation of HIV-1 Protease Inhibitory Activity in Betalain Pigments] The HIV-1 protease used was recombinant HIV-1 protease (ab84117) from Abcam, and the substrate peptide for HIV-1 protease was HIV-1 protease substrate (Lys-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nleamide) from Sigma-Aldrich. HIV-1 protease activity was assessed according to a previously reported method (Boso, G., Orvell, C. and Somia, NV (2015) The nature of the N-terminal amino acid residue of HIV-1 RNase H is critical for the stability of reverse transcriptase in viral particles. J. Virol. 89, 1286-1297.). Amaranthine, betanine, or cerocyanine II was added to 16 pmol of HIV-1 protease and 4 nmol of HIV-1 protease substrate. Buffer (25 mM NaCl, 25 mM NaHPO, 1 mM dithiothreitol, pH 4.7) was added to make the reaction volume 30 μl, and the reaction was allowed to proceed at 25°C for 2 hours. Amaranthine, betanine, and cerocyanine II were added at a 100-fold excess relative to the amount of HIV-1 protease. Cerocyanine II was also evaluated at 25- and 50-fold excess. The HIV-1 inhibitor saquinavir was used as a positive control. After the reaction, the amount of remaining substrate peptide in the reaction mixture was measured using HPLC, and the remaining ratio was calculated relative to the substrate before the reaction. HPLC was performed using a Shimadzu LC-20AD with a Shim-pack GWS C18 column (5 μm; 200 × 4.6 mmi.d.; Shimadzu GLC) as the analytical column. The analytical solvent system was water with 0.05% TFA in solution A and acetonitrile with 0.05% TFA in solution B. The HPLC program was set to a linear gradient of 0% solution B at 0 min and 50% solution B at 25°C, with a flow rate of 0.5 mL / min. The analysis wavelength was set to 260 nm, which allows detection of HIV-1 protease substrate peptides.

[0061] [result] It was revealed that the addition of 25-fold more cerocyanine II to HIV-1 protease had the same inhibitory activity as the addition of 100-fold more amaranthin (Fig. 4). In other words, cerocyanine II was confirmed to have four times the inhibitory effect on HIV-1 protease activity as amaranthin. Furthermore, the inhibitory activity of cerocyanine II against HIV-1 protease was dose-dependent (Fig. 4). [Example]

[0062] [Effect of betalain dyes on amyloid-β polymerization] There have been no reports on betalain pigments with respect to the polymerization of amyloid beta. The present inventors evaluated whether cerocyanine II, amaranthin, betanin pigment, and ferulic acid, which constitutes cerocyanine II, inhibit the polymerization of amyloid beta using the ThT assay.

[0063] [Evaluation of amyloid-β polymerization inhibitory activity in the ThT (thioflavin T) assay] The ThT assay was performed using SensoLyte ThioflavinT β-Amyloid (1-40) Aggregation Kit (ANASPEC) to evaluate polymerization according to the attached protocol. The fluorescence derived from amyloid-β polymerization was measured using a VarioskanLUX (ThermoFisherScientific) at 5-minute intervals for 4 hours. The sample was stirred for 15 seconds before each measurement. The excitation wavelength was 440 nm and the detection wavelength was 484 nm. Samples used in the ThT assay were adjusted to a final concentration of 50 μM. Tannic acid (Ono, K., Hasegawa, K., Naiki, H. & Yamada, M. (2004) Anti-amyloidogenic activity of tannic acid and its activity to destabilize Alzheimer's beta-amyloid fibrils in vitro. Biochim Biophys Acta 1690, 193-202) was used as an inhibitor control.

[0064] [result] Cercyanine II and betanin showed polymerization inhibitory activity equal to or greater than that of existing inhibitors (tannic acid) (Figure 5). Furthermore, amaranthine retained inhibitory activity, although its effect was less than that of cerocyanine II and betanin (Figure 5). On the other hand, when ferulic acid, which is a component of cerocyanine II, was used alone, its polymerization inhibitory effect was weaker than that of cerocyanine II, betanin pigment, and amaranthine (Figure 5). From the above, it was confirmed that betalain dyes have the activity of inhibiting amyloid polymerization. [Example]

[0065] <Search for the cerocyanine II synthase gene> To isolate the gene synthesizing celosianin II from the quinoa genome, we identified the celosianin II synthetase gene through subcellular localization prediction and gene expression analysis. Celocyanin II is a compound formed by the conjugation of ferulic acid to the glucuronic acid present in the amaranthin molecule (Figure 1). For flavonoids, another plant pigment, enzymes that add ferulic acid analogs such as sinapic acid, p-coumaric acid, and gallic acid to flavonoids have already been isolated (Bontpart, T., Cheynier, V., Ageorges, A. & Terrier, N. (2015) BAHD or SCPLacyltransferase? What a dilemma for acylation in the world of plant phenolic compounds. New Phytol 208, 695-707.). The enzymes that add these molecules (acyltransferases) belong to the SCPL (Serine CarboxyPeptidase Like) group and are known to be localized in the vacuole (Bontpart, T., Cheynier, V., Ageorges, A. & Terrier, N. (2015) BAHD or SCPL acyltransferase? What adilemma for acylation in the world of plant phenolic compounds. New Phytol 208,695-707.). To isolate the genes responsible for synthesizing cerocyanin II from quinoa, we first analyzed 87 SCPL group proteins present in the quinoa genome using the subcellular localization prediction program (WoLF PSORT).

[0066] [Vacuole localization prediction] We analyzed SCPL group proteins registered in NCBI using a subcellular localization prediction program (WoLF PSORT, https: / / wolfpsort.hgc.jp / ). As a result of the analysis, proteins with a vacuolar localization score of 4 or higher were selected as vacuolar-localized proteins.

[0067] [Comprehensive expression analysis in quinoa hypocotyls] RNA was extracted from quinoa (Chenopodium quinoa) hypocotyls 5 days after germination using the RNeasy Plant Mini kit (Qiagen). Next-generation sequencing libraries were constructed from 1 μg of extracted RNA using the NEBNextPoly(A) mRNA Magnetic Isolation Module (NEB). The constructed libraries were sequenced using a MiSeq (Illumina). The resulting next-generation sequencing data were aligned to the quinoa genome using HISAT2 (Kim, D., Langmead, B. & Salzberg, SL (2015) HISAT: a fast spliced ​​aligner with low memory requirements. Nat Methods 12, 357-360.). Gene prediction was performed using String Tie (Pertea, M. et al. (2015) String Tie enables improved reconstruction of a transcriptome from RNA-seq reads. NatBiotechnol33, 290-295.), and expression levels were determined using feature Counts (Liao, Y., Smyth, GK & Shi, W. (2014) Feature Counts: an unefficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930.).

[0068] [result] As a result of vacuolar localization prediction, 26 proteins were predicted to be localized in the vacuole (Fig. 6). Since cerocyanin II accumulates in the hypocotyl, it is predicted that the gene synthesizing cerocyanin II is also expressed in the hypocotyl. Therefore, as a next step, we investigated the expression in the hypocotyl of 26 genes predicted to be localized in the vacuole. For this selection, we performed RNA-seq on quinoa hypocotyls. Based on the obtained RNA-seq data, we selected genes expressed in the hypocotyl (transcripts per kilobase million (TPM) > 5). As a result, we found that 15 genes were expressed in the hypocotyl (Figure 6). [Example]

[0069] <Evaluation of cerocyanine II synthesis activity of the cerocyanine II synthesis gene> The 15 selected candidate genes were compared for the strength of their activity to synthesize cerocyanin II using an evaluation system using quinoa and amaranth.

[0070] [Construction of plant expression plasmids] The betalain pigment biosynthesis genes used in this experiment were expressed using the modified pCAMBIA1301 vector (Imamura, T., Takagi, H., Miyazato, A., Ohki, S., Mizukoshi, H., and Mori, M. (2018) Isolation and characterization of the betalain biosynthesis gene involved in hypocotyl pigmentation of the allotetraploid Chenopodiumquinoa. Biochem. Biophys. Res. Commun. 496, 280-286.). Restriction enzyme sites were added to the 5' and 3' ends of each gene to synthesize fragments by PCR. The resulting PCR fragments were then cleaved at the added sites and inserted into a plant-modified vector to construct plant expression vectors.

[0071] [Agrobacterium transformation method] The plasmid was introduced from E. coli harboring the constructed plasmid into Agrobacterium using the triparental mating method (Wise, AA, Liu, Z. and Binns, AN (2006) Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol. Biol. 343, 43-53.), and transformed Agrobacterium was produced.

[0072] [Evaluation of cerocyanine synthesis activity using a transient plant expression system] The selected candidate genes were evaluated to see if they retained the ability to synthesize cerocyanin II. For the experiment, quinoa and amaranth, a plant in the same Amaranthaceae family as quinoa that synthesizes amaranthin, the precursor to cerocyanin II (but not cerocyanin II), were used. These plants were infected with transformed Agrobacterium, and the candidate genes were transiently expressed in the plants, and the synthesis of cerocyanin II in the infected plants was evaluated. A plasmid expressing the selected candidate gene in plants was constructed and introduced into Agrobacterium. The resulting transformed Agrobacterium was then infiltrated in 3 mL of LB medium at 26°C for 1 day at 120 rpm. After sufficient Agrobacterium growth, the 3 mL culture was diluted to 20 mL with sterile water and supplemented with 20 μL of 10 mg / mL acetosyringone and 10 μL of Tween 20 (surfactant) to prepare an infection solution. Quinoa and amaranth plant tissues were placed in the infection solution and the pressure was reduced in a desiccator for 10–15 minutes to allow the bacteria to infiltrate into the plant tissue. The infiltrated plant tissues were transferred to MS plates and cultured statically in the dark at 23°C for 3–4 days. Betalain pigments were extracted from the cultured plant tissues and analyzed by HPLC to evaluate the ability of the candidate genes to synthesize cerocyanin II.

[0073] [result] Pigment analysis revealed that transient expression of the gene (XM_021915966: SEQ ID NOs: 56 and 57) in amaranth resulted in the accumulation of cerocyanine II, which is not normally observed in amaranth (Figure 14). Furthermore, transient expression in quinoa resulted in an approximately two-fold increase in the amount of cerocyanine II accumulated compared to the control (Figure 15). In addition, XM_021905266 (SEQ ID NOs: 58 and 59) is highly homologous to XM_021915966 (amino acid identity: 94.75%) and therefore has a similar effect. Furthermore, 13 other genes (XM_021889932, XM_021888895, XM_021888617, XM_021884200, XM_021884203, XM_021877796, XM_021900658, XM_021887381, XM_021868667, XM_021915969, XM_021891169, XM_021879737, XM_021868668) belonging to the same SCPL gene family as XM_021915966 also have similar effects. [Example]

[0074] <Mass production of cerocyanine II in tobacco cultured cells (BY-2 cells)> Having successfully isolated the celosianin II synthase gene, we attempted to mass-produce celosianin II in tobacco BY-2 cells. To produce amaranthin, we constructed a vector for expression in BY-2 cells and created transformants by introducing five betalain biosynthesis genes (CqCYP76AD1-1, CqDODA-1, CqCDOPA5GT, CqAmaSy1, and celosianin II synthetase). From the resulting transformed BY-2 lines, we selected lines with a strong red color.

[0075] [Constitutive expression analysis in tobacco BY2 cells] The plasmid contained in the transformed Agrobacterium is introduced into tobacco BY2 cells using the Agrobacterium method (Hagiwara, Y., Komoda, K., Yamanaka, T., Tamai, A., Meshi, T., Funada, R., Tsuchiya, T., Naito, S. and Ishikawa, M. (2003) Subcellular localization of host and viral proteins associated with tobamovirus RNA replication. EMBO J. 22, 344-353.). The resulting transformed lines are maintained and used for other analyses.

[0076] [result] The selected cerocyanine II-producing strains will be subjected to HPLC and mass spectrometry to confirm the production of cerocyanine II. The amount of cerocyanine II produced in a 150 mL culture will also be examined. This experiment will enable the mass production of betalain pigments (amaranthin and betanin) in cultured cells of tobacco BY-2, a plant that does not produce betalains. [Example]

[0077] <Betaxanthin production in tobacco cultured cells (BY-2 cells)> Using the amino acid sequences of similar genes from beet (Beta vulgaris) {BvCYP76AD5 (NCBI accession number: AJD87473) and BvCYP76AD6 (AJD87474)}, we selected and identified CqCYP76AD5 (XM_021920495, XM_021861483) and CqCYP76AD6 (XM_021861500) by homology search (Blastp). Of these, XM_021920495 was used for expression in BY-2 cells. To produce betaxanthin, we constructed a vector for expression in BY-2 cells and transformed them with two betaxanthin biosynthetic genes (CqCYP76AD5 and CqDODA-1). From the resulting transformed BY-2 lines, we selected lines that exhibited yellow to orange color.

[0078] [Constitutive expression analysis in tobacco BY2 cells] The plasmids contained in the transformed Agrobacterium were introduced into tobacco BY2 cells using the Agrobacterium method (Hagiwara, Y., Komoda, K., Yamanaka, T., Tamai, A., Meshi, T., Funada, R., Tsuchiya, T., Naito, S. and Ishikawa, M. (2003) Subcellular localization of host and viral proteins associated with tobamovirus RNA replication. EMBO J. 22, 344-353.). The resulting transformed lines were maintained and used for analysis.

[0079] [result] The results are shown in Figure 10. Tobacco BY-2 cells transformed with CqCYP76AD5 and CqDODA1-1 turned yellow or orange, indicating the synthesis of betaxanthin. The expression levels of CqCYP76AD5 and CqDODA1-1 varied depending on the number and location of the target genes inserted into the BY-2 cell genome during transformation, resulting in differences in the yellow or orange color of each line. Orange lines accumulated more betaxanthin than yellow lines. Tobacco BY2 cells transfected with an empty vector remained white, indicating the lack of betaxanthin synthesis. Since the synthesized betaxanthin is a mixture of betaxanthins, HPLC and mass spectrometry are performed to identify the betaxanthin. [Example]

[0080] <Evaluation of the activity of betaxanthin synthesized in cells> The activity of the extracted betaxanthin was evaluated for the betaxanthin-synthesizing tobacco BY-2 cells prepared in Example 7.

[0081] [Betaxanthin extract] BY-2 cell samples in which betaxanthin had accumulated in the plant body were disrupted, ethanol was added, and the mixture was centrifuged (20,000 g, 10 minutes, 15 minutes). The supernatant was collected, and four volumes of ethanol were added, followed by centrifugation (20,000 g, 10 minutes, 15 minutes). The supernatant obtained by centrifugation was concentrated using a concentrating centrifuge (CC-105, Tomy Seiko). The resulting concentrate was used as a betaxanthin extract.

[0082] [Effect of betaxanthin on amyloid-β polymerization] There have been no reports on betaxanthins in relation to amyloid-β polymerization. Therefore, we evaluated whether betaxanthins inhibit amyloid-β polymerization using the ThT assay.

[0083] [Evaluation of amyloid-β polymerization inhibitory activity in the ThT (thioflavin T) assay] The ThT assay was performed using SensoLyte Thioflavin Tβ-Amyloid (1-40) Aggregation Kit (ANASPEC) to evaluate polymerization according to the attached protocol. The fluorescence derived from amyloid-β polymerization was measured using a VarioskanLUX (Thermo Fisher Scientific) at 5-minute intervals for 4 hours. The sample was stirred for 15 seconds before each measurement. The excitation wavelength was 440 nm and the detection wavelength was 484 nm. Samples used in the ThT assay were adjusted to a final concentration of 50 μM. An extract of empty vector-transfected BY-2 cells (VC) and water (PC) were used as controls. Betanin and curcumin were used as positive controls. Curcumin is a plant-derived substance reported to inhibit amyloid beta polymerization (Yang F, Lim GP, Begum AN, et al. Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo. J Biol Chem. 2005;280(7):5892-5901. doi:10.1074 / jbc.M404751200).

[0084] [result] The results of the ThT assay showed that betaxanthin had inhibitory activity equivalent to that of betanin (Figure 11). From the above, it was confirmed that betaxanthin has the activity of inhibiting amyloid polymerization. [Example]

[0085] <Observation of amyloid peptides using a transmission electron microscope> The effect of betalain dyes on amyloid peptide aggregation was observed using a transmission electron microscope. Lyophilized amyloid peptide (human amyloid-β 1-40, Aβ40) was purchased from Peptide Institute, Inc., dissolved in dimethyl sulfoxide at a concentration of 1 mM to prepare a stock solution, and stored at −20° C. until use. As the betaxanthin, betaxanthin extracted from betaxanthin-producing BY2 cells (FIG. 10) was used. The reaction conditions were as follows: Aβ40 was prepared at a final concentration of 25 μM, and the samples (betanin, amaranthin, cerocyanine II, ferulic acid, and betaxanthin) were prepared at a final concentration of 50 μM in PBS (pH 7.4). As a control, distilled water was used instead of the samples. The prepared samples were left at 37°C for 5 days in the dark. After the reaction, approximately 2 μL of 2% uranyl acetate solution was added to the sample, and the sample was stained on a grid mesh for transmission electron microscopy. After the solution dried, amyloid peptide aggregation was observed using a transmission electron microscope (Hitachi 7650 TEM).

[0086] [result] When amyloid peptide aggregation was observed using a transmission electron microscope, amorphous amyloid peptide aggregation (Figure 12A) and amyloid fibrils (Figure 12B) were observed in the control sample, in which water was added instead of betalain dye. On the other hand, when betanin, amaranthin, cerocyanin II, or ferulic acid (a component of cerocyanin II) was added, only short amyloid fibrils were observed, and aggregation of amyloid peptides was inhibited (FIGS. 12C to 12G). Furthermore, when betaxanthin was added, amorphous aggregation was observed, but no amyloid fibrils were observed (Figure 12H). From the above results, it was confirmed that betanin, amaranthin, cerocyanine, and ferulic acid have the effect of inhibiting both the polymerization and amorphous aggregation of amyloid fibrils, and that betaxanthin has the effect of inhibiting the polymerization of amyloid fibrils. [Example]

[0087] <Test using nematodes> In transgenic nematodes expressing amyloid peptide, amyloid peptide aggregates form in the body over the growth period, causing the nematodes to become paralyzed. Therefore, we evaluated whether the rate of paralysis was affected by feeding on betanin. Nematodes were grown on NGM plates (nematode growth medium) and fed Escherichia coli (OP50 strain) at 15°C unless otherwise specified. Transgenic nematodes (CL2006) expressing human amyloid peptide (human amyloid-β 1-42, Aβ42) were obtained from the Caenorhabditis Genetics Center (CGC) in the United States. CL2006 expresses Aβ42 in the cells of the nematode's body wall muscles, which subsequently leads to the aggregation of amyloid peptide in the muscles. As a result, the CL2006 strain develops paralysis (C.D. Link, Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans, Proc. Natl. Acad. Sci. USA, 92 (1995) 9368-9372.). To ensure that the nematode populations used in the experiments were at the same developmental stage, they were grown on NGM plates for two or three generations without starvation. Young adult nematodes were collected and washed, then lysed using a lysis solution (0.6% sodium hypochlorite, 200 mM sodium hydroxide) to separate the eggs. After 12–14 hours at 20°C, the separated eggs were hatched, and synchronized L1 larvae were used for subsequent experiments. The synchronized L1 stage larvae were fed and raised at 20°C until they became young adults. During the raising period, 5-fluorodeoxyuridine (0.5 mg / ml) was added to prevent the production of the next generation. Adult worms grown in synchronous culture were transferred to NGM plates containing diet containing various concentrations (2, 10, 50 μM) of dextrin or betanin. After transfer, the paralysis of the worms was evaluated at regular intervals. 20 synchronized adults were transferred to each plate. Dextrin was used as a control. Paralysis was assessed by tapping the worms' nose with a platinum wire and observing their behavior afterward. According to a previous report, worms that were unable to move their noses but could not move their bodies after being tapped were considered paralyzed (E. Cohen, J. Bieschke, RM Perciavalle et al., Opposing activities protect against age-onset proteotoxicity, Science, 313 (2006) 1604-1610.).

[0088] [result] In experiments using transgenic nematodes overexpressing amyloid peptide, the percentage of paralyzed nematodes fed 50 μM betanin was significantly improved (reduced) compared to the control group fed dextrin (Figure 13B). Figure 13C shows a representative example of a paralyzed nematode fed dextrin. Figure 13D shows a representative example of a normal nematode fed betanin. These results demonstrate that betanin inhibits the aggregation of amyloid peptides in vivo. [Industrial Applicability]

[0089] The present invention makes it possible to provide a method for synthesizing cerocyanine II and a method for synthesizing betaxanthin.

Claims

1. A method for synthesizing cerocyanine II, comprising the steps of: 1) Culturing a host into which the following genes have been introduced, the host having the ability to produce tyrosine or 3-hydroxy-L-tyrosine (L-DOPA), including a gene encoding cerocyanine II synthase, a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine, a gene encoding an enzyme having L-DOPA oxidase activity, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid, and the host having the ability to produce tyrosine or 3-hydroxy-L-tyrosine (L-DOPA), and extracting cerocyanine II from the cultured host; or 2) culturing a host having the ability to produce tyrosine or 3-hydroxy-L-tyrosine, into which have been introduced the following genes: a gene encoding cerocyanine II synthase; a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine; a gene encoding an enzyme having L-DOPA oxidase activity; a gene encoding an enzyme having DOPA 4,5-dioxygenase activity; a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group; and a gene encoding an enzyme having the activity of adding glucuronic acid; and extracting cerocyanine II from the cultured host; or 3) culturing a host into which the following genes have been introduced, the host having the ability to produce tyrosine or 3-hydroxy-L-tyrosine (L-DOPA), including a gene encoding cerocyanine II synthase, a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine, a gene encoding an enzyme having DOPA 4,5-dioxygenase activity, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid, and the host having the ability to produce tyrosine or 3-hydroxy-L-tyrosine (L-DOPA), and extracting cerocyanine II from the host after cultivation; Or, 1) Culturing a host into which a gene encoding the following cerocyanine II synthase has been introduced and which has an enzyme activity having the activity of hydroxylating the 3-position of the phenol ring of tyrosine, an L-DOPA oxidase activity, an enzyme activity having the activity of adding sugar to a phenolic hydroxyl group, an enzyme activity having the activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine, and extracting cerocyanine II from the cultured host; or 2) Culturing a host into which a gene encoding the following cerocyanine II synthase has been introduced and which has an enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine, an L-DOPA oxidase activity, a DOPA 4,5-dioxygenase activity, an enzyme activity of adding sugar to a phenolic hydroxyl group, an enzyme activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine, and extracting cerocyanine II from the cultured host; or 3) culturing a host into which a gene encoding the following cerocyanine II synthase has been introduced and which has the enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine, DOPA 4,5-dioxygenase activity, enzyme activity of adding sugar to a phenolic hydroxyl group, enzyme activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine, and extracting cerocyanine II from the cultured host; wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having a substitution, deletion, insertion and / or addition of 1 to 20 amino acids in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and having the ability to synthesize cerocyanine II of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16; (3) A gene encoding a polypeptide having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II having the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (5) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (6) A gene consisting of a DNA having 90% or more identity to a DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15; and (7) A gene consisting of DNA comprising degenerate bases of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, A method for synthesizing cerocyanine II, characterized by:

2. A method for synthesizing cerocyanine II, comprising the steps of: a host into which a gene encoding the following cerocyanine II synthase has been introduced and which has the ability to produce amaranthin is cultured, and cerocyanine II is extracted from the cultured host; wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having a substitution, deletion, insertion and / or addition of 1 to 20 amino acids in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and having the ability to synthesize cerocyanine II of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16; (3) A gene encoding a polypeptide having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II having the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (5) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (6) SEQ ID NOs: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11 a gene consisting of a DNA having 90% or more identity with the DNA consisting of the base sequence set forth in 13 or 15, and (7) A gene consisting of DNA comprising degenerate bases of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, A method for synthesizing cerocyanine II, characterized by:

3. A method for synthesizing cerocyanine II, comprising the steps of: a host having the ability to produce betanin, into which a gene encoding the cerocyanine II synthase and a gene encoding an enzyme having the activity of adding glucuronic acid are introduced, and cerocyanine II is extracted from the host after the culture; Or, a host into which a gene encoding the following cerocyanine II synthase has been introduced and which has an enzyme activity having an activity of adding glucuronic acid and an ability to produce betanin, and then cerocyanine II is extracted from the cultured host; wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having a substitution, deletion, insertion and / or addition of 1 to 20 amino acids in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and having the ability to synthesize cerocyanine II of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16; (3) A gene encoding a polypeptide having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II having the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (5) In DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15, 1 to 50 bases are substituted or deleted. , a gene consisting of inserted and / or added DNA, (6) A gene consisting of a DNA having 90% or more identity to a DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15; and (7) A gene consisting of DNA comprising degenerate bases of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, A method for synthesizing cerocyanine II, characterized by:

4. A method for synthesizing cerocyanine II, comprising the steps of: a host having the ability to produce betanidin, into which a gene encoding cerocyanine II synthase, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid are introduced, and cerocyanine II is extracted from the host after the culture; Or, a host into which a gene encoding the following cerocyanine II synthase has been introduced, and which has an enzyme activity of adding sugar to a phenolic hydroxyl group, an enzyme activity of adding glucuronic acid, and an ability to produce betanidin, and then extracting cerocyanine II from the cultured host; wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having a substitution, deletion, insertion and / or addition of 1 to 20 amino acids in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and having the ability to synthesize cerocyanine II of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16; (3) A gene encoding a polypeptide having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II having the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (5) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (6) A gene consisting of a DNA having 90% or more identity to a DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15; and (7) A gene consisting of DNA comprising degenerate bases of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, A method for synthesizing cerocyanine II, characterized by:

5. The method for synthesizing cerocyanine II according to any one of claims 1 to 4, wherein the gene encoding the cerocyanine II synthase is selected from any one or more of genes consisting of DNA having the base sequence set forth in SEQ ID NOs: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 and 15.

6. A cerocyanine II synthetic composition comprising any one of the following genes or a vector carrying said gene: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having a substitution, deletion, insertion and / or addition of 1 to 20 amino acids in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and having the ability to synthesize cerocyanine II of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16; (3) A gene encoding a polypeptide having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II having the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (5) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (6) A gene consisting of a DNA having 90% or more identity to a DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13, or 15; and (7) A gene consisting of DNA consisting of degenerate bases of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15.

7. A synthetic composition of cerocyanine II having a peptide represented by any one of the following amino acid sequences: (1) an amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) An amino acid sequence in which 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and which forms a polypeptide having the ability to synthesize cerocyanine II of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16; and (3) An amino acid sequence that has 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and forms a polypeptide capable of synthesizing cerocyanin II of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16.

8. A cerocyanine II-producing host into which the synthetic composition according to claim 6 or 7 has been introduced.

9. A method for synthesizing cerocyanine II according to the following (1) or (2): (1) contacting amaranthin with the cerocyanine II synthesis composition of claim 7; (2) (a) contacting betanin with an enzyme having glucuronic acid-adding activity; (b) contacting the amaranthin obtained in (a) with the cerocyanine II synthesis composition of claim 7;

10. A cerocyanine II-producing host, The host is 1) A gene encoding the following enzymes has been introduced: a gene encoding cerocyanine II synthase; a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine; a gene encoding an enzyme having L-DOPA oxidase activity; a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group; and a gene encoding an enzyme having the activity of adding glucuronic acid, and the gene has the ability to produce tyrosine or L-DOPA, or 2) A gene encoding any of the following enzymes has been introduced: a gene encoding cerocyanine II synthase; a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine; a gene encoding an enzyme having L-DOPA oxidase activity; a gene encoding an enzyme having DOPA 4,5-dioxygenase activity; a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group; and a gene encoding an enzyme having the activity of adding glucuronic acid, and the gene has the ability to produce tyrosine or L-DOPA, or 3) A gene encoding the following enzymes, namely, a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenolic ring of tyrosine, a gene encoding an enzyme having DOPA 4,5-dioxygenase activity, a gene encoding an enzyme having the activity of adding sugar to a phenolic hydroxyl group, and a gene encoding an enzyme having the activity of adding glucuronic acid, has been introduced, and the gene has the ability to produce tyrosine or L-DOPA, or Or, 1) A gene encoding the following cerocyanine II synthase has been introduced, and the strain has an enzyme activity of hydroxylating the 3-position of the phenolic ring of tyrosine, an L-DOPA oxidase activity, an enzyme activity of adding sugar to a phenolic hydroxyl group, an enzyme activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine, or 2) A gene encoding the following cerocyanine II synthase has been introduced, and the enzyme has the activity of hydroxylating the 3-position of the phenol ring of tyrosine, L-DOPA oxidase activity, DOPA 4,5-dioxygenase activity, the activity of adding sugar to a phenolic hydroxyl group, the activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine, or 3) A gene encoding the following cerocyanine II synthase has been introduced, and the strain has an enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine, a DOPA 4,5-dioxygenase activity, an enzyme activity of adding sugar to a phenolic hydroxyl group, an enzyme activity of adding glucuronic acid, and the ability to produce tyrosine or 3-hydroxy-L-tyrosine; wherein the gene encoding the cerocyanine II synthase is selected from any one or more of the following: (1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (2) A gene encoding a polypeptide having a substitution, deletion, insertion and / or addition of 1 to 20 amino acids in the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16, and having the ability to synthesize cerocyanine II of the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14 or 16; (3) A gene encoding a polypeptide having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16 and having the ability to synthesize cerocyanine II having the amino acid sequence set forth in SEQ ID NO: 57, 59, 61, 63, 65, 67, 69, 2, 4, 6, 8, 10, 12, 14, or 16; (4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, (5) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, and (6) A gene consisting of a DNA having 90% or more identity to a DNA consisting of the base sequence set forth in SEQ ID NO: 56, 58, 60, 62, 64, 66, 68, 1, 3, 5, 7, 9, 11, 13 or 15, A host producing cerocyanine II.

11. An amyloid beta polymerization inhibitor or an agent for treating or preventing Alzheimer's disease, comprising any one of the following: (1) Celocyanine II (2) Amaranthine

12. An amyloid peptide aggregation inhibitor comprising any one of the following: (1) Celocyanine II (2) Amaranthine (3) Betanin (4) Betaxanthin

13. A preventive and / or therapeutic agent for neurodegenerative diseases, Alzheimer's disease, systemic amyloidosis, Parkinson's disease, Huntington's disease, prion disease, multiple sclerosis, and amyotrophic lateral sclerosis, comprising the amyloid peptide aggregation inhibitor according to claim 12 as an active ingredient.

14. Inhibitors of HIV-1 protease activity, including: (1) Celocyanine II Here, the HIV-1 protease inhibitory activity of Celocyanine II is four times that of the same amount of amaranthin. Inhibitor of HIV-1 protease activity.

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