JP2019106980A - Betalain pigment synthesis method - Google Patents

Abstract

To provide a single (R body) betalain when a betalain extract/purified product is used as a pharmaceutical, and a function thereof is analyzed.SOLUTION: The invention is configured to perform isolation of a gene related to a betalain which is synthesized specifically to a hypocotyl of a quinua, then analyze a function of the gene. In the analysis, in a betalain pigment synthesis in the quinua, isobetanin is not substantially synthesized.SELECTED DRAWING: None

Description

  The present invention relates to a method for synthesizing betalain pigment, an agent for converting L-DOPA to 4,5-seco-DOPA, a host producing betalain pigment, and an amarantine synthetase.

(Betalain)
Betalain is a compound containing red and yellow nitrogen which is synthesized by plants of the order Nadeshikoda. It is known to have high antioxidant power and various functions. A lot of betalain is accumulated in the root of red sugar beet and it is noted as a health food. Betalain contained in sugar beet is synthesized and accumulated to the same extent as betatin and its optical isomer isobetanin.

Patent Document 1 discloses a microorganism or microorganism having “an enzyme activity for hydroxylating position 3 of the phenol ring of tyrosine, a DOPA 4, 5-dioxygenase activity, an L-DOPA oxidase activity, and an enzyme activity for adding a sugar to a phenolic hydroxyl group. Disclosed is a method for producing betacyanins having a step (conversion step) of converting a raw material into betacyanins in an aqueous medium in the presence of the treated product.
Non-Patent Document 1 reports that "the betanin synthesis system of ochreiana synthesizes 88% of betaine betaine which is S form and 12% of isobetanine which is tetragonal form".
Non-Patent Document 2 reports that "the potato betanin synthesis system synthesizes both S-form and R-form".
Non-Patent Document 3 reports that “the betanine synthesis system of Bensminana tobacco synthesizes both an S form and an R form”.
However, none of the documents disclose or suggest the quinoa-derived 4,5-dihydroxy-phenylalanine dioxygenase extradiol {4,5-DOPA (dihydroxy- phenylalanine) dioxygenase extradiol} of the present invention.

JP-A-2016-182044

Sasaki, N., Abe, Y., Goda, T., Adachi, T., Kasahara, K. and Ozeki, Y. 2009, Detection of DOPA 4, 5-dioxygenase (DOD) activity using recombinant protein prepared from Escherichia coli cellsharboring cDNA encoding DOD from Bilitis jalapa Plant Cell Physiol., 50, 1012-6. Polturak, G., Grossman, N., Vela-Corcia, D., et al. . Polturak, G., Breitel, D., Grossman, N., etal.

  Since there is an optical isomer in organisms producing betalain, it is required to synthesize single (R-form) betalain when using betalain extracted / purified product as a pharmaceutical or analyzing its function. It is done.

In order to solve the above-mentioned problems, the present inventors have isolated a gene involved in betalain which is specifically synthesized in hypocotyl of quinoa, and analyzed the function of the gene. In the synthetic system, it was confirmed that isobetanin was not substantially synthesized.
By the above, the synthesis method of the betalain pigment of the present invention, the conversion agent of L-DOPA to 4, 5-seco-DOPA, and the betalain pigment production host were completed.

That is, the present invention is as follows.
1. A method of synthesizing betalain dyes,
A gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having an L-DOPA oxidase activity, a gene encoding an enzyme having an activity of adding a sugar to a phenolic hydroxyl group The following gene groups of genes encoding enzymes having DOPA 4,5-dioxygenase activity have been introduced and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) production Cultivating a host having the ability to extract betalain pigment from the host after cultivation,
Or
The following genes encoding an enzyme having DOPA 4,5-dioxygenase activity have been introduced, and an enzyme activity having an activity of hydroxylating position 3 of the phenol ring of tyrosine, an enzyme activity having L-DOPA oxidase activity (B) culturing a host having an enzyme activity having an activity of adding a sugar to a phenolic hydroxyl group and capable of producing tyrosine or 3-hydroxy-L-tyrosine to extract betalain dye from the host after culture;
Here, a gene encoding an enzyme having the DOPA 4,5-dioxygenase 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 NOs: 17, 19, 21, 23, 25 or 27;
(2) In the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, 21. A gene encoding a polypeptide having the ability to convert L-DOPA substantially equivalent to the amino acid sequence set forth in 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) An amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, and an amino acid set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27 A gene encoding a polypeptide having the ability to convert L-DOPA substantially homologous to the sequence to 4,5-seco-DOPA,
(4) a gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28;
(5) Hybridizes with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28 under a stringent condition, and L-DOPA A gene comprising a DNA encoding a polypeptide having the ability to convert to 5-seco-DOPA,
(6) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28 ,
(7) A gene consisting of a DNA having a homology of 90% or more with the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28; and (8) the amino acid sequence of SEQ ID NO: 33 Any one or more genes of (1) to (7) above, comprising a gene encoding a polypeptide consisting of
A method for producing a betalain pigment characterized in that
2. The method for synthesizing a betalain dye according to item 1 above, wherein the betalain dye is betanine.
3. The method for synthesizing the betalain pigment according to the above 1 or 2, wherein the betalain pigment is substantially free of isobetanine.
4. An agent for converting L-DOPA containing the gene shown in any one of the following (1) to (8) or a vector carrying the gene into 4, 5-seco-DOPA:
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27;
(2) In the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, 21. A gene encoding a polypeptide having the ability to convert L-DOPA substantially equivalent to the amino acid sequence set forth in 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) An amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, and an amino acid set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27 A gene encoding a polypeptide having the ability to convert L-DOPA substantially homologous to the sequence to 4,5-seco-DOPA,
(4) a gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28;
(5) Hybridizes with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28 under a stringent condition, and L-DOPA A gene comprising a DNA encoding a polypeptide having the ability to convert to 5-seco-DOPA,
(6) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28 ,
(7) A gene consisting of a DNA having a homology of 90% or more with the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28; and (8) the amino acid sequence of SEQ ID NO: 33 The gene of any one or more of the above (1) to (7), which comprises a gene encoding a polypeptide consisting of
5. An agent for converting L-DOPA having a peptide represented by the amino acid sequence of any one of (1) to (4) below into 4,5-seco-DOPA;
(1) the amino acid sequence as set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27,
(2) In the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, An amino acid sequence having the ability to convert L-DOPA substantially homologous to the amino acid sequence set forth in 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) L-DOPA substantially homologous to the amino acid sequence of SEQ ID NO: 1 and having a homology of 90% or more with the amino acid sequence of SEQ ID NO: 17, 19, 21, 23, 25 or 27 An amino acid sequence having the ability to convert to 4,5-seco-DOPA, and (4) an amino acid sequence of any one or more of the above (1) to (3) comprising the amino acid sequence set forth in SEQ ID NO: 33.
6. The betalain pigment production host which the conversion agent of the preceding clause 4 or 5 was introduce | transduced.
7. Betalein pigment producing host,
The host is
A gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having an L-DOPA oxidase activity, a gene encoding an enzyme having an activity of adding a sugar to a phenolic hydroxyl group The following gene groups of genes encoding enzymes having DOPA 4,5-dioxygenase activity have been introduced and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) production Have the ability to
Or
The following genes encoding an enzyme having DOPA 4,5-dioxygenase activity have been introduced, and an enzyme activity having an activity of hydroxylating position 3 of the phenol ring of tyrosine, an enzyme activity having L-DOPA oxidase activity , An enzyme activity having an activity of adding a sugar to a phenolic hydroxyl group and an ability to produce tyrosine or 3-hydroxy-L-tyrosine,
Here, a gene encoding an enzyme having the DOPA 4,5-dioxygenase 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 NOs: 17, 19, 21, 23, 25 or 27;
(2) In the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, 21. A gene encoding a polypeptide having the ability to convert L-DOPA substantially equivalent to the amino acid sequence set forth in 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) An amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, and an amino acid set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27 A gene encoding a polypeptide having the ability to convert L-DOPA substantially homologous to the sequence to 4,5-seco-DOPA,
(4) a gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28;
(5) Hybridizes with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28 under a stringent condition, and L-DOPA A gene comprising a DNA encoding a polypeptide having the ability to convert to 5-seco-DOPA,
(6) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28 ,
(7) A gene consisting of a DNA having a homology of 90% or more with the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28; and (8) the amino acid sequence of SEQ ID NO: 33 Any one or more genes of (1) to (7) above, comprising a gene encoding a polypeptide consisting of
A betalain pigment production host characterized in that.
8. The betalein pigment-producing host according to the above 7, wherein the betalain pigment is substantially free of isobetanine.

  The present invention has been able to provide a method for synthesizing betalain pigment, an agent for converting L-DOPA to 4,5-seco-DOPA, a host producing betalain pigment, and an amarantine synthetase.

Betalain pigment synthesis. Amino acid sequence of the active site of DODA1. A: Conserved sequence of DODA1 active site (Christinet, L., Burdet, FX, Zaiko, M., Hinz, U. and Zlyd, JP 2004, Characterization and functional identification of a novel plant , 134, 265-74.). B: Sequence of the active site of Quinua DODA1. Betalain synthetic gene expression vector. A: Diagram of pCAMBIA_CqCYP76AD1-ox. B: Diagram of pCAMBIA_CqDODA1-1 to 1-6-ox. C: Diagram of pCAMBIA_CqCDOPA5GT-ox. D: Diagram of pCAMBIA1301M which is an empty vector (vector control). HPT indicates hygromycin resistant gene, 35S indicates enhanced CaMV 35 Spromoter, NosT indicates Nopaline synthe sis Terminater, LB indicates Left Border, and RB indicates Right Border. Activity evaluation 1 in Bensminana tobacco. A: Green leaves of N. benthamiana transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. B: Green leaves of C. camphorata transformed with CqCYP76AD1, empty vectors (vector control) pCAMBIA1301M and CqCDOPA5GT. C: HPLC analysis result. Betanin indicates a standard product (betanin, isobetanine mixture). Activity evaluation 2 in Bensminana tobacco. A: Green leaves of N. benthamiana transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. B: Green leaves of N. benthamiana transformed with CqCYP76AD1, CqDODA1-2 and CqCDOPA5GT. C: HPLC analysis result. Betanin indicates a standard product (betanin, isobetanine mixture). Activity evaluation in tobacco BY2 cells. A: Tobacco BY2 cells transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. B: Tobacco BY2 cells transformed with CqCYP76AD1, empty vector (vector control) pCAMBIA1301M and CqCDOPA5GT. C: HPLC analysis result. Synthetic route of betalamic acid from L-DOPA. A phylogenetic tree using a neighbor joining algorithm (neighbor joiningalgorithm). Scale bars indicate 50% differences in amino acid sequences. Evaluation of amarantine synthetase activity in Bensminana tobacco. "-" Shows pCAMBIA1301M which is an empty vector (vector control). HPLC analysis results in evaluation of amarantine synthesis ability. Evaluation of the activity of DODIA gene of Oshiroibana in Bensminana tobacco. The HPLC analysis result in evaluation of the activity of DODA1 gene of Oshiroibana.

  The present invention relates to a method for synthesizing betalain pigment, an agent for converting L-DOPA to 4,5-seco-DOPA, a host producing betalain pigment, and an amarantine synthetase. Hereinafter, the present invention will be described in detail.

(Betalain pigment)
The betalain dyes in the present invention are classified into betaxanthins and betacyanins based on structural features. In addition, since betaxanthin develops yellow and betacyanins develop reddish purple, it has been conventionally used as a natural coloring agent. The term "betacyanins" is a generic name of a group of compounds in which saccharides are glycosidically bonded to the phenolic hydroxyl group of bethanidine.

(Betalain pigment synthesis system)
A summary of the betalain pigment synthesis system derived from the hypocotyl of quinoa according to the present invention is shown in FIG. As apparent from the description of FIG. 1, tyrosine (Tyrosine) or 3-hydroxy-L-tyrosine (3-hydroxy-L-tyrosine: L-DOPA), further, an amino acid, an amine (eg, putrescine), Hosts capable of producing spermidine (spermine) (including hosts that can be imported from the outside), but enzymes that hydroxylate position 3 of the phenol ring of tyrosine (eg, CqCYP76AD1) activity, L -An enzyme having DOPA oxidase activity (eg, CqCYP76AD1) activity, an enzyme having an activity of adding a sugar to a phenolic hydroxyl group {eg, an enzyme having betanidin 5-O-glucosyltransferase activity (Cyclo-DOPA 5-O-glucosyltransferase, CqCDOPA5GT etc.) activity, 4,5-dihydroxy-phenylalanine dioxygenase extradiol of the present invention {4, 5-DOPA (dihydroxy-phenylalanine) dioxygenase estradiol If you have a 4,5-DOPA dioxygenase extradiol 1} activity, it can be synthesized solid xanthine and betacyanin acids (eg, bethanidine, betanin) a.
CqCYP76AD1 has properties of both an enzyme activity of hydroxylating the 3-position of the phenol ring of tyrosine and an enzyme activity of having L-DOPA oxidase activity.
From the results of Example 8 below, a gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having L-DOPA oxidase activity, DOPA 4, 5-dioxygenase activity A plant body which is a host into which a gene group of a gene encoding an enzyme having an enzyme having an activity of adding a sugar to a phenolic hydroxyl group and a gene encoding an amarantine synthetase has been introduced is one of betalain pigments. It has the ability to synthesize one of amarantine.

(An enzyme that hydroxylates the 3-position of the phenol ring of tyrosine)
The enzyme for hydroxylating the 3-position of the phenol ring of tyrosine in the present invention has an activity capable of adding a hydroxyl group to the 3-position of the phenol ring possessed by tyrosine, and any kind of such an activity can be used. The origin of
Examples of the enzyme for hydroxylating the 3-position of the phenol ring of tyrosine include tyrosinase, cytochrome P450 (particularly, CYP76AD1, CYP76AD2, CYP76AD3 etc.), catechol oxidase etc., preferably CqCYP76AD1 (SEQ ID NO: 29, accession It is number XP_021769302).

(Enzyme having L-DOPA oxidase activity)
The enzyme having L-DOPA oxidase activity in the present invention has an activity of converting L-DOPA into cyclo-DOPA, and may have any activity as long as it has the activity.
Examples of enzymes having L-DOPA oxidase activity include tyrosinase, CYP76AD1 that is cytochrome P450, CYP76AD2, CYP76AD3, CYP76AD5, CYP76AD6 and the like as the enzyme having L-DOPA oxidase activity, preferably CqCYP76AD1 (sequence Number 29).

(Enzyme that adds sugar to phenolic hydroxyl group)
In the present invention, an enzyme that adds a sugar to a phenolic hydroxyl group has an activity of adding a sugar to a phenolic hydroxyl group present at the 5th or 6th position of the cyclo-DOPA skeleton, and it has such activity. It may be derived from any species.
Examples of enzymes that add a sugar to a phenolic hydroxyl group include cyclo-DOPA 5-O-glucosyltransferase, betanidine 5-O-glucosyltransferase (cyclo-DOPA 5-O-glucosyltransferase), betanidine 6-O-glucosyltransferase, etc. However, preferably, it is CqCDOPA5GT (SEQ ID NO: 31, Accession No. XP_021748306).
The enzyme that synthesizes amaranthin (Amaranthin) from betanin (amaranthine synthetase) is SEQ ID NO: 36 (Accession No. XP 021754077).

If the enzyme that adds a sugar to a phenolic hydroxyl group is bethanidine 6-O-glucosyltransferase, betanin is synthesized.
If the enzyme that adds sugar to phenolic hydroxyl group is betanidin 6-O-glucosyltransferase, Gomphrenin-I is synthesized.
If the enzyme that adds a sugar to a phenolic hydroxyl group is betanidin 5-O-glucosyltransferase and betanin beta-D-glucuronosyltransferase, amaranthine (especially 15S form) is synthesized.
If the enzyme that adds a sugar to the phenolic hydroxyl group is bethanidine 6-O-glucosyltransferase and there is an amarantine synthetase, amarantine is synthesized.
If the enzyme that adds a sugar to a phenolic hydroxyl group is betanidin 5-O-glucosyltransferase and betanin feruloyltransferase, Lampranthin II (especially 15S form) is synthesized.
If the enzyme that adds a sugar to the phenolic hydroxyl group is betanidin 5-O-glucosyltransferase, betanin beta-D-glucuronosyltransferase, and betanin feruloyl transferase, Celosianin II (in particular, 15S form) is synthesized.

(An enzyme having DOPA 4,5-dioxygenase activity)
The enzyme having DOPA 4,5-dioxygenase activity of the present invention has the ability to convert L-DOPA to 4,5-seco-DOPA by activity catalysing extradiol cleavage of L-3,4-dihydroxyphenylalanine. Have. In addition, 4,5-seco-DOPA is converted to betalamic acid via a spontaneous reaction (see: FIG. 7).

The gene encoding the enzyme having DOPA 4,5-dioxygenase activity of the present invention 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: 17 (Accession No. XP_021769303), 19 (Accession No. XP_021769301), 21, 23, 25, 27 or 34.
(2) 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, and SEQ ID NO: 17, A gene encoding a polypeptide having the ability to convert L-DOPA substantially equivalent to the amino acid sequence set forth in 19, 21, 23, 25, 27, or 34 into 4,5-seco-DOPA.
(3) It has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, and SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34 A gene encoding a polypeptide having the ability to convert L-DOPA substantially homologous to the amino acid sequence described in 4,5-seco-DOPA.
(4) A gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NOs: 18, 20, 22, 24, 26, 28 or 35.
(5) It hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35, and L-DOPA A gene consisting of a DNA encoding a polypeptide having the ability to convert 4,5-seco-DOPA.
(6) In the DNA consisting of the nucleotide sequence set forth in SEQ ID NOs: 18, 20, 22, 24, 26, 28 or 35, from DNA in which 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added Genes that
(7) A gene consisting of a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35 and a DNA having a homology of 90% or more.
(8) The gene of any one or more of the above (1) to (7), which comprises a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 33.

The gene of the above (2) is a gene encoding a polypeptide into which a mutation has been introduced to such an extent that the ability to convert L-DOPA to 4,5-seco-DOPA is not lost. Such mutations include artificial mutations as well as mutations that occur in nature. Examples of means for generating artificial mutations include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982) and the like. The number of mutated amino acids is usually 20 or less, preferably 10 or less, more preferably 5 or less, and most preferably 3 amino acids. Whether the mutated polypeptide retains the ability to convert L-DOPA to 4,5-seco-DOPA can be obtained, for example, by introducing a gene encoding the mutated polypeptide into a plant or the like, This can be seen by confirming the ability to convert L-DOPA in the plant into 4,5-seco-DOPA.
Regarding the gene of (3) above, “the ability to convert L-DOPA substantially homologous to the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34 into 4, 5-seco-DOPA” Is stronger than the ability to convert L-DOPA of the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34 into 4, 5-seco-DOPA. You may also be weak. For example, about 50%, about 60% as compared to the ability to convert L-DOPA of the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34 into 4, 5-seco-DOPA About 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%.
Also, identity can be calculated using BLAST (Basic Local Alignment Search Tool at National Center for Biological Information) and the like (for example, using default or default parameters).
The gene of (5) above is a gene obtained by utilizing hybridization of DNAs. The term "stringent conditions" in this gene refers to conditions under which only specific hybridization can occur and nonspecific hybridization can not occur. Such conditions are usually hybridization at 37 ° C. in a buffer containing 5 × SSC, 1% SDS, and washing at 37 ° C. with a buffer containing 1 × SSC, 0.1% SDS. Preferably, conditions such as hybridization at 42 ° C. in a buffer containing 5 × SSC, 1% SDS and washing at 42 ° C. with a buffer containing 0.5 × SSC, 0.1% SDS, more preferably Conditions such as hybridization at 65 ° C. in a buffer containing 5 × SSC, 1% SDS and washing at 65 ° C. with a buffer containing 0.2 × SSC, 0.1% SDS. Whether or not the DNA obtained by utilizing hybridization encodes a polypeptide having activity can be obtained, for example, by introducing the DNA into a plant or the like, and using L-DOPA of the plant as 4,5-seco. -By knowing the ability to convert to DOPA. The DNA obtained by hybridization usually has high identity to the gene of (4) above (SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35). High identity refers to 90% identity or more, preferably 95% identity or more, more preferably 98% identity or more.
In the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35, the gene of (6) above is 1 to 50, preferably 1 to 30, and more preferably 1 to 10. It is a gene consisting of DNA in which 20, most preferably 1 to 10, and most preferably 1 to 5 base sequences are substituted, deleted, inserted and / or added.
The gene of the above (7) is 90% or more, preferably 93% or more, more preferably 95% or more, and DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35 Preferably, it is a gene consisting of DNA having an identity of 98% or more.

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 as set forth in SEQ ID NOs: 17, 19, 21, 23, 25, 27 or 34.
(2) 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, and SEQ ID NO: 17, An amino acid sequence having the ability to convert L-DOPA substantially identical to the amino acid sequence set forth in 19, 21, 23, 25, 27, or 34 into 4,5-seco-DOPA.
(3) L- having a homology of 90% or more with the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25, 27 or 34, and substantially homologous to the amino acid sequence set forth in SEQ ID NO: 1 Amino acid sequence with the ability to convert DOPA to 4,5-seco-DOPA.
(4) The amino acid sequence of any one or more of the above (1) to (3), which comprises the amino acid sequence set forth in SEQ ID NO: 33.
In addition, in introducing a mutation of a peptide, for example, homologous amino acids (polar amino acids, nonpolar amino acids, Mutual substitution between hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids etc. is easily envisioned.
In particular, the amino acid sequence set forth in SEQ ID NO: 33 includes the sequence of the active site. Mutations are preferably introduced into amino acids other than these domains, as mutations in this sequence are likely to lose the ability to convert L-DOPA to 4,5-seco-DOPA.

(Method of synthesizing betalain pigment)
The synthesis method of the betalain dye of the present invention can be exemplified as follows.
(1) A gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having an L-DOPA oxidase activity, and an enzyme having an activity of adding a sugar to a phenolic hydroxyl group And a gene having a gene encoding a gene encoding an enzyme having DOPA 4,5-dioxygenase activity, and capable of producing tyrosine or 3-hydroxy-L-tyrosine. Extract betalain dye from the host.
(2) A gene encoding an enzyme having DOPA 4,5-dioxygenase activity has been introduced, and an enzyme activity having the activity of hydroxylating the 3-position of the phenol ring of tyrosine, an enzyme activity having the L-DOPA oxidase activity A host having an enzyme activity having an activity of adding a sugar to a phenolic hydroxyl group and capable of producing tyrosine or 3-hydroxy-L-tyrosine is cultured to extract betalain dye from the host after culture.
In the method for synthesizing betalain dyes of the present invention, various betacyanins can be synthesized depending on the type of enzyme having an activity of adding a sugar to a phenolic hydroxyl group.
In addition, in the method for synthesizing betalain dye of the present invention, it is confirmed in the following examples that isobetanin is not substantially synthesized.
The phrase "does not substantially synthesize isobetanine" means that the isobetanine synthesis amount is 3 parts by weight or less, 2 parts by weight or less, 1 part by weight or less, and 0.5 parts by weight or less per 100 parts by weight of betanine synthesis 0.3 parts by weight or less, 0.2 parts by weight or less, 0.1 parts by weight or less, 0.01 parts by weight or less, 0.001 parts by weight or less, 0.0001 parts by weight or less, or 0 parts by weight .
In addition, “does not substantially synthesize isoamarantin” means that the synthesized amount of isoamaranthine is 3 parts by weight or less, 2 parts by weight or less, 1 part by weight or less, with respect to 100 parts by weight of amaranthine synthesized. .5 parts by weight or less, 0.3 parts by weight or less, 0.2 parts by weight or less, 0.1 parts by weight or less, 0.01 parts by weight or less, 0.001 parts by weight or less, 0.0001 parts by weight or less, or 0 Means parts by weight.

(Host)
The host used in the method for synthesizing the betalain pigment of the present invention is not particularly limited as long as it has tyrosine or 3-hydroxy-L-tyrosine producing ability, and, for example, a recombinant Escherichia coli protein synthesis system known per se, an insect protein synthesis system Yeast protein synthesis systems, plant cell protein synthesis systems, cell-free protein synthesis systems, plant protein synthesis systems, animal culture cells and the like can be used.
A method known per se can be used to introduce each gene into a host used in the method for synthesizing betalain pigment of the present invention. For example, a vector carrying the gene can be used to introduce it into a host.
As vectors, viral vectors known per se, particularly plant virus vectors (eg, vectors derived from viruses belonging to the genus Tobamo virus, tobacco mosaic virus vectors, tomato mosaic virus vectors) can be used.
In addition, the Agrobacterium method using a Ti plasmid can be used.

(How to introduce each gene into the host)
In the method of synthesizing betalain pigment of the present invention, the method of introducing each gene into a host can introduce the gene into a plant by a method known per se. For example, each gene can be introduced into a host by applying a solution containing a plant virus vector carrying each gene to leaves, stems, roots, ears and the like of a plant body. As another method, particle gun method and Agrobacterium method can be exemplified.

(How to introduce each protein into the host)
In the method for synthesizing betalain pigment of the present invention, the method for introducing each protein into a host (in particular, a plant) can introduce the protein into a plant by a method known per se. For example, each protein can be introduced into a host by applying a solution containing each protein to leaves, stems, roots, ears and the like of a plant body. As another method, particle gun method and Agrobacterium method can be exemplified.

(Betalain pigment production host)
The betalain pigment production host of the present invention has, for example, any one or more of the following constitutions / features.
(1) A gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having an L-DOPA oxidase activity, and an enzyme having an activity of adding a sugar to a phenolic hydroxyl group Genes and genes encoding an enzyme having DOPA 4,5-dioxygenase activity have been introduced, and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) It has a production ability.
(2) An enzyme having introduced therein a gene encoding an enzyme having DOPA 4,5-dioxygenase activity and having an activity of hydroxylating the 3-position of the phenol ring of tyrosine and an L-DOPA oxidase activity It has an activity, an enzyme activity having an activity of adding a sugar to a phenolic hydroxyl group, and an ability to produce tyrosine or 3-hydroxy-L-tyrosine.
(3) A conversion agent of L-DOPA to 4,5-seco-DOPA is introduced.
In the betalain pigment production host of the present invention, various betacyanines can be synthesized depending on the type of enzyme having an activity of adding sugar to phenolic hydroxyl group.
In addition, it is confirmed in the following example that the betalain dye-producing host of the present invention does not substantially synthesize isobetanine.

(Converter for L-DOPA to 4,5-seco-DOPA)
The agent for converting L-DOPA of the present invention to 4,5-seco-DOPA comprises the gene shown in any one of the following (1) to (8) or a vector carrying the gene.
(1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25, 27 or 34.
(2) 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, and SEQ ID NO: 17, A gene encoding a polypeptide having the ability to convert L-DOPA substantially equivalent to the amino acid sequence set forth in 19, 21, 23, 25, 27, or 34 into 4,5-seco-DOPA.
(3) It has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, and SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34 A gene encoding a polypeptide having the ability to convert L-DOPA substantially homologous to the amino acid sequence described in 4,5-seco-DOPA.
(4) A gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NOs: 18, 20, 22, 24, 26, 28 or 35.
(5) It hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35, and L-DOPA A gene consisting of a DNA encoding a polypeptide having the ability to convert 4,5-seco-DOPA.
(6) In the DNA consisting of the nucleotide sequence set forth in SEQ ID NOs: 18, 20, 22, 24, 26, 28 or 35, from DNA in which 1 to 50 nucleotide sequences are substituted, deleted, inserted and / or added Genes that
(7) A gene consisting of a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35 and a DNA having a homology of 90% or more.
(8) The gene of any one or more of the above (1) to (7), which comprises a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 33.

Moreover, the conversion agent of L-DOPA of the present invention to 4,5-seco-DOPA has a peptide represented by the amino acid sequence of any one of the following (1) to (4).
(1) The amino acid sequence as set forth in SEQ ID NOs: 17, 19, 21, 23, 25, 27 or 34.
(2) 1 to 20 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, and SEQ ID NO: 17, An amino acid sequence having the ability to convert L-DOPA substantially identical to the amino acid sequence set forth in 19, 21, 23, 25, 27, or 34 into 4,5-seco-DOPA.
(3) L- having a homology of 90% or more with the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25, 27 or 34, and substantially homologous to the amino acid sequence set forth in SEQ ID NO: 1 Amino acid sequence with the ability to convert DOPA to 4,5-seco-DOPA.
(4) The amino acid sequence of any one or more of the above (1) to (3), which comprises the amino acid sequence set forth in SEQ ID NO: 33.

  The present invention will be described in detail by way of specific examples below, but the present invention is not limited to these examples.

<Preparation of Agrobacterium transformed with quinea-derived betalain synthesis ability gene>
CqCYP76AD1 (an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine and an enzyme having an L-DOPA oxidase activity) which is a gene encoding an enzyme used in the method for synthesizing a betalain pigment according to the present invention; The following method is applied to DOPA 4,5-dioxygenase activity enzyme, CqDODA1-2 (DOPA 4,5-dioxygenase activity enzyme) and CqCDOPA5GT (enzyme having activity to add sugar to phenolic hydroxyl group) An expression plasmid was constructed by the method described above and transformed into Agrobacterium. Transformed Agrobacterium was used for evaluation of betalain synthesis ability described later.

[Construction of Expression Plasmid for Quinoa-Derived Betalain Synthesis Gene]
(1) RNA was extracted from the hypocotyl of quinoa (Chenopodium quinoa) germination day 4 using RNeasy Plant Mini kit (Qiagen). The extracted RNA was used to synthesize cDNA using the HighCapacity cDNA Reverse Transcription Kit. The procedure followed the attached instructions.
(2) Using the synthesized cDNA as a template, the full-length ORF of the betalain synthesis gene was amplified by PCR using an enzyme of Prime STAR GXL (TaKaRa). The primers used for PCR were CqCYP76AD1 (SEQ ID NO: 30, Accession No. XM-021913610), SEQ ID NOs: 1 and 2, CqDODA1-1 (SEQ ID NO: 18, Accession No. XM-021913611), SEQ ID NOs: 3 and 4, CqDODA1-2 (Sequence) SEQ ID NOS: 5 and 6 for C. ID No. 20, Accession No. XM_021913609), and SEQ ID NOs. The amplified PCR fragment was digested with restriction enzymes (CqCYP67AD1: SpeI and BstPI, CqDODA1-1 and CqDODA1-2: EcoRI and BamHI, CqCDOPA5GT: BamHI and BstPI).
(3) A plasmid pCAMBIA1301M which constitutively expresses in the plant a PCR fragment which has been subjected to restriction enzyme treatment (Imamura, T., Kusano, H., Kajigaya, Y., Ichikawa, M. and Shimada, H. 2007, A rice dihydrosphingosine C4 Plant cell Physiol., 48, 1108-20.) was introduced downstream of the CaMV 35 S promoter (FIG. 3). H. xylase (DSH1) gene, which is abundantly expressed in the stigmas, vascular cellsandapical meristem, may be involuted. The constructed vectors were named as pCAMBIA_CqCYP76AD1-ox, pCAMBIA_CqDODA1-1-ox, pCAMBIA_CqDODA1-2-ox, pCAMBIA_CqDOPA5GT-ox, respectively.

[Agrobacterium transformation method]
Using E. coli carrying the above-constructed plasmid, the try-pa rental mating method (Wise, AA, Liu, Z. and Binns, AN 2006, Three methods for the introduction offoreign DNA into Agrobacterium. Methods Mol. Biol., 343, 43 According to -53), a plasmid was introduced into Agrobacterium to prepare a transformed Agrobacterium.

<Evaluation of betalain synthesis ability in plants>
The ability to synthesize betalain in the host plant was evaluated using the non-competitive Benzamica tobacco according to the following method.

[Transient expression analysis in bensamiana tobacco]
The transformed Agrobacterium prepared in Example 1 was cultured in 3 ml of LB medium, respectively, and the following combination of "Evaluation of activity 1 with Bensminana tobacco" and "Activity evaluation 2 with Bensminana tobacco" The culture broths were mixed with each other, and the green leaves of C. benthamiana tobacco were inoculated using agroinfiltration method (Shamloul, M., Trusa, J., Mett, V. and Yusibov, V. 2014, Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana. Journal of visualized experiments: JOVE. After growing for 3 to 5 days after inoculation, green leaves of the grown Bensamiana tobacco were used for the following analysis.
(1) Activity evaluation 1 in Bensamine tobacco 1
One C. benthamiana tobacco was transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. CqCYP76AD1 and pCAMBIA1301M and CqCDOPA5GT, which are empty vectors (vector control), were introduced into the other N. benthamiana tobacco.
(2) Activity evaluation 2 in bensamiana tobacco 2
One C. benthamiana tobacco was transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. CqCYP76AD1, CqDODA1-2 and CqCDOPA5GT were introduced into the other benthamiana tobacco.

[Betalain pigment extraction and HPLC analysis]
(1) A transiently expressed green leaf sample of N. benthamiana was crushed, water was added, and centrifugation (20,000 g, 10 minutes, 15 minutes) was performed. The centrifuged supernatant was collected and the same operation was performed once more. The supernatant obtained by centrifugation was subjected to HPLC analysis as a pigment extract.
(2) HPLC analysis using reverse phase column (5C18-AR-300, Nacalai Tesque, Inc.), using water and acetonitrile as the analysis solvent, flow rate 1 ml / min, analysis program to analyze the concentration of acetonitrile The analysis wavelength was set to 536 nm at which betacyanin can be detected by setting the condition to reach 50% linearly from 0% to 60 minutes at time 0 minutes. A beet extract (betanin, isobetanin mixture, Tokyo Chemical Industry Co., Ltd.) was used as a standard.

[result]
The result of "the activity evaluation 1 in bensamiana tobacco" is shown in FIG.
The leaves of Bensminaria tobacco transiently expressing CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT turned red, indicating that a red pigment was synthesized (FIG. 4A). The benthamiana tobacco in which the empty vector was introduced instead of CqDODA1-1, the leaves remained green, indicating that the red pigment was not synthesized (FIG. 4B).
As a result of HPLC analysis, a single peak of betanine betanine, which is one of the betalain dyes, was detected in a sample extracted from N. benthamiana transiently expressing CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT (FIG. 4C) . No peak was detected in the sample extracted from N. benthamiana tobacco in which an empty vector was introduced instead of CqDODA1-1 (FIG. 4C).
The result of "activity evaluation 2 in bensamiana tobacco" is shown in FIG.
The leaves of the leaves turn red and the red pigment turns red for both CsqYP76AD1, CqDODA1-1 and CqCDOPA5GT transiently expressed as well as CqCYP76AD1, CqDODA1-2 and CqCDOPA5GT transiently expressed respectively It showed that it synthesize | combined (FIG. 5A and B).
As a result of HPLC analysis, S was obtained in samples extracted from both of the benthamiana tobacco that transiently expressed CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT, and the bentom tobacco that transiently expressed CqCYP76AD1, CqDODA1-2 and CqCDOPA5GT. A single peak of body betanin was detected (FIG. 5C).
As described above, a gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having L-DOPA oxidase activity, and an enzyme having DOPA 4,5-dioxygenase activity A plant body which is a host into which a gene group of a gene and a gene group encoding a gene encoding an enzyme having an activity of adding a sugar to a phenolic hydroxyl group is introduced has the ability to synthesize betanine betaine which is one of betalain dyes.

<Evaluation of betalain synthesis ability in cells>
The betalain synthesis ability in the host cell was evaluated in tobacco BY2 cells not having the synthetic ability according to the following method.

[Constant expression analysis in tobacco BY2 cells]
The plasmid held by the transformed Agrobacterium was introduced into tobacco BY2 cells using the Agrobacterium method in the following combination (Hagiwara, Y., Komoda, K., Yamanaka, T., et al. 2003, Subcellularlocalization of host and viral proteins associated with tobamovirus RNA replication. EMBO J., 22, 344-53.). The constant expression system using tobacco BY2 cells of the obtained transformed strain was maintained and used for the following analysis.
The combination of plasmids transformed CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT into one tobacco BY2 cell. The other tobacco BY2 cells were transformed with CqCYP76AD1, pCAMBIA1301M and CqCDOPA5GT as vector control (vector control).

[Betalain pigment extraction and HPLC analysis]
(1) A tobacco BY2 cell sample of the transformed strain was disrupted, water was added, and centrifugation (20,000 g, 10 minutes, 15 minutes) was performed. The centrifuged supernatant was collected and the same operation was performed once more. The supernatant obtained by centrifugation was subjected to HPLC analysis as a pigment extract.
(2) HPLC analysis was performed in the same manner as in Example 2.

[result]
The results are shown in FIG.
The tobacco BY2 cells transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT turned red in color, indicating that betalain pigment was synthesized (FIG. 6A). The tobacco BY2 cells into which empty vector was introduced instead of CqDODA1-1 remained white, indicating that betalain dye was not synthesized (FIG. 6B).
As a result of HPLC analysis, a single peak of beta-nin of S form was detected in a sample extracted from tobacco BY2 cells transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT (FIG. 6C). No peak was detected in the sample extracted from tobacco BY2 cells into which empty vector was introduced instead of CqDODA1-1 (FIG. 6C).
As described above, a gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having L-DOPA oxidase activity, and an enzyme having DOPA 4,5-dioxygenase activity A cell, which is a host into which a gene group of a gene and a gene group encoding a gene encoding an enzyme having an activity of adding a sugar to a phenolic hydroxyl group, has the ability to synthesize betanine betaine which is one of betalain dyes.

  In the past reports, results of betalain synthesis using three synthetic genes (CYP76AD1, DODA1-1, CDOPA5GT) derived from plants other than quinua have been reported (Non-patent Documents 2 and 3). However, in those reports, simultaneously with the production of betanin (S form), the stereoisomer isobetanine (R form) is simultaneously synthesized. The appearance of stereoisomers is due to the reaction involving DODA1, and it has been found in the past that DODA1 of Oshiroibana synthesizes 88% of betanine precursor (S form) and 12% isobetanine precursor (R form) It reports that it does (nonpatent literature 1). From these reports and the results of this example, DODA1-1 and DODA1-2 isolated from the quinoa according to the present invention are novel DOPA 4,5-dioxygenases capable of substantially synthesizing only betanin. It turned out that it is an enzyme having activity.

<Preparation of Agrobacterium transformed with Quinoa DODA1 Homolog>
The ability to synthesize betarein is confirmed in the same manner as in Example 1 with respect to CqDODA1 homologs CqDODA1-3, CqDODA1-4, CqDODA1-5 and CqDODA1-6.

The following primers are used for PCR, and the amplified PCR fragment is digested with the following restriction enzymes in the same manner as in Example 1.
The PCR primers are CqDODA1-3 (SEQ ID NO: 22) in SEQ ID NOs: 9 and 10, CqDODA1-4 (SEQ ID NO: 24) in SEQ ID NOs: 11 and 12, CqDODA1-5 (SEQ ID NO: 26) in SEQ ID NOs: 13 and 14, CqDODA1. -6 (SEQ ID NO: 28) uses SEQ ID NOs: 15 and 16.
The amplified PCR fragment is digested with restriction enzymes (CqDODA1-3 to 1-6: EcoRI and BamHI).
The constructed vectors are pCAMBIA_CqDODA1-3-ox, pCAMBIA_CqDODA1-4-ox, pCAMBIA_CqDODA1-5-ox, pCAMBIA_CqDODA1-6-ox as described in FIG.

[Agrobacterium transformation method]
The above constructed plasmid is introduced into Agrobacterium in the same manner as in Example 1 to prepare a transformed Agrobacterium.

<Evaluation of the betalain synthesis ability in Bensamiana tobacco by quinua DODA1 homolog>
The betalain synthesis ability of CqDODA1-3, CqDODA1-4, CqDODA1-5 or CqDODA1-6 is evaluated by introducing into bensamiana tobacco having no synthetic ability according to the following method.

[Transient expression analysis in bensamiana tobacco]
CqCYP76AD1 and CqCDOPA5GT transformed Agrobacterium prepared in Example 1, and CqDODA1-3, CqDODA1-4, CqDODA1-5 or CqDODA1-6 transformed Agrobacterium prepared in Example 4 in 3 ml of LB medium Incubate with
CqCYP76AD1, CqDODA1-3 and CqCDOPA5GT combination, CqCYP76AD1, CqDODA1-4 and CqCDOPA5GT combination, CqCYP76AD1, CqDODA1-5 and CqCDOPA5GT combination, or CqCYP76AD1, CqDODA1-6 and CqCDOPA5GT combination solution are prepared.
The prepared mixed solution is used to inoculate benthamiana tobacco in the same manner as in Example 2. After growing for 3 to 5 days after inoculation, green leaves of the grown Bensamiana tobacco are used for the following analysis.

[Betalain pigment extraction and HPLC analysis]
The betalain dye extraction and HPLC analysis are carried out in the same manner as in Example 2.

[result]
CqCYP76AD1, CqDODA1-3 and CqCDOPA5GT combinations, CqCYP76AD1, CqDODA1-4 and CqCDOPA5GT combinations, CqCYP76AD1, CqDODA1-5 and CqCDOPA5GT combinations, or CqCYP76AD1, CqDODA1-6 and CqCDOPA5GT combinations The leaves of Samiana tobacco turn red to confirm that a red pigment is synthesized.
As a result of HPLC analysis, a combination of CqCYP76AD1, CqDODA1-3 and CqCDOPA5GT, a combination of CqCYP76AD1, CqDODA1-4 and CqCDOPA5GT, a combination of CqCYP76AD1, CqDODA1-5 and CqCDOPA5GT, or a combination of CqCYP76AD1, CqDODA1-6 and CqCDOP4 A single peak of betanine betanine, which is one of the betalain dyes, is confirmed in a sample extracted from overexpressed bensamiana tobacco.
Therefore, all of CqDODA1-3, CqDODA1-4, CqDODA1-5, and CqDODA1-6 can be used for S-betanine production in all CqDODA1.

<Confirmation of homology between quinoa-derived betalain synthesis gene and other plant-derived betalain synthesis gene>
The proteins encoded by CqCYP76AD1, CqDODA1-1, CqDODA1-2 and CqCDOPA5GT were confirmed to be homologous to proteins from other plants. Furthermore, the homology between CqDODA1-1 to 1-6 was confirmed.

[result]
CqCYP76AD1 showed 88% homology to beet CYP76AD1 and CqCDOPA5GT showed 77% homology to beet CDOPA5GT. On the other hand, CqDODA1-1 and CqDODA1-2 both had a low homology of 69% to beet DODA1. Furthermore, CqDODA1-1 and CqDODA1-2 showed lower homology with 54% and 53% when compared with Oshiroiwana DODA1.
The sequence of the active site of DONA1 (CqDODA1-1 to 1-6) of the quinoa isolated this time is HPLDETPHYHGGVAPWAAX ₁ FDSWLDX ₂ ALTX ₃ GRX ₄ EEVNTYETKAPNWEL (X ₁ denotes D or E, X ₂ is L, V or T X ₃ represents K or N and X ₄ represents F or I: SEQ ID NO: 33) (FIG. 2B). In the past reports, the conserved sequence of the active site of DODA1 derived from other plants has been reported (Fig. 2A) (Christinet, L., Burdet, FX, Zaiko, M., Hinz, U. and Zryd, JP2004, Characterization and functional identification of anovelplant 4, 5-extradioldioxygenase Involved in betalain pigment biosynthesisinPortulaca grandiflora. Plant Physiol., 134, 265-74.).
From the above, it was confirmed that the sequence of DODA1 (in particular, active site) of the quinua isolated this time was clearly different from the sequence of DODA1 (in particular, active site) derived from other plants (FIGS. 2A and B).

<Pigment analysis of quinoa hypocotyl>
The pigment accumulated in quinoa hypocotyls with red color was analyzed.
Quinoa strains Kd and CQ127 were used. Betaurein pigment extraction and HPLC analysis were carried out from quinoa hypocotyls in the same manner as in Example 2. More specifically, the apparatus was performed at 25 ° C. using LC-20AD (Shimadzu Corporation). As an analysis solvent, water containing 0.05% trifluoroacetic acid and 0.05% acetonitrile was used.
As a result of HPLC analysis, amaranthine and cerocyanine II were detected as main peaks, and a peak of betanin was slightly detected.

<Isolation of quintua-derived amarantine synthetase gene and evaluation of amarantine synthesis ability>
From the results of Example 7, phylogenetic analysis for isolation of amarantine synthetase was performed, and the ability to synthesize amarantine of the candidate gene thus obtained was evaluated by N. benthamiana.

Phylogenetic analysis for isolation of amarantine synthetase
Amarantine is a betalain pigment in which sugar is added to betanine. ClustalW algorithm (JD Thompson, DG Higgins, TJ Gibson, CLUSTAL W: using flavonoid 3-O-glucoside, 2'-O-glucosyltransferase (UGT79B6, At5g54010, NP — 200212), which is a glycosidation enzyme of flavonol, a plant pigment in Arabidopsis We performed phylogenetic tree analysis by improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res., 22 (1994) 4673-4680. Note that the neighbor joining algorithm by Mega7 software (using neighbor joiningalgorithm) A phylogenetic tree was constructed.

[result]
The constructed phylogenetic tree is shown in FIG. There were 17 homologs in quinoa, which were divided into two groups.
Flavonoid-modifying flavonoid 3-O-glucoside: As a result of examining the expression in quinoa hypocotyls for the group to which 2 "-O-glucosyltransferase does not belong, the expression of accession numbers XP 021754077.1, XP 021753589.1 and XP 021735840.1 It could be confirmed.

[Transient expression analysis in bensamiana tobacco]
The amarantine synthetase activity was evaluated for the above three genes and the gene of XP_021743749.
Agrobacterium was transformed in the same manner as in Example 1. Using the agroinfiltration method, green leaves of N. benthamiana tobacco were inoculated in the same manner as in Example 2 in the following combinations. For each green leaf sample and quinoa hypocotyl-extracted sample (control) of the combination, betalain dye extraction and HPLC analysis were performed in the same manner as in Example 2.
(1) Combination Containing XP_021754077.1 CsqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 (Protein 19), and XP_021754077.1 were transiently introduced into the benthamiana tobacco.
(2) Combinations Containing XP_221735839. 1 CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 and XP_021735839.1 were transiently introduced into C. benthamiana tobacco.
(3) Combinations Containing XP_021735840.1 CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 and XP_021735840.1 were transiently introduced into C. benthamiana tobacco.
(4) Combinations Containing XP_021743749 Cs_CYP76AD1, CqCDOPA5GT, CqDODA1, P19 and XP_021743749 were transiently introduced into C. benthamiana tobacco.
(5) Combination Including Empty Vector CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 and pCAMBIA1301M which is an empty vector (vector control) were transiently introduced into C. benthamiana tobacco.

[result]
The results of the agroinfiltration are shown in FIG.
As a result of HPLC analysis, a peak of amarantine is detected in samples extracted from C. camphorata transformed with CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 (Protein 19) and XP 021754077.1 and in control quinoa hypocotyl extract samples (Hypocotyl extract) And isoamaranthine was not detected (FIG. 10). In the sample (-) extracted from N. benthamiana to which an empty vector was introduced, a peak of betanin was detected, and a peak of amarantine was not detected (FIG. 10).
Therefore, it was confirmed that XP — 021754077.1 (SEQ ID NO: 37) is a gene encoding an amarantine synthetase and has an enzyme activity of adding sugar to betanin. The homology between XP 0 217 54 077 and Arabidopsis flavonoid 3-O-glucoside: 2 "-O-glucosyltransferase was 46%, that is, it was confirmed that the amarantine synthetase was completely different from known enzymes.
As described above, a gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having L-DOPA oxidase activity, and an enzyme having DOPA 4,5-dioxygenase activity A plant body which is a host into which a gene group of a gene, a gene encoding an enzyme having an activity of adding a sugar to a phenolic hydroxyl group, and a gene group of a gene encoding an amaranthine synthetase is synthesized Have a

<Evaluation of betalain synthesis ability of betalain synthesis ability gene derived from Oshiroiwana>
It was investigated whether DODI of Oshiroiwana could substantially synthesize only betanin.

[Transient expression analysis in bensamiana tobacco]
The DODA1 gene (MjDODA1; SEQ ID NO: 35) of Oshiroiwana was transformed into an Agrobacterium in the same manner as in Example 1. Using the agroinfiltration method, green leaves of N. benthamiana tobacco were inoculated in the same manner as in Example 2 in the following combinations. For each green leaf sample of the combination, betalain dye extraction and HPLC analysis were performed in the same manner as in Example 2.
(1) Combination Containing MjDODA1 CqCYP76AD1, CqCDOPA5GT, P19 and MjDODA1 were transiently introduced into C. benthamiana tobacco.
(2) Combination Including CqDODA-1 (CqDODA1-1) CqCYP76AD1, CqCDOPA5GT, P19 and CqDODA-1 were transiently introduced into C. benthamiana tobacco.
(3) Combinations Containing CqDODA-5 (CqDODA1-5) CqCYP76AD1, CqCDOPA5GT, P19 and CqDODA-5 were transiently introduced into C. benthamiana tobacco.
(4) Combination Containing CqDODA-6 (CqDODA1-6) CqCYP76AD1, CqCDOPA5GT, P19 and CqDODA-6 were transiently introduced into C. benthamiana tobacco.

[result]
As a result of agroinfiltration, the leaves turned red in all combinations, indicating that a red pigment was synthesized (FIG. 11).
As a result of HPLC analysis, a single peak of betanin is detected and a sample of isobetanin is detected also in samples extracted from Bensminaria tobacco transiently expressing MjDODA1, as well as CqDODA-1, CqDODA-5 and CqDODA-6. There was no (Figure 12).
Therefore, it became clear that DODAI (MjDODAl) of Oshiroiwana is an enzyme having DOPA 4, 5-dioxygenase activity that can substantially synthesize only betanin.

<Preparation of ghy mutant and functional evaluation of causative gene>
A mutant of a gene involved in betalain pigment synthesis in quinoa was prepared, and the functional analysis of betalain pigment synthesis was performed using the mutant.

[Mutagenesis by ethyl methane sulfonic acid (EMS)]
Seeds of quinoa strain Kd (obtained from Kyoto University) and CQ 127 (obtained from United States Department of Agriculture (USDA)) were grown at 23 ° C. for 12 hours light / 12 hours dark. EMS treatment of grown plants was commissioned to Inplanta Innovations Co., Ltd. The EMS-treated seeds were grown to obtain M1 and grown to the M3 generation. M3 was used for mutation analysis.

[Mutation analysis]
The library was constructed using TruSeq library preparation kit (Illumina). Candidate SNPs were analyzed using MutMap pipeline ver. 1.4.4 (http://genome-e.ibrc.or.jp/home/bioinformatics-team/mutmap) (see: H. Li, R. Durbin, Fast Li, B. Handsaker, A. Wysoker, T. Fennell, J. Ruan, N. Homer, G. Marth, G. and accurate short read alignment with Burrows-Wheelertransform, Bioinformatics, 25 (2009) 1754-1760. Abecasis, R. Durbin, The Sequence Alignment / Map format and SAMtools, Bioinformatics, 25 (2009) 2078-2079.).

[result]
As a result of mutational analysis, two green hypocotyl mutant (ghy) lines were discovered and named ghy1 and ghy2. As a result of pigment analysis, accumulation of betacyanin pigment was not observed in both mutants, and it was predicted that the ghy mutant had an abnormality in the betalain pigment synthesis system.

[Identification of causative gene]
The ghy1 and ghy2 M3 seeds were separated into wild type: ghy = 3: 1 by the separation test. Both phenotypes were sequenced by sequencing and subjected to the MutMap ⁺ method (R. Fekih, H. Takagi, M. Tamiru et al, MutMap +: genetic mapping and mutant identification without crossing in rice, PLoS One, 8 (2013) e68529.) , SNPs of ghy mutant causative genes were identified.
Among these identified SNPs, the point mutation of CqCYPAD1-1 (Gene ID: 110735347) has replaced the 371 arginine with cysteine in ghy 1 and the 124 tryptophan with termination codon in ghy 2 It was confirmed.
That is, CqCYPAD1-1 in which the 371st arginine was substituted by cysteine and CqCYPAD1-1 in which the 124th tryptophan was substituted by the termination codon were confirmed.

[Ghy1-ghy2 hybridization test]
In order to confirm that CqCYPAD1-1 is the causative gene, ghy1 and ghy2 were crossed. As a result, all F1 plants showed a green hypocotyl phenotype (n = 6).
Therefore, it was confirmed that the mutation of CqCYPAD1-1 is responsible for the green hypocotyl phenotype and CqCYPAD1-1 is involved in betacyanin dye synthesis.

[CqCYPAD1-1 functional analysis]
(1) Overexpression of CqCYPAD1-1 For functional analysis, the ghy1 mutant CqCYPAD1-1 was overexpressed. A CqCYPAD1-1 overexpression vector was constructed and introduced into Rhizobium rhizogenes (ATCC 15834). Rhizobium rhizogenes transformants were infected into the hairy roots of the ghy1 mutant. As a result, red pigmentation was observed in the hairy roots.
(2) Evaluation of CYP76AD1 Activity in Bensminaria Tobacco CqCYP76AD1-1, a combination of CqDODA1-1 and CqCDOPA5GT, or CqCYP76AD1-1_R371C (an arginine at position 371) was used in green leaves of Bensminaria tobacco in the same manner as in Example 2. The combination of CqCYP76AD1-1), CqDODA1-1 and CqCDOPA5GT substituted with cysteine was transiently expressed. As a result, the leaves in the former combination turned red while the leaves did not discolor in the latter combination. Thus, it was confirmed that CqCYP76AD1-1 has CYP76AD1 activity. In addition, accumulation of betalain was not observed in the CqCYP76AD1-1 — R371C transformant.
(3) CqCYP76AD1-1 expression analysis in quinoa seeds CqCYP76AD1-1 expression analysis was performed in quinoa seeds. As a result, CqCYP76AD1-1 showed high expression in cotyledons and hypocotyls in which a red pigment was observed under light and dark conditions. On the other hand, CqCYP76AD1-1 was found to be expressed in the cotyledons two days after sowing under constant dark conditions, but not in the hypocotyls. Thus, it was revealed that expression of CqCYP76AD1-1 in hypocotyls is light-dependent.

[CqCYPAD1-2 functional analysis]
(1) CqCYP76AD1-2 expression analysis in hypocotyl It was predicted that CqCYP76AD1-2 had an enzyme activity based on sequence analysis. As a result of analyzing CqCYP76AD1-2 expression in hypocotyls by RT-PCR, an amplified fragment derived from the ORF of CqCYP76AD1-2 was confirmed in the Kd strain, but an amplified fragment derived from the CqCYP76AD1-2 genome was confirmed in the CQ127 strain . The splice junction of CqCYP76AD1-2 was also confirmed. In the CQ127 strain, a SNP occurred in the splicing region, and a 23-bp deletion was present near the 3 'splicing region.
(2) Evaluation of CYP76AD1 activity in Bensminaria tobacco A genomic region of CqCYP76AD1-2 derived from Kd strain, a combination of CqDODA1-1 and CqCDOPA5GT, or Kd strain in green leaves of Bensminaria tobacco by the same method as Example 2. The combination of CqCYP76AD1-2 cDNA, CqDODA1-1 and CqCDOPA5GT, was transiently expressed. As a result, both of the leaves turned red. As a result of HPLC analysis and MS analysis, it was confirmed that this pigmentation was due to betanin. On the other hand, as a result of transiently expressing the combination of the CqCYP76AD1-2 genome region derived from CQ127 strain, CqDODA1-1 and CqCDOPA5GT in green leaves of C. benthamiana by the same method as Example 2, no red pigmentation was observed The Thus, it was revealed that CqCYP76AD1-2 normally functions in the genome derived from the Kd strain and does not function by mis-splicing in the genome derived from the CQ127 strain.

[Utility of ghy mutant]
Expression of CqCYP76AD1-1 occurs on exposure to light in the hypocotyl. As a result, betalain pigment is accumulated in the hypocotyl immediately after germination. This is to protect plants from environmental stress and biological stress (G. Polturak, N. Grossman, D. Vela-Corcia, Y. Dong, A. Nudel, M. Pliner, M. Levy, I. Rogachev, A. Aharoni, Engineered gray mold resistance, antioxidant capacity, and pigmentation in betalain-producing crops and ornamentals, Proc. Natl. Acad. Sci. USA, 114 (2017) 9062-9067, G. Jain, KE Schwinn, KS Gould , Betalain induction by l-DOPA application confers photoprotection tosaline-exposed leaves of Disphymaaustrale, New Phytol., 207 (2015) 1075-1083.). Red pigmentation in the hypocotyl was observed in all of the 75 Chenopodium species, including quinoa. Thus, betalain dye is a component of environmental stress tolerance.
The ghy mutant does not show accumulation of betacyanin and is useful for elucidating the physiological function of betacyanin in the hypocotyl.
The ghy phenotype appears in the M3 generation, but in allogeneic tetraploid quinua, it is believed that this generation of mutations will not appear. Quinoa may have lost one pair of homologous genes. In fact, the homologous genes of CqCYP76AD1-1 and CqCYP76AD1-2 lost their function and became pseudogenes. In the CQ127 strain, CqCYP76AD1-2 lost its function by mis-splicing, and only CqCYP76AD1-1 functioned normally. The characteristic of the M3 mutation in ghy mutants is expected to be controlled by a single gene in allogeneic tetraploid quinua.
The ghy1 mutant is effective for CYP76AD1 activity analysis. This is also supported by the fact that the residue at position 371 of CqCYP76AD1-1 having a mutation of ghy1 in 3D structural modeling is close to the active center.
Transformation of plants that normally make betalains is difficult. On the other hand, ghy is easily infected with Agrobacterium, and transformation is possible by using ghy mutants.
Betalain is likely to inhibit transformation. The ghy mutant has high redifferentiation ability because it does not synthesize betalain.
That is, the present invention is also directed to green hypocotyl mutant (ghy) lines ghy1 and ghy2.
The present invention is also directed to CqCYPAD1-1 in which arginine at position 371 is substituted with cysteine, and CqCYPAD1-1 in which tryptophan at position 124 is substituted for codon throughout.

  According to the present invention, it has become possible to provide a method for synthesizing betalain dye, an agent for converting L-DOPA to 4,5-seco-DOPA, and a host producing betalain dye.

Claims (8)

A method of synthesizing betalain dyes,
A gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having an L-DOPA oxidase activity, a gene encoding an enzyme having an activity of adding a sugar to a phenolic hydroxyl group The following gene groups of genes encoding enzymes having DOPA 4,5-dioxygenase activity have been introduced and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) production Cultivating a host having the ability to extract betalain pigment from the host after cultivation,
Or
The following genes encoding an enzyme having DOPA 4,5-dioxygenase activity have been introduced, and an enzyme activity having an activity of hydroxylating position 3 of the phenol ring of tyrosine, an enzyme activity having L-DOPA oxidase activity (B) culturing a host having an enzyme activity having an activity of adding a sugar to a phenolic hydroxyl group and capable of producing tyrosine or 3-hydroxy-L-tyrosine to extract betalain dye from the host after culture;
Here, a gene encoding an enzyme having the DOPA 4,5-dioxygenase 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 NOs: 17, 19, 21, 23, 25 or 27;
(2) In the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, 21. A gene encoding a polypeptide having the ability to convert L-DOPA substantially equivalent to the amino acid sequence set forth in 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) An amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, and an amino acid set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27 A gene encoding a polypeptide having the ability to convert L-DOPA substantially homologous to the sequence to 4,5-seco-DOPA,
(4) a gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28;
(5) Hybridizes with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28 under a stringent condition, and L-DOPA A gene comprising a DNA encoding a polypeptide having the ability to convert to 5-seco-DOPA,
(6) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28 ,
(7) A gene consisting of a DNA having a homology of 90% or more with the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28; and (8) the amino acid sequence of SEQ ID NO: 33 Any one or more genes of (1) to (7) above, comprising a gene encoding a polypeptide consisting of
A method for producing a betalain pigment characterized in that
The method for synthesizing a betalain dye according to claim 1, wherein the betalain dye is betanine.
The method for synthesizing betalain dyes according to claim 1 or 2, wherein the betalain dyes are substantially free of isobetanine.
An agent for converting L-DOPA containing the gene shown in any one of the following (1) to (8) or a vector carrying the gene into 4, 5-seco-DOPA:
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27;
(2) In the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, 21. A gene encoding a polypeptide having the ability to convert L-DOPA substantially equivalent to the amino acid sequence set forth in 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) An amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, and an amino acid set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27 A gene encoding a polypeptide having the ability to convert L-DOPA substantially homologous to the sequence to 4,5-seco-DOPA,
(4) a gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28;
(5) Hybridizes with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28 under a stringent condition, and L-DOPA A gene comprising a DNA encoding a polypeptide having the ability to convert to 5-seco-DOPA,
(6) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28 ,
(7) A gene consisting of a DNA having a homology of 90% or more with the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28; and (8) the amino acid sequence of SEQ ID NO: 33 The gene of any one or more of the above (1) to (7), which comprises a gene encoding a polypeptide consisting of
An agent for converting L-DOPA having a peptide represented by the amino acid sequence of any one of (1) to (4) below into 4,5-seco-DOPA;
(1) the amino acid sequence as set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27,
(2) In the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, An amino acid sequence having the ability to convert L-DOPA substantially homologous to the amino acid sequence set forth in 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) L-DOPA substantially homologous to the amino acid sequence of SEQ ID NO: 1 and having a homology of 90% or more with the amino acid sequence of SEQ ID NO: 17, 19, 21, 23, 25 or 27 An amino acid sequence having the ability to convert to 4,5-seco-DOPA, and (4) an amino acid sequence of any one or more of the above (1) to (3) comprising the amino acid sequence set forth in SEQ ID NO: 33.
A betalain dye production host into which the conversion agent according to claim 4 or 5 is introduced.
Betalein pigment producing host,
The host is
A gene encoding an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, a gene encoding an enzyme having an L-DOPA oxidase activity, a gene encoding an enzyme having an activity of adding a sugar to a phenolic hydroxyl group The following gene groups of genes encoding enzymes having DOPA 4,5-dioxygenase activity have been introduced and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) production Have the ability to
Or
The following genes encoding an enzyme having DOPA 4,5-dioxygenase activity have been introduced, and an enzyme activity having an activity of hydroxylating position 3 of the phenol ring of tyrosine, an enzyme activity having L-DOPA oxidase activity , An enzyme activity having an activity of adding a sugar to a phenolic hydroxyl group and an ability to produce tyrosine or 3-hydroxy-L-tyrosine,
Here, a gene encoding an enzyme having the DOPA 4,5-dioxygenase 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 NOs: 17, 19, 21, 23, 25 or 27;
(2) In the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, 21. A gene encoding a polypeptide having the ability to convert L-DOPA substantially equivalent to the amino acid sequence set forth in 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) An amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27, and an amino acid set forth in SEQ ID NOs: 17, 19, 21, 23, 25 or 27 A gene encoding a polypeptide having the ability to convert L-DOPA substantially homologous to the sequence to 4,5-seco-DOPA,
(4) a gene consisting of DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28;
(5) Hybridizes with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28 under a stringent condition, and L-DOPA A gene comprising a DNA encoding a polypeptide having the ability to convert to 5-seco-DOPA,
(6) A gene consisting of DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added in the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28 ,
(7) A gene consisting of a DNA having a homology of 90% or more with the DNA consisting of the nucleotide sequence of SEQ ID NO: 18, 20, 22, 24, 26 or 28; and (8) the amino acid sequence of SEQ ID NO: 33 Any one or more genes of (1) to (7) above, comprising a gene encoding a polypeptide consisting of
A betalain pigment production host characterized in that.
  The betalain dye producing host according to claim 7, wherein the betalain dye is substantially free of isobetanine.