JP6986973B2 - Method for synthesizing betalain pigment - Google Patents

Description

The present invention relates to a method for synthesizing betalain dye, a conversion agent for L-DOPA to 4,5-seco-DOPA, a host for producing betalain dye, and an amaranthin synthase.

(Betalain)
Betalain is a red or yellow nitrogen-containing compound synthesized by plants of the order Caryophyllales. It is known to have high antioxidant power and various functionalities. Betalain is accumulated in the roots of red sugar beet and is attracting attention as a healthy food. Betalain contained in sugar beet has the same degree of synthesis and accumulation of betanin and its optical isomer, isobetanin.

Patent Document 1 describes a microorganism having "enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine, DOPA4,5-dioxygenase activity, L-DOPA oxidase activity, and enzyme activity for adding a sugar to a phenolic hydroxyl group". 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 is disclosed.
Non-Patent Document 1 reports that "the betanin synthesis system of marvel-of-peru synthesizes 88% of S-form betanin and 12% of R-form isobetanin".
Non-Patent Document 2 reports that "the betanin synthesis system of potato synthesizes both S-form and R-form".
Non-Patent Document 3 reports that "the betanin synthesis system of Nicotiana benthamiana synthesizes both S-form and R-form".
However, neither document discloses or suggests 4,5-dihydroxy-phenylalanine dioxygenase extradiol {4,5-DOPA (dihydroxy-phenylalanine) dioxygenase extradiol} derived from quinoa of the present invention.

JP-A-2016-182044

Sasaki, N., Abe, Y., Goda, Y., Adachi, T., Kasahara, K. and Ozeki, Y. 2009, Detection of DOPA4,5-dioxygenase (DOD) activityusing recombinant protein preparedfromEscherichia colicellsharboring cDNA encoding DOD from Mirabilis jalapa .PlantCell Physiol., 50, 1012-6. Polturak, G., Grossman, N., Vela-Corcia, D., et al. 2017, Engineered gray mold resistance, antioxidantcapacity, andpigmentationinbetalain-producing crops and ornamentals. Proc.Natl.Acad.Sci. USA, 114,9062-7 .. Polturak, G., Breitel, D., Grossman, N., et al. 2016, Elucidation of the first committed stepinbetalainbiosynthesisenablesthe heterologous engineering of betalainpigmentsinplants. NewPhytol., 210,269-83.

Since optical isomers are present in organisms that produce betalain, it is necessary to synthesize a single (S-form ) betalain when using betalain extract / purified products as pharmaceuticals or when analyzing their functions. Has been done.

In order to solve the above problems, the present inventors isolated a gene involved in betalain synthesized hypocotyl-specifically in quinoa, and analyzed the function of the gene. As a result, the betalain pigment in quinoa was analyzed. In the synthetic system, it was confirmed that isobetanin was not substantially synthesized.
From the above, the method for synthesizing the betalain dye of the present invention, the conversion agent for L-DOPA to 4,5-seco-DOPA, and the host for producing the betalain dye were completed.

That is, the present invention is as follows.
1. 1. It is a method of synthesizing betalain pigment.
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, and a gene encoding an enzyme. The following genes encoding genes encoding enzymes with DOPA 4,5-dioxygenase activity have been introduced and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) production. Cultivate a capable host and extract the tyrosine pigment from the cultured host,
Or,
The following genes encoding enzymes having DOPA 4,5-dioxygenase activity have been introduced, and the enzyme activity has the activity of hydroxylating the 3-position of the phenol ring of tyrosine, and the enzyme activity having L-DOPA oxidase activity. , A host having an enzymatic activity having an activity of adding a sugar to a phenolic hydroxyl group and a host having a tyrosine or 3-hydroxy-L-tyrosine producing ability is cultured, and a betalein pigment is extracted from the host after the culture.
Here, the gene encoding the 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 NO: 17, 19, 21, 23, 25 or 27.
(2) In the amino acid sequence set forth in SEQ ID NO: 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, A gene encoding a polypeptide having the ability to convert L-DOPA substantially identical to the amino acid sequence according to 21, 23, 25 or 27 to 4,5-seco-DOPA.
(3) The amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25 or 27 and the amino acid set forth in SEQ ID NO: 17, 19, 21, 23, 25 or 27. A gene encoding a polypeptide capable of converting L-DOPA substantially homologous to a sequence to 4,5-seco-DOPA,
(4) A gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28.
(5) Hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28, and L-DOPA is 4 , A gene consisting of DNA encoding a polypeptide capable of converting 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 DNA having 90% or more homology with the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28, and (8) the amino acid sequence set forth in SEQ ID NO: 33. One or more genes of any one of (1) to (7) above, which comprises a gene encoding a polypeptide consisting of
A method for producing a betalain pigment, which is characterized by the fact that.
2. 2. The method for synthesizing a betalain pigment according to item 1 above, wherein the betalain pigment is betanin.
3. 3. The method for synthesizing a betalain dye according to the above item 1 or 2, wherein the betalain dye does not substantially contain isobetanin.
4. A conversion agent for L-DOPA containing the gene shown in any one of (1) to (8) below or a vector carrying the gene to 4,5-seco-DOPA;
(1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25 or 27.
(2) In the amino acid sequence set forth in SEQ ID NO: 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, A gene encoding a polypeptide having the ability to convert L-DOPA substantially identical to the amino acid sequence according to 21, 23, 25 or 27 to 4,5-seco-DOPA.
(3) The amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25 or 27 and the amino acid set forth in SEQ ID NO: 17, 19, 21, 23, 25 or 27. A gene encoding a polypeptide capable of converting L-DOPA substantially homologous to a sequence to 4,5-seco-DOPA,
(4) A gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28.
(5) Hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28, and L-DOPA is 4 , A gene consisting of DNA encoding a polypeptide capable of converting 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 DNA having 90% or more homology with the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28, and (8) the amino acid sequence set forth in SEQ ID NO: 33. A gene according to any one of (1) to (7) above, which comprises a gene encoding a polypeptide comprising.
5. A conversion agent for L-DOPA having a peptide represented by the amino acid sequence of any one of (1) to (4) below to 4,5-seco-DOPA;
(1) The amino acid sequence according to SEQ ID NO: 17, 19, 21, 23, 25 or 27,
(2) In the amino acid sequence set forth in SEQ ID NO: 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 capable of converting L-DOPA substantially identical to the amino acid sequence according to 21, 23, 25 or 27 into 4,5-seco-DOPA,
(3) L-DOPA having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25 or 27 and substantially the same as the amino acid sequence set forth in SEQ ID NO: 1. An amino acid sequence having the ability to convert to 4,5-seco-DOPA, and (4) an amino acid sequence according to any one of (1) to (3) above, which comprises the amino acid sequence set forth in SEQ ID NO: 33.
6. A betalain pigment-producing host into which the conversion agent according to the above item 4 or 5 has been introduced.
7. Betalain pigment production 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, and a gene encoding an enzyme. The following genes encoding genes encoding enzymes with DOPA 4,5-dioxygenase activity have been introduced and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) production. Has the ability,
Or,
The following genes encoding enzymes having DOPA 4,5-dioxygenase activity have been introduced, and the enzyme activity has the activity of hydroxylating the 3-position of the phenol ring of tyrosine, and the enzyme activity having L-DOPA oxidase activity. , Enzymatic activity having activity to add sugar to phenolic hydroxyl group and tyrosine or 3-hydroxy-L-tyrosine producing ability.
Here, the gene encoding the 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 NO: 17, 19, 21, 23, 25 or 27.
(2) In the amino acid sequence set forth in SEQ ID NO: 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, A gene encoding a polypeptide having the ability to convert L-DOPA substantially identical to the amino acid sequence according to 21, 23, 25 or 27 to 4,5-seco-DOPA.
(3) The amino acid having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25 or 27 and the amino acid set forth in SEQ ID NO: 17, 19, 21, 23, 25 or 27. A gene encoding a polypeptide capable of converting L-DOPA substantially homologous to a sequence to 4,5-seco-DOPA,
(4) A gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28.
(5) Hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28, and L-DOPA is 4 , A gene consisting of DNA encoding a polypeptide capable of converting 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 DNA having 90% or more homology with the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26 or 28, and (8) the amino acid sequence set forth in SEQ ID NO: 33. One or more genes of any one of (1) to (7) above, which comprises a gene encoding a polypeptide consisting of
A host that produces betalain pigments.
8. The host for producing betalain pigment according to item 7 above, wherein the betalain pigment is substantially free of isobetanin.

The present invention was able to provide a method for synthesizing betalain dye, a conversion agent for L-DOPA to 4,5-seco-DOPA, a host for producing betalain dye, and an amaranthin synthase.

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 Zryd, JP 2004, characterizationandfunctional identification of a novel plant 4,5-extradioldioxygenase involvedinbetalainpigmentbiosynthesis in Portulaca grandiflora.Plant Physiol. , 134,265-74.). B: Sequence of the active site of quinoa DODA1. Betalain synthetic gene expression vector. A: Figure of pCAMBIA_CqCYP76AD1-ox. B: Figure of pCAMBIA_CqDODA1-1 ~ 1-6-ox. C: Figure of pCAMBIA_CqCDOPA5GT-ox. D: Figure of pCAMBIA1301M which is an empty vector (vector control). HPT indicates hygromycin resistant gene, 35S indicates enhancedCaMV35Spromoter, NosT indicates Nopaline synthaseTerminater, LB indicates Left Border, and RB indicates Right Border. Activity evaluation with Nicotiana benthamiana 1. A: Green leaves of Nicotiana benthamiana transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. B: Green leaves of Nicotiana benthamiana transformed with CqCYP76AD1, pCAMBIA1301M which is an empty vector (vector control) and CqCDOPA5GT. C: HPLC analysis results. Betanin indicates a standard (a mixture of betanin and isobetanin). Activity evaluation with Nicotiana benthamiana 2. A: Green leaves of Nicotiana benthamiana transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. B: Green leaves of Nicotiana benthamiana transformed with CqCYP76AD1, CqDODA1-2 and CqCDOPA5GT. C: HPLC analysis results. Betanin indicates a standard (a mixture of betanin and isobetanin). Evaluation of activity in tobacco BY2 cells. A: Tobacco BY2 cells transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. B: Tobacco BY2 cells transformed with CqCYP76AD1, the empty vector pCAMBIA1301M and CqCDOPA5GT. C: HPLC analysis results. Synthetic pathway of betaramic acid from L-DOPA. A phylogenetic tree using a neighbor joining algorithm. Scale bars show a 50% difference in amino acid sequence. Evaluation of amaranthin synthase activity in Nicotiana benthamiana. "-" Indicates pCAMBIA1301M which is an empty vector (vector control). HPLC analysis results in the evaluation of amaranthin synthesis ability. Evaluation of the activity of the DODA1 gene in Marvel-of-peru in Bensamiana tobacco. HPLC analysis results in the activity evaluation of the DODA1 gene of Marvel-of-peru.

The present invention relates to a method for synthesizing betalain dye, a conversion agent for L-DOPA to 4,5-seco-DOPA, a host for producing betalain dye, and an amaranthin synthase. Hereinafter, the present invention will be described in detail.

(Betalain pigment)
The betalain pigment in the present invention is classified into betaxanthine and betacyanins based on their structural characteristics. Since betaxanthine develops a yellow color and betacyanins develop a magenta color, it has been conventionally used as a natural colorant. The betacyanins are a general term for a group of compounds in which a saccharide is glycosidic bonded to a phenolic hydroxyl group of betanidin.

(Betalain pigment synthesis system)
A summary of the betalain pigment synthesis system derived from the hypocotyl of quinoa in the present invention is shown in FIG. As is apparent from the description in FIG. 1, tyrosine or 3-hydroxy-L-tyrosine (L-DOPA), as well as amino acids, amines (eg, putrescine), Hosts capable of producing spermidine and spermine (including hosts that can be taken in from the outside) have the activity of an enzyme that hydroxylates the 3-position of the phenol ring of tyrosine (eg, CqCYP76AD1), L. -An enzyme having DOPA oxidase activity (eg, CqCYP76AD1) activity, an enzyme having an activity of adding sugar to a phenolic hydroxyl group {for example, an enzyme having betanidin 5-O-glucosyltransferase activity (for example, Cyclo-DOPA 5-O-glucosyltransferase, CqCDOPA5GT, etc.) Activity, 4,5-dihydroxy-phenylalanine dioxygenase extradiol of the present invention {4,5-DOPA (dihydroxy-phenylalanine) dioxygenase extradiol, 4,5-DOPA dioxygenase extradiol 1} If active, betaxanthine And betacyanins (eg, betanidin, betanin) can be synthesized.
In addition, CqCYP76AD1 has both the property of the enzyme activity which hydroxylates the 3-position of the phenol ring of tyrosine and the enzyme activity which has the 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 an L-DOPA oxidase activity, and a DOPA 4,5-dioxygenase activity The plant body, which is the host into which the gene encoding the enzyme having the It has the ability to synthesize one of the amaranthins.

(Enzyme that hydroxylates the 3-position of the phenol ring of tyrosine)
The enzyme that hydroxylates 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 of tyrosine, and any species having the activity. It may be the origin of.
Examples of the enzyme that hydroxylates the 3-position of the phenol ring of tyrosine include tyrosinase, cytochrome P450 (particularly CYP76AD1, CYP76AD2, CYP76AD3, etc.), catechol oxidase, etc., but CqCYP76AD1 (SEQ ID NO: 29, accession) is preferable. The number is 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 to cyclo-DOPA, and any species may be derived as long as it has the activity.
Examples of the enzyme having L-DOPA oxidase activity include tyrosinase, CYP76AD1, CYP76AD2, CYP76AD3, CYP76AD5, CYP76AD6 and the like, which are cytochrome P450, and CqCYP76AD1 (sequence) is preferable. The number 29).

(Enzyme that adds sugar to phenolic hydroxyl group)
The enzyme that adds a sugar to a phenolic hydroxyl group in the present invention has an activity of adding a sugar to a phenolic hydroxyl group existing at the 5-position or the 6-position of the cyclo-DOPA skeleton, and what if it has this activity? It may be derived from various species.
Examples of the enzyme that adds a sugar to the phenolic hydroxyl group include cyclo-DOPA5-O-glucosyltransferase, betanidin 5-O-glucosyltransferase, betanidin 6-O-glucosyltransferase and the like. However, it is preferably CqCDOPA5GT (SEQ ID NO: 31, accession number XP_021748306).
The enzyme that synthesizes Amaranthin from betanin (Amaranthin synthase) is SEQ ID NO: 36 (Axon No. XP_021754077).

If the enzyme that adds sugar to the phenolic hydroxyl group is betanidine 6-O-glucosyltransferase, betanin is synthesized.
If the enzyme that adds sugar to the phenolic hydroxyl group is betanidin 6-O-glucosyltransferase, Gomphrenin-I is synthesized.
If the enzymes that add sugar to the phenolic hydroxyl group are betanidin 5-O-glucosyltransferase and betanin beta-D-glucuronosyltransferase, amaranthin (particularly, 15S form) is synthesized.
If the enzyme that adds sugar to the phenolic hydroxyl group is betanidin 6-O-glucosyltransferase and there is an amaranthin synthase, amaranthin is synthesized.
If the enzymes that add sugar to the phenolic hydroxyl group are betanidin 5-O-glucosyltransferase and betanin feruloyltransferase, Lampranthin II (particularly, 15S form) is synthesized.
If the enzymes that add sugar to the phenolic hydroxyl group are betanidin 5-O-glucosyltransferase, betanin beta-D-glucuronosyltransferase and betanin feruloyltransferase, Celosianin II (particularly, 15S form) is synthesized.

(Enzyme with 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 the activity of catalyzing the extradiol cleavage of L-3,4-dihydroxyphenylalanine. Have. In addition, 4,5-seco-DOPA undergoes a spontaneous reaction to convert to betaramic acid (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 according to SEQ ID NO: 17 (accession number XP_021769303), 19 (accession number XP_021769301), 21, 23, 25, 27 or 34.
(2) In the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 17, A gene encoding a polypeptide having the ability to convert L-DOPA substantially identical to the amino acid sequence according to 19, 21, 23, 25, 27 or 34 to 4,5-seco-DOPA.
(3) 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 has SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34. A gene encoding a polypeptide having the ability to convert L-DOPA substantially identical to the amino acid sequence described in 4,5-seco-DOPA.
(4) A gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35.
(5) Hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35, and L-DOPA. A gene consisting of DNA encoding a polypeptide having the ability to convert 4,5-seco-DOPA.
(6) In the DNA consisting of the base sequences set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35, from the DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added. Gene.
(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: 18, 20, 22, 24, 26, 28 or 35.
(8) One or more genes according to any one of (1) to (7) above, which comprises a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 33.

The gene (2) above is a gene encoding a polypeptide having a mutation introduced to the extent that the ability to convert L-DOPA to 4,5-seco-DOPA is not lost. Such mutations include human-induced mutations as well as those that occur in nature. As a means for causing an artificial mutation, a site-directed mutagenesis method (Nucleic Acids Res. 10, 6487-6500, 1982) and the like can be mentioned. The number of mutated amino acids is usually 20 amino acids or less, preferably 10 amino acids or less, more preferably 5 amino acids or less, and most preferably 3 amino acids or less. Whether or not the mutated polypeptide retains the ability to convert L-DOPA to 4,5-seco-DOPA is determined, for example, by introducing a gene encoding the mutated polypeptide into a plant or the like. It can be seen by confirming the ability of the plant to convert L-DOPA to 4,5-seco-DOPA.
Regarding the gene of (3) above, "the ability to convert L-DOPA substantially the same as the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34 to 4,5-seco-DOPA". The degree of action 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 to 4,5-seco-DOPA. May be weak. For example, about 50%, about 60% 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 to 4,5-seco-DOPA. , About 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150% can be exemplified.
Identity can also be calculated using BLAST (BasicLocalAlignmentSearchToolat the National Center for Biological Information) and the like (eg, using default or default parameters).
The gene of (5) above is a gene obtained by utilizing hybridization between DNAs. The "stringent condition" in this gene means a condition in which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually conditions such as hybridization at 37 ° C. in a buffer containing 5 × SSC, 1% SDS and washing treatment at 37 ° C. with a buffer solution containing 1 × SSC, 0.1% SDS. , Preferably, hybridization at 42 ° C. in a buffer solution containing 5 × SSC, 1% SDS and washing treatment at 42 ° C. with a buffer solution containing 0.5 × SSC, 0.1% SDS, more preferably. , Hybridization at 65 ° C. in a buffer containing 5 × SSC, 1% SDS and washing treatment at 65 ° C. with a buffer solution containing 0.2 × SSC, 0.1% SDS. Whether or not the DNA obtained by utilizing hybridization encodes an active polypeptide, for example, the DNA is introduced into a plant or the like, and the L-DOPA of the plant is 4,5-seco. -It can be found by confirming the ability to convert to DOPA. The DNA obtained by hybridization usually has a high identity with the gene of (4) above (SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35). High identity refers to 90% or more identity, preferably 95% or more identity, and more preferably 98% or more identity.
The gene of (6) is 1 to 50, preferably 1 to 30, more preferably 1 to 20 in the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35. It is a gene consisting of 20 DNAs, most preferably 1 to 10, and even more preferably 1 to 5 base sequences substituted, deleted, inserted and / or added.
The gene of (7) is 90% or more, preferably 93% or more, more preferably 95% or more, most of the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35. It is preferably a gene consisting of DNA having 98% or more identity.

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 according to SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34.
(2) In the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 17, An amino acid sequence having the ability to convert L-DOPA substantially identical to the amino acid sequence according to 19, 21, 23, 25, 27 or 34 into 4,5-seco-DOPA.
(3) L- having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34 and substantially the same as the amino acid sequence set forth in SEQ ID NO: 1. An amino acid sequence capable of converting DOPA to 4,5-seco-DOPA.
(4) An amino acid sequence according to any one of (1) to (3) above, which comprises the amino acid sequence set forth in SEQ ID NO: 33.
From the viewpoint of not changing the basic properties (physical properties, function, physiological activity, immunological activity, etc.) of the peptide when introducing a mutation in the peptide, for example, homologous amino acids (polar amino acids, non-polar amino acids, etc.) Mutual substitutions between hydrophobic amino acids, hydrophilic amino acids, positive charged amino acids, negative charged amino acids, aromatic amino acids, etc.) are easily assumed.
In particular, the amino acid sequence set forth in SEQ ID NO: 33 includes the sequence of the active site. It is preferred that the mutation be introduced into an amino acid other than these domains, as the ability to convert L-DOPA to 4,5-seco-DOPA is likely to be lost if a mutation is introduced into this sequence.

(Method of synthesizing betalain pigment)
The method for synthesizing 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 are encoded. Genes for genes and genes encoding enzymes having DOPA 4,5-dioxygenase activity have been introduced, and a host capable of producing tyrosine or 3-hydroxy-L-tyrosine has been cultured and post-cultured. Extract the tyrosine pigment from the host.
(2) An enzyme activity having an enzyme encoding an enzyme having DOPA 4,5-dioxygenase activity, an enzyme activity having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, and an enzyme activity having an L-DOPA oxidase activity. , A host having an enzymatic activity having an activity of adding a sugar to a phenolic hydroxyl group and a host having a tyrosine or 3-hydroxy-L-tyrosine producing ability is cultured, and the betalein pigment is extracted from the host after the 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.
Further, it has been confirmed in the following examples that the method for synthesizing betalain dye of the present invention does not substantially synthesize isobetanin.
In addition, "substantially does not synthesize isobetanin" means that the amount of isobetanin synthesized is 3 parts by weight or less, 2 parts by weight or less, 1 part by weight or less, 0.5 parts by weight or less with respect to 100 parts by weight of betanin. , 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, "substantially does not synthesize isoamaranthin" means that the amount of isoamaranthin synthesized is 3 parts by weight or less, 2 parts by weight or less, 1 part by weight or less, 0 with respect to 100 parts by weight of amaranthin 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 the weight part.

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

(Method of introducing each gene into the host)
In the method for synthesizing the betalain dye of the present invention, the gene can be introduced into a plant by a method known per se as a method for introducing each gene into a host. For example, each gene can be introduced into a host by applying a solution containing a plant virus vector carrying each gene to the leaves, stems, roots, ears, etc. of a plant. As other methods, the particle gun method and the Agrobacterium method can be exemplified.

(Method of introducing each protein into the host)
In the method for synthesizing the betalain dye of the present invention, the protein can be introduced into a plant by a method known per se as a method for introducing each protein into a host (particularly, a plant). For example, each protein can be introduced into a host by applying a solution containing each protein to the leaves, stems, roots, ears, etc. of a plant. As other methods, the particle gun method and the Agrobacterium method can be exemplified.

(Host for producing betalain pigment)
The betalain dye-producing host of the present invention has, for example, any one or more of the following configurations / characteristics.
(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 are encoded. A gene group of genes encoding an enzyme having DOPA 4,5-dioxygenase activity has been introduced, and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) has been introduced. Has production ability.
(2) An enzyme having a gene encoding an enzyme having DOPA 4,5-dioxygenase activity, an enzyme activity having an activity of hydroxylating the 3-position of the phenol ring of tyrosine, and an enzyme having an L-DOPA oxidase activity. It has an activity, an enzymatic activity having an activity of adding a sugar to a phenolic hydroxyl group, and a tyrosine or 3-hydroxy-L-tyrosine producing ability.
(3) A conversion agent for converting L-DOPA to 4,5-seco-DOPA has been introduced.
In the betalain pigment producing host 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.
Further, it has been confirmed in the following examples that the host for producing the betalain pigment of the present invention does not substantially synthesize isobetanin.

(Conversion agent for L-DOPA to 4,5-seco-DOPA)
The converting agent for converting L-DOPA to 4,5-seco-DOPA of the present invention includes the gene shown in any one of (1) to (8) below or a vector carrying the gene.
(1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34.
(2) In the amino acid sequence set forth in SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34, 1 to 20 amino acids are substituted, deleted, inserted and / or added, and SEQ ID NO: 17, A gene encoding a polypeptide having the ability to convert L-DOPA substantially identical to the amino acid sequence according to 19, 21, 23, 25, 27 or 34 to 4,5-seco-DOPA.
(3) 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 has SEQ ID NO: 17, 19, 21, 23, 25, 27 or 34. A gene encoding a polypeptide having the ability to convert L-DOPA substantially identical to the amino acid sequence described in 4,5-seco-DOPA.
(4) A gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35.
(5) Hybridizes under stringent conditions with a DNA having a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35, and L-DOPA. A gene consisting of DNA encoding a polypeptide having the ability to convert 4,5-seco-DOPA.
(6) In the DNA consisting of the base sequences set forth in SEQ ID NO: 18, 20, 22, 24, 26, 28 or 35, from the DNA in which 1 to 50 base sequences are substituted, deleted, inserted and / or added. Gene.
(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: 18, 20, 22, 24, 26, 28 or 35.
(8) One or more genes according to any one of (1) to (7) above, which comprises a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 33.

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

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

<Preparation of Agrobacterium transformed with quinoa-derived betalain synthetic 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), CqDODA1-1 (an enzyme having an activity of hydroxylating the 3-position of the phenol ring of tyrosine), which is a gene encoding an enzyme used in the method for synthesizing a betalein dye according to the present invention. DOPA 4,5-dioxygenase activity enzyme), CqDODA1-2 (DOPA 4,5-dioxygenase activity enzyme) and CqCDOPA5GT (enzyme having activity to add sugar to phenolic hydroxyla), the following methods An expression plasmid was constructed with the above-mentioned enzyme and transformed into agrobacterium. Transformed Agrobacterium was used to evaluate the ability to synthesize betalain, which will be described later.

[Construction of expression plasmid for quinoa-derived betalain synthetic gene]
(1) RNA was extracted from the hypocotyl on the 4th day of germination of Quinopodium quinoa using the RNeasy Plant Mini kit (Qiagen). The extracted RNA was synthesized into cDNA using the High Capacity 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 synthetic gene was amplified by PCR using the enzyme of Prime STAR GXL (TaKaRa). The primers used for PCR are SEQ ID NOs: 1 and 2 for CqCYP76AD1 (SEQ ID NO: 30, accession number XM_021913610), SEQ ID NOs: 3 and 4 for CqDODA1-1 (SEQ ID NO: 18, accession number XM_021913611), and CqDODA1-2 (sequence number 18). SEQ ID NOs: 20 and XM_021913609) were used as SEQ ID NOs: 5 and 6, and SEQ ID NOs: 7 and 8 were used for CqCDOPA5GT (SEQ ID NO: 32, accession numbers XM_021892614). The amplified PCR fragment was cleaved with restriction enzymes (CqCYP67AD1: SpeI and BstPI, CqDODA1-1 and CqDODA1-2: EcoRI and BamHI, CqCDOPA5GT: BamHI and BstPI).
(3) A plasmid pCAMBIA1301M (Imamura, T., Kusano, H., Kajigaya, Y., Ichikawa, M. and Shimada, H. 2007, A rice dihydrosphingosine C4) that constitutively expresses PCR fragments treated with restriction enzymes in plants. It was introduced downstream of the CaMV35S promoter of hydroxylase (DSH1) gene, which isabundantly expressed in the stigmas, vascular cells andapicalmeristem, may beinvolved in fertility. Plant Cell Physiol., 48, 1108-20.) (Fig. 3). The constructed vectors were named pCAMBIA_CqCYP76AD1-ox, pCAMBIA_CqDODA1-1-ox, pCAMBIA_CqDODA1-2-ox, and pCAMBIA_CqDOPA5GT-ox, respectively.

[Agrobacterium transformation method]
Wise, AA, Liu, Z. and Binns, AN 2006, Three methods for the introduction offoreignDNAintoAgrobacterium. Methods Mol. Biol., 343, 43 using E. coli carrying the above constructed plasmid. By -53), a plasmid was introduced into Agrobacterium to prepare transformed Agrobacterium.

<Evaluation of betalain synthesis ability in plants>
The ability to synthesize betalain in the host plant was evaluated by the following method in Nicotiana benthamiana which does not have the ability to synthesize.

[Transient expression analysis in Nicotiana benthamiana]
The transformed Agrobacterium prepared in Example 1 was cultured in 3 ml of LB medium, respectively, and combined with the following "Activity evaluation 1 in Nicotiana benthamiana" and "Activity evaluation 2 in Nicotiana benthamiana". The culture medium was mixed with Nicotiana benthamiana and the green leaves of Nicotiana benthamiana were inoculated by the 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 3 to 5 days after inoculation, the green leaves of Nicotiana benthamiana grown were used for the following analysis.
(1) Activity evaluation with Nicotiana benthamiana 1
One of Nicotiana benthamiana was transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. CqCYP76AD1, the empty vector pCAMBIA1301M and CqCDOPA5GT were introduced into the other Nicotiana benthamiana.
(2) Activity evaluation with Nicotiana benthamiana 2
One of Nicotiana benthamiana was transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT. CqCYP76AD1, CqDODA1-2 and CqCDOPA5GT were introduced into the other Nicotiana benthamiana.

[Betalain dye extraction and HPLC analysis]
(1) A transiently expressed green leaf sample of Nicotiana 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 used as a dye extract for HPLC analysis.
(2) HPLC analysis uses a reverse phase column (5C18-AR-300, Nakaraitesk), the analysis solvent is water and acetonitrile, the flow velocity is 1 ml / min, and the analysis program analyzes the concentration of acetonitrile. The analysis was performed by setting the condition to reach 50% linearly from 0% in 0 minutes to 50% in 60 minutes, and setting the analysis wavelength to 536 nm, which can detect acetonitrile. A betan extract (betanin, isobetanin mixture, Tokyo Kaseisha) was used as a standard.

[result]
The result of "activity evaluation 1 in Nicotiana benthamiana" is shown in FIG.
The leaves of Nicotiana benthamiana that transiently expressed CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT turned red, indicating that a red pigment was synthesized (Fig. 4A). Nicotiana benthamiana into which an empty vector was introduced instead of CqDODA1-1 showed that the leaves remained green and no red pigment was synthesized (Fig. 4B).
As a result of HPLC analysis, a single peak of S-form betanin, which is one of the betalain pigments, was detected in a sample extracted from Nicotiana benthamiana that transiently expressed CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT (Fig. 4C). .. No peak was detected in the sample extracted from Nicotiana benthamiana into which an empty vector was introduced instead of CqDODA1-1 (Fig. 4C).
The result of "activity evaluation 2 in Nicotiana benthamiana tobacco" is shown in FIG.
For both Nicotiana benthamiana that transiently expressed CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT, and Nicotiana benthamiana that transiently expressed CqCYP76AD1, CqDODA1-2 and CqCDOPA5GT, the leaves turned red and the red pigment It was shown that it was synthesized (FIGS. 5A and B).
As a result of HPLC analysis, S A single peak of body betanin was detected (Fig. 5C).
From the above, the gene encoding the enzyme having the activity of hydroxylating the 3-position of the phenol ring of tyrosine, the gene encoding the enzyme having the L-DOPA oxidase activity, and the gene encoding the enzyme having the DOPA 4,5-dioxygenase activity are encoded. The plant body, which is the host into which the gene group of the gene encoding the enzyme having the activity of adding sugar to the phenolic hydroxyl group is introduced, has the ability to synthesize S-form betanin, which is one of the betalein pigments.

<Evaluation of betalain synthesis ability in cells>
The ability to synthesize betalain in the host cell was evaluated by the following method in tobacco BY2 cells having no such ability.

[Constant expression analysis in tobacco BY2 cells]
The plasmid carried by the transformed Agrobacterium was introduced into tobacco BY2 cells by the Agrobacterium method in the following combination (Hagiwara, Y., Komoda, K., Yamanaka, T., et al. 2003, Subcellularlocalizationof). host and viral proteins associated with tobamovirusRNA replication.EMBO J., 22, 344-53.). The constitutive expression system using the obtained transformed line tobacco BY2 cells was maintained and used for the following analysis.
The plasmid combination transformed CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT into one tobacco BY2 cell. The other tobacco BY2 cells were transformed with CqCYP76AD1, the empty vector pCAMBIA1301M and CqCDOPA5GT.

[Betalain dye extraction and HPLC analysis]
(1) A tobacco BY2 cell sample of a transformed line 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 used as a dye extract for HPLC analysis.
(2) HPLC analysis was performed by the same method as in Example 2.

[result]
The results are shown in FIG.
Tobacco BY2 cells transformed with CqCYP76AD1, CqDODA1-1 and CqCDOPA5GT turned red, indicating that betalain pigment was synthesized (Fig. 6A). Tobacco BY2 cells introduced with an empty vector instead of CqDODA1-1 remained white, indicating that betalain pigment was not synthesized (Fig. 6B).
As a result of HPLC analysis, a single peak of S-form betanin 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 an empty vector was introduced instead of CqDODA1-1 (Fig. 6C).
From the above, the gene encoding the enzyme having the activity of hydroxylating the 3-position of the phenol ring of tyrosine, the gene encoding the enzyme having the L-DOPA oxidase activity, and the gene encoding the enzyme having the DOPA 4,5-dioxygenase activity are encoded. The host cell into which the gene and the gene group of the gene encoding the enzyme having the activity of adding sugar to the phenolic hydroxyl group have been introduced has the ability to synthesize S-form betanin, which is one of the betarain pigments.

In past reports, the results of betalain synthesis using three synthetic genes (CYP76AD1, DODA1-1, CDOPA5GT) derived from plants other than quinoa have been reported (Non-Patent Documents 2 and 3). However, in those reports, betanin (S-form) is produced and at the same time, the stereoisomer isobetanin (R-form) is synthesized at the same time. The appearance of steric isomers is due to a reaction involving DODA1, and past findings indicate that DODA1 in Marvel-of-peru is 88% synthetic of betanin precursor (S-form) and 12% of isobetanin precursor (R-form). It is reported that this is done (Non-Patent Document 1). From these reports and the results of this example, DODA1-1 and DODA1-2 isolated from quinoa according to the present invention are novel DOPA 4,5-dioxygenases capable of synthesizing substantially only betanin. It became clear that it is an active enzyme.

<Preparation of Agrobacterium transformed with quinoa DODA1 homolog>
For the CqDODA1 homologs CqDODA1-3, CqDODA1-4, CqDODA1-5 and CqDODA1-6, the betalain synthesis ability is confirmed by the same method as in Example 1.

The following primers are used for PCR, and the amplified PCR fragment is cleaved with the following restriction enzymes by the same method as in Example 1.
The PCR primers are SEQ ID NOs: 9 and 10 for CqDODA1-3 (SEQ ID NO: 22), SEQ ID NOs: 11 and 12 for CqDODA1-4 (SEQ ID NO: 24), SEQ ID NOs: 13 and 14 for CqDODA1-5 (SEQ ID NO: 26), and CqDODA1. For -6 (SEQ ID NO: 28), SEQ ID NOs: 15 and 16 are used.
The amplified PCR fragment is cleaved 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, and pCAMBIA_CqDODA1-6-ox, as shown in FIG.

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

<Evaluation of betalain synthesis ability in Nicotiana benthamiana by quinoa DODA1 homolog>
The betalain synthesizing ability of CqDODA1-3, CqDODA1-4, CqDODA1-5 or CqDODA1-6 is evaluated by introducing it into Bensamiana tobacco which does not have the synthesizing ability by the following method.

[Transient expression analysis in Nicotiana benthamiana]
3 ml of LB medium for each of the CqCYP76AD1 and CqCDOPA5GT transformed Agrobacterium prepared in Example 1 and the CqDODA1-3, CqDODA1-4, CqDODA1-5 or CqDODA1-6 transformed Agrobacterium prepared in Example 4. Incubate in.
Combination of CqCYP76AD1, CqDODA1-3 and CqCDOPA5GT, combination of CqCYP76AD1, CqDODA1-4 and CqCDOPA5GT, combination of CqCYP76AD1, CqDODA1-5 and CqCDOPA5GT, or combination of CqCYP76AD1, CqDODA1-6 and CqCDOPA5.
The prepared mixed bacterial solution is inoculated into Nicotiana benthamiana by the same method as in Example 2. After growing 3 to 5 days after inoculation, the green leaves of Nicotiana benthamiana grown are used for the following analysis.

[Betalain dye extraction and HPLC analysis]
Betalain dye extraction and HPLC analysis are performed by the same method as in Example 2.

[result]
Combination of CqCYP76AD1, CqDODA1-3 and CqCDOPA5GT, combination of CqCYP76AD1, CqDODA1-4 and CqCDOPA5GT, combination of CqCYP76AD1, CqDODA1-5 and CqCDOPA5GT, or all combinations of CqCYP76AD1, CqDODA1-6 and CqCDOPA5 The leaves of benthamiana turn red, confirming that the red pigment was synthesized.
As a result of HPLC analysis, the combination of CqCYP76AD1, CqDODA1-3 and CqCDOPA5GT, the combination of CqCYP76AD1, CqDODA1-4 and CqCDOPA5GT, the combination of CqCYP76AD1, CqDODA1-5 and CqCDOPA5GT, or the combination of all CqCYP76AD1, CqDODA1-6 and CqDODA1-6 A single peak of S-form betanin, one of the betalain pigments, is confirmed in a sample extracted from overexpressed Nicotiana benthamiana.
Therefore, all CqDODA1 of CqDODA1-3, CqDODA1-4, CqDODA1-5 and CqDODA1-6 can be used for the production of S-form betanin.

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

[result]
CqCYP76AD1 showed 88% homology to beat CYP76AD1 and CqCDOPA5GT showed 77% homology to beat CDOPA5GT. On the other hand, both CqDODA1-1 and CqDODA1-2 had a low homology of 69% with respect to beat DODA1. Furthermore, when CqDODA1-1 and CqDODA1-2 were compared with Oshiroybana DODA1, they showed low homology at 54% and 53%.
_{The sequence of the active site of DODA 1} (CqDODA1-1 to 1-6) of quinoa isolated this time is HPLDETPHYHGGVAPWAAX 1 FDSWLDX ₂ ALTX ₃ GRX ₄ EEVNTYETKAPNWEL (X ₁ indicates D or E, X ₂ indicates L, V or T). , X ₃ indicates K or N, and X ₄ indicates F or I: SEQ ID NO: 33) (FIG. 2B). Previous reports have reported conserved sequences of the active site of DODA1 from other plants (Fig. 2A) (Christinet, L., Burdet, FX, Zaiko, M., Hinz, U. and Zryd, JP2004, characterization and). functional identification of anovelplant4,5-extradioldioxygenase involved in betalain pigment biosynthesisinPortulacagrandiflora.Plant Physiol., 134, 265-74.).
From the above, it was confirmed that the sequence of DODA1 (particularly the active site) of quinoa isolated this time is clearly different from the sequence of DODA1 (particularly the active site) derived from other plants (FIGS. 2A and B).

<Dye analysis of quinoa hypocotyl>
The pigments accumulated in the red quinoa hypocotyl were analyzed.
Quinoa strains Kd and CQ127 were used. Betalain dye extraction and HPLC analysis were performed from the quinoa hypocotyl by the same method as in Example 2. More specifically, the apparatus was carried out using LC-20AD (Shimadzu Corporation) under the condition of 25 ° C. Water containing 0.05% trifluoroacetic acid and 0.05% acetonitrile was used as the analysis solvent.
As a result of HPLC analysis, amaranthin and cerosinin II were detected as main peaks, and a slight peak of betanin was detected.

<Isolation of quinoa-derived amaranthin synthase gene and evaluation of amaranthin synthesis ability>
From the results of Example 7, a phylogenetic analysis for isolation of amaranthin synthase was performed, and the ability of the obtained candidate gene to synthesize amaranthin was evaluated with Nicotiana benthamiana.

[Physical analysis for isolation of amaranthin synthase]
Amaranthin is a betalain pigment in which sugar is added to betanin. Clustal W algorithm (JD Thompson, DG Higgins, TJ Gibson, CLUSTAL W: A phylogenetic tree analysis was performed using improvingthesensitivityof progressive multiple sequence alignmentthroughsequenceweighting, position-specific gap penalties and weight matrixchoice, NucleicAcids Res., 22 (1994) 4673-4680.). A phylogenetic tree was constructed.

[result]
The constructed phylogenetic tree is shown in FIG. There were 17 homologs in Quinoa, divided into two groups.
As a result of examining the expression in the quinoa hypocotyl in the group to which flavonoid3-O-glucoside: 2 ″ -O-glucosyltransferase that modifies flavonols does not belong, the expression of accession numbers XP_021754077.1, XP_021735839.1 and XP_021735840.1 was observed. It could be confirmed.

[Transient expression analysis in Nicotiana benthamiana]
The amaranthin synthase activity was evaluated for the above three genes and the gene of XP_021743749.
It was transformed into Agrobacterium by the same method as in Example 1. The green leaves of Nicotiana benthamiana were inoculated using the agroinfiltration method in the same manner as in Example 2 with the following combinations. For each green leaf sample and quinoa hypocotyl extraction sample (control) of the combination, betalain dye extraction and HPLC analysis were performed by the same method as in Example 2.
(1) Combinations containing XP_021754077.1 CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 (Protein19) and XP_021754077.1 were transiently introduced into Bensamiana benthamiana.
(2) Combinations containing XP_021735839.1 CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 and XP_021735839.1 were transiently introduced into Bensamiana benthamiana.
(3) Combinations containing XP_021735840.1 CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 and XP_021735840.1 were transiently introduced into Bensamiana benthamiana.
(4) Combinations containing XP_021743749 CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 and XP_021743749 were transiently introduced into Bensamiana benthamiana.
(5) Combination containing empty vector CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 and pCAMBIA1301M which is an empty vector (vector control) were transiently introduced into Bensamiana tobacco.

[result]
The results of agroinfiltration are shown in FIG.
As a result of HPLC analysis, the peak of amaranthin was detected in the sample extracted from Bensamiana tobacco transformed with CqCYP76AD1, CqCDOPA5GT, CqDODA1, P19 (Protein 19) and XP_021754077.1, and the control quinoa hypocotyl extract. However, isoamarantin was not detected (Fig. 10). A peak of betanin was detected and a peak of amaranthin was not detected in the sample (-) extracted from Nicotiana benthamiana into which an empty vector was introduced (FIG. 10).
Therefore, it was confirmed that XP_021754077.1 (SEQ ID NO: 37) is a gene encoding amaranthin synthase and has an enzymatic activity of adding sugar to betanin. The homology between XP_021754077 and Arabidopsis flavonoid 3-O-glucoside: 2 ″ -O-glucosyltransferase was 46%. That is, it was confirmed that the amaranthin synthase was completely different from the known enzyme.
From the above, the gene encoding the enzyme having the activity of hydroxylating the 3-position of the phenol ring of tyrosine, the gene encoding the enzyme having the L-DOPA oxidase activity, and the gene encoding the enzyme having the DOPA 4,5-dioxygenase activity are encoded. The plant body, which is the host into which the gene, the gene encoding the enzyme having the activity of adding sugar to the phenolic hydroxyl group, and the gene group of the gene encoding the amaranthin synthase are introduced, is the synthesis of amaranthin, which is one of the betalein pigments. Has the ability.

<Evaluation of betalain synthesis ability of betalain synthesis ability gene derived from Marvel-of-peru>
We investigated whether Marvel-of-peru DODA1 could synthesize substantially only betanin.

[Transient expression analysis in Nicotiana benthamiana]
The DODA1 gene (MjDODA1; SEQ ID NO: 35) of Marvel-of-peru was transformed into Agrobacterium by the same method as in Example 1. The green leaves of Nicotiana benthamiana were inoculated using the agroinfiltration method in the same manner as in Example 2 with the following combinations. For each green leaf sample of the combination, betalain dye extraction and HPLC analysis were performed by the same method as in Example 2.
(1) Combinations containing MjDODA1 CqCYP76AD1, CqCDOPA5GT, P19 and MjDODA1 were transiently introduced into Bensamiana benthamiana.
(2) Combinations containing CqDODA-1 (CqDODA1-1) CqCYP76AD1, CqCDOPA5GT, P19 and CqDODA-1 were transiently introduced into Bensamiana benthamiana.
(3) Combinations containing CqDODA-5 (CqDODA1-5) CqCYP76AD1, CqCDOPA5GT, P19 and CqDODA-5 were transiently introduced into Bensamiana benthamiana.
(4) Combinations containing CqDODA-6 (CqDODA1-6) CqCYP76AD1, CqCDOPA5GT, P19 and CqDODA-6 were transiently introduced into Bensamiana benthamiana.

[result]
As a result of agroinfiltration, it was shown that the leaves turned red in all combinations and the red pigment was synthesized (Fig. 11).
As a result of HPLC analysis, a single peak of betanin was detected and isobetanin was detected not only in CqDODA-1, CqDODA-5 and CqDODA-6 but also in a sample extracted from Nicotiana benthamiana that transiently expressed MjDODA1. There was no (Fig. 12).
Therefore, it was clarified that DODA1 (MjDODA1) of Marvel-of-peru is an enzyme having DOPA 4,5-dioxygenase activity capable of synthesizing substantially only betanin.

<Preparation of ghy mutant and functional evaluation of causative gene>
Mutants of genes involved in betalain pigment synthesis were prepared in quinoa, and functional analysis related to betalain pigment synthesis was performed using the mutants.

[Mutagenesis with ethyl methanesulfonic acid (EMS)]
Seeds of the quinoa line Kd (obtained from Kyoto University) and CQ127 {obtained from the United States Department of Agriculture (USDA)} were grown at 23 ° C. for 12 hours light and 12 hours dark. The EMS treatment of the grown plants was outsourced to Implanta Innovations, Inc. EMS-treated seeds were grown, M1 was obtained, and the seeds were grown up to the M3 generation. M3 was used for mutation analysis.

[Mutation analysis]
A library was constructed using the 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, Fastmap). and accurate short read alignment with Burrows-Wheelertransform, Bioinformatics, 25 (2009) 1754-1760.; H. Li, B. Handsaker, A.Wysoker, T. Fennell, J. Ruan, N. Homer, G.Marth, G. Abecasis, R.Durbin, TheSequence Alignment / Map format and SAMtools, Bioinformatics, 25 (2009) 2078-2079.).

[result]
As a result of mutation analysis, two green hypocotyl mutant (ghy) lines were discovered and named ghy1 and ghy2. As a result of pigment analysis, no accumulation of betacyanin pigment was 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]
M3 seeds of ghy1 and ghy2 were isolated in wild type: ghy = 3: 1 by isolation test. Both phenotypes were sequenced ^{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 the causative gene of the ghy variant were identified.
Of these identified SNPs, a point mutation in CqCYPAD1-1 (Gene ID: 110733547) replaced arginine at position 371 with cysteine in ghy1 and tryptophan at position 124 in ghy2 from beginning to end. It was confirmed.
That is, CqCYPAD1-1 in which arginine at position 371 was replaced with cysteine and CqCYPAD1-1 in which tryptophan at position 124 was replaced with a stop codon were confirmed.

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

[CqCYPAD1-1 functional analysis]
(1) Overexpression of CqCYPAD1-1 The ghy1 mutant CqCYPAD1-1 was overexpressed for functional analysis. A CqCYPAD1-1 overexpression vector was constructed and introduced into Rhizobium rhizogenes (ATCC 15834). The Rhizobium rhizogenes transformant was infected with the hairy root of the ghy1 mutant. As a result, red pigment formation was observed in the hairy root.
(2) Evaluation of CYP76AD1 activity in Nicotiana benthamiana By the same method as in Example 2, the green leaves of Nicotiana benthamiana were combined with CqCYP76AD1-1, CqDODA1-1 and CqCDOPA5GT, or CqCYP76AD1-1_R371C (371st arginine). The combination of CqCYP76AD1-1), CqDODA1-1 and CqCDOPA5GT substituted with cysteine was transiently expressed. As a result, the leaves turned red in the former combination, whereas the leaves did not turn red in the latter combination. Therefore, it was confirmed that CqCYP76AD1-1 has CYP76AD1 activity. In addition, no accumulation of betalain was 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 the cotyledons and hypocotyls in which red pigment was observed under light and dark conditions. On the other hand, CqCYP76AD1-1 was expressed in the cotyledons 2 days after sowing under constant dark conditions, but not in the hypocotyl. Therefore, it was clarified that the expression of CqCYP76AD1-1 in the hypocotyl is photodependent.

[CqCYPAD1-2 Function analysis]
(1) Analysis of CqCYP76AD1-2 expression in the hypocotyl Based on the nucleotide sequence analysis, it was predicted that CqCYP76AD1-2 would have enzymatic activity. As a result of analyzing the expression of CqCYP76AD1-2 in the hypocotyl 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. .. A splicing junction of CqCYP76AD1-2 was also confirmed. In the CQ127 strain, SNP occurred in the splicing region and a 23-bp deletion was present near the 3'splicing region.
(2) Evaluation of CYP76AD1 activity in Nicotiana benthamiana By the same method as in Example 2, the genomic region of CqCYP76AD1-2 derived from the Kd strain, the combination of CqDODA1-1 and CqCDOPA5GT, or the Kd strain was added to the green leaves of Nicotiana benthamiana. The combination of CqCYP76AD1-2 cDNA, CqDODA1-1 and CqCDOPA5GT from which it was derived was transiently expressed. As a result, the leaves of both turned red. As a result of HPLC analysis and MS analysis, it was confirmed that this pigment formation was due to betanin. On the other hand, as a result of transiently expressing the combination of CqCYP76AD1-2 genomic region, CqDODA1-1 and CqCDOPA5GT derived from CQ127 strain in the green leaves of Nicotiana benthamiana by the same method as in Example 2, no red pigment formation was observed. rice field. Therefore, it was clarified that CqCYP76AD1-2 functions normally in the genome derived from the Kd lineage and does not function by missplicing in the genome derived from the CQ127 lineage.

[Usefulness of ghy mutant]
Expression of CqCYP76AD1-1 occurs by light exposure in the hypocotyl. As a result, betalain pigment is accumulated in the hypocotyl immediately after germination. This is to protect plants from environmental and biological stresses (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-DOPAapplication confers photoprotection tosaline-exposed leaves of Disphymaaustrale, New Phytol., 207 (2015) 1075-1083.). Red pigment formation in the hypocotyl was observed in all 75 species of Chenopodium, including quinoa. Therefore, betalain pigment is one element 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 appeared in the M3 generation, but it is thought that mutations in this generation do not appear in the heteropolyploid quinoa. Quinoa may have lost a pair of homologous genes. In fact, the homologous genes of CqCYP76AD1-1 and CqCYP76AD1-2 lost their functions and became pseudogenes. In the CQ127 strain, CqCYP76AD1-2 lost its function due to missplicing, and only CqCYP76AD1-1 functioned normally. The characteristic of M3 mutations in ghy mutants is expected to be regulated by a single gene in heteropolyploid quinoa.
The ghy1 mutant is useful for CYP76AD1 activity analysis. This is also supported by the fact that the 371st residue of CqCYP76AD1-1 with a mutation in ghy1 is close to the active center in 3D structural modeling.
Transformation of plants that normally produce betalain is difficult. On the other hand, ghy becomes more susceptible to Agrobacterium and can be transformed by using ghy mutants.
Betalain is likely to inhibit transformation. Since the ghy mutant does not synthesize betalain, it has a high redifferentiation ability.
That is, the present invention also covers ghy1 and ghy2, which are the green hypocotyl mutant (ghy) series.
The present invention also covers CqCYPAD1-1 in which arginine at position 371 is replaced with cysteine and CqCYPAD1-1 in which tryptophan at position 124 is replaced with a stop codon from beginning to end.

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

Claims (5)

It is a method for synthesizing S-form betanin that does not contain isobetanin.
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, and a gene encoding an enzyme. The following genes encoding genes encoding enzymes with DOPA 4,5-dioxygenase activity have been introduced and tyrosine or 3-hydroxy-L-tyrosine (L-DOPA) production. Cultivate a capable host and extract the tyrosine pigment from the cultured host,
Or,
The following genes encoding enzymes having DOPA 4,5-dioxygenase activity have been introduced, and the enzyme activity has the activity of hydroxylating the 3-position of the phenol ring of tyrosine, and the enzyme activity having L-DOPA oxidase activity. , A host having an enzymatic activity having an activity of adding a sugar to a phenolic hydroxyl group and a host having a tyrosine or 3-hydroxy-L-tyrosine producing ability is cultured, and a betalein pigment is extracted from the host after the culture.
Here, the gene encoding the 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 NO: 17, 19, 25 or 27.
(2) In the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27 , 1 to 20 amino acids are substituted, deleted, inserted and / or added, and in SEQ ID NO: 17, 19, 25 or 27 . A gene encoding a polypeptide having the ability to convert L-DOPA, which is homogeneous with the polypeptide consisting of the described amino acid sequence, to 4,5-seco-DOPA.
(3) SEQ ID NO: to 17,19,25 or 27 having an amino acid sequence identity of 90% or more of the described and set forth in SEQ ID NO: 17,19,25 or 27 comprising the amino acid sequence polypeptides and homogenous A gene encoding a polypeptide capable of converting L-DOPA to 4,5-seco-DOPA,
(4) A gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28,
(5) Hybrid under stringent conditions with a DNA having a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28 , and SEQ ID NO: 17, 19, 25 or 27. A gene consisting of DNA encoding a polypeptide having the ability to convert L-DOPA homologous to the polypeptide consisting of the amino acid sequence described in 1.5-seco-DOPA.
(6) In the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28 , 1 to 50 base sequences are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, A gene consisting of DNA encoding a polypeptide having the ability to convert L-DOPA homologous to the polypeptide consisting of the amino acid sequence set forth in 25 or 27 to 4,5-seco-DOPA, and (7) SEQ ID NO: 18. L-DOPA having 90% or more homology with the DNA consisting of the base sequence set forth in 20, 26 or 28 and the same quality as the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27. A method for producing an isobetanin-free S-form betanin , which comprises a gene consisting of DNA encoding a polypeptide having the ability to convert to 4,5-seco-DOPA.
L-DOPA for isobetanin-free S-form betanin synthesis containing the gene shown in any one of (1) to (7) below or the vector carrying the gene to 4,5-seco-DOPA. Converter;
(1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27.
(2) In the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27 , 1 to 20 amino acids are substituted, deleted, inserted and / or added, and in SEQ ID NO: 17, 19, 25 or 27 . A gene encoding a polypeptide having the ability to convert L-DOPA, which is homogeneous with the polypeptide consisting of the described amino acid sequence, to 4,5-seco-DOPA.
(3) SEQ ID NO: to 17,19,25 or 27 having an amino acid sequence identity of 90% or more of the described and set forth in SEQ ID NO: 17,19,25 or 27 comprising the amino acid sequence polypeptides and homogenous A gene encoding a polypeptide capable of converting L-DOPA to 4,5-seco-DOPA,
(4) A gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28,
(5) Hybrid under stringent conditions with a DNA having a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28 , and SEQ ID NO: 17, 19, 25 or 27. A gene consisting of DNA encoding a polypeptide having the ability to convert L-DOPA homologous to the polypeptide consisting of the amino acid sequence described in 1.5-seco-DOPA.
(6) In the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28 , 1 to 50 base sequences are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, A gene consisting of DNA encoding a polypeptide having the ability to convert L-DOPA homologous to the polypeptide consisting of the amino acid sequence set forth in 25 or 27 to 4,5-seco-DOPA, and (7) SEQ ID NO: 18. L-DOPA having 90% or more homology with the DNA consisting of the base sequence set forth in 20, 26 or 28 and the same quality as the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27. A gene consisting of DNA encoding a polypeptide capable of converting to 4,5-seco-DOPA.
L-DOPA for isobetanin-free S-form betanin synthesis having a peptide represented by the amino acid sequence of any one of (1) to (3) below was added to 4,5-seco-DOPA. Converter;
(1) The amino acid sequence according to SEQ ID NO: 17, 19, 25 or 27.
(2) In the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27 , 1 to 20 amino acids are substituted, deleted, inserted and / or added, and in SEQ ID NO: 17, 19, 25 or 27 . 90% or more of the amino acid sequence having the ability to convert L-DOPA equivalent to the amino acid sequence described to 4,5-seco-DOPA, and (3) the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27. An amino acid sequence having the same identity and capable of converting L-DOPA substantially identical to the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27 into 4,5-seco-DOPA.
A betalain dye-producing host into which the conversion agent according to claim 2 or 3 has been introduced.
An isobetanin-free S-form betanin- producing host,
The host is
L, a gene encoding an enzyme having the activity of hydroxylating the 3-position of the phenol ring of tyrosine
-The gene encoding the enzyme having DOPA oxidase activity, the gene encoding the enzyme having the activity of adding sugar to the phenolic hydroxyl group, and the gene group of the gene encoding the enzyme having the following DOPA 4,5-dioxygenase activity are Introduced and tyrosine or 3-
Has the ability to produce hydroxy-L-tyrosine (L-DOPA),
Or,
The following gene encoding an enzyme having DOPA 4,5-dioxygenase activity has been introduced, and the enzyme activity L-DO has the activity of hydroxylating the 3-position of the phenol ring of tyrosine.
It has an enzyme activity having PA oxidase activity, an enzyme activity having an activity of adding a sugar to a phenolic hydroxyl group, and a tyrosine or 3-hydroxy-L-tyrosine producing ability.
Here, the gene encoding the 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 NO: 17, 19, 25 or 27.
(2) In the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27 , 1 to 20 amino acids are substituted, deleted, inserted and / or added, and in SEQ ID NO: 17, 19, 25 or 27 . A gene encoding a polypeptide having the ability to convert L-DOPA, which is homogeneous with the polypeptide consisting of the described amino acid sequence, to 4,5-seco-DOPA.
(3) SEQ ID NO: to 17,19,25 or 27 having an amino acid sequence identity of 90% or more of the described and set forth in SEQ ID NO: 17,19,25 or 27 comprising the amino acid sequence polypeptides and homogenous A gene encoding a polypeptide capable of converting L-DOPA to 4,5-seco-DOPA,
(4) A gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28,
(5) Hybrid under stringent conditions with a DNA having a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28 , and SEQ ID NO: 17, 19, 25 or 27. A gene consisting of DNA encoding a polypeptide having the ability to convert L-DOPA homologous to the polypeptide consisting of the amino acid sequence described in 1.5-seco-DOPA.
(6) In the DNA consisting of the base sequence set forth in SEQ ID NO: 18, 20, 26 or 28 , 1 to 50 base sequences are substituted, deleted, inserted and / or added, and SEQ ID NOs: 17, 19, A gene consisting of DNA encoding a polypeptide having the ability to convert L-DOPA homologous to the polypeptide consisting of the amino acid sequence set forth in 25 or 27 to 4,5-seco-DOPA, and (7) SEQ ID NO: 18, L-DOPA having 90% or more homology with the DNA consisting of the base sequence set forth in 20, 26 or 28 and the same quality as the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 17, 19, 25 or 27. An isobetanin-free S-form betanin- producing host characterized by a gene consisting of DNA encoding a polypeptide capable of converting to 4,5-seco-DOPA.

Images