JP2015192668A - Betaxanthin production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a betaxanthin production method by which betaxanthin can be supplied steadily and which can be industrialized.SOLUTION: A betaxanthin production method comprises a step of converting a raw material into betaxanthin (conversion step) in an aqueous medium under the presence of microorganisms having enzyme activity which hydroxylates the 3-position of the phenol ring of tyrosine, and DOPA 4,5-dioxygenase activity, or a processed product thereof.

Description

  The present invention relates to a method for producing betaxanthin, which is a group of betalain pigments.

A betalain pigment is a water-soluble pigment characterized by containing nitrogen in its chemical structure, which is naturally contained only in plants of the order Dianthera and certain higher fungi. Betalaine pigments are classified into betaxanthines and betacyanins based on their structural characteristics. Betaxanthin is colored yellow and betacyanins are reddish purple, so that they are conventionally used as natural colorants.

  In recent years, it has been found that betalaine dyes have a strong antioxidant effect, and their use as an antioxidant is also being studied. Furthermore, it has been found that betalaine dyes have the property of efficiently absorbing solar energy, and their use in sensitizing dyes for dye-sensitized solar cells is also expected. As a method for producing this betalein pigment, a method of extracting and purifying from a plant is generally used, but plants containing betalain pigments are deciduous species such as pine bug, beet, cactus, etc. It is limited to the cirrus family, cactiaceae, red crustaceae, and white-spotted family). Therefore, it is expensive and difficult to supply stably.

In recent years, as a new method for producing betalain pigments, tyrosinase genes derived from fungi such as basidiomycetes containing various mushrooms and L-3,4-dihydroxyphenylalanine (DOPA) 4,5-dioxygenase genes derived from Osileubana, A method for producing betaxanthin using transformed plant cells introduced under the control of a promoter has been reported (Patent Document 1).

In addition, many genes derived from plants and microorganisms are known as tyrosinase-encoding genes (Non-patent Document 1), and genes encoding DOPA 4,5-dioxygenase include those derived from Osileubana or sugar beet. It is known (Non-Patent Documents 2 and 3).

JP 2012-55208 A

Michael Fairhead et al, New Biotechnology, Volume 29, Issue 2, 15 January 2012, p.183-191 Sasaki N. et al, Plant Cell Physiol. 50 (5): 1012-1026 (2009) Fernando Gandia-Herrero et al, Planta (2012) 236: 91-100

  At present, in the production method for extracting betalaine pigments from commonly used plants, as described above, it is necessary to extract from a very limited plant, and the content of these plants is extremely small. It is difficult to supply stably and continuously, and the manufacturing cost is high. In addition, in the method using plant cells described in Patent Document 1, since the cell growth rate of plant cells is extremely slow, production efficiency is low, and industrialization is considered difficult.

In addition, a gene encoding tyrosinase (Non-patent Document 1) and a gene encoding DOPA 4,5-dioxygenase (Non-patent Documents 2 and 3) are introduced individually into microorganisms such as Escherichia coli and yeast. Examples in which the enzyme activity is confirmed individually are known. However, there is no known example in which these two types of genes are introduced into a microorganism at the same time, and betaxanthin, which is a group of betalaine pigments, is produced from a substrate such as glucose using a microorganism into which these two types of genes have been introduced. There are no known examples that have confirmed this.

  This invention makes it a subject to provide the manufacturing method of the betaxanthin which can be supplied stably and can be industrialized.

  As a result of intensive studies to solve the above problems, the present inventors have found that the enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine, which is essential for the biosynthesis of betaxanthin, and the DOPA4,5-dioxygenase activity The present inventors have succeeded in producing a microorganism having the same, found that it is possible to efficiently produce betaxanthin using the microorganism, and completed the present invention.

That is, the gist of the present invention resides in the following (1) to (8).
(1) A step of converting a raw material into betaxanthin in an aqueous medium in the presence of a microorganism having hydroxylase at the 3-position of the phenol ring of tyrosine and DOPA4,5-dioxygenase activity or a treated product thereof ( A method for producing betaxanthin, comprising a conversion step).
(2) Before the conversion step, a step of culturing a microorganism having an enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine and DOPA 4,5-dioxygenase activity in an aqueous medium (cultivation step) The method for producing betaxanthin according to (1), wherein: (3) The method for producing betaxanthin according to (1) or (2), further comprising a step of recovering betaxanthin from the aqueous medium after the conversion step.
(4) The method for producing betaxanthin according to any one of (1) to (3), wherein the enzyme that hydroxylates the 3-position of the phenol ring of tyrosine is tyrosinase.
(5) The method for producing betaxanthin according to any one of (1) to (4), wherein a resting cell is used as the microorganism in the conversion step.
(6) The method for producing betaxanthin according to any one of (1) to (5), wherein the raw material is tyrosine and / or a saccharide raw material.
(7) The method for producing betaxanthin according to any one of (1) to (6), wherein an amino acid or amine other than tyrosine is added in addition to the raw material.
(8) In the conversion step, the aqueous medium contains at least one selected from the group consisting of ascorbic acid, copper and iron, and the betaxanthin according to any one of (1) to (7), A method of manufacturing a kind.

According to the present invention, it is possible to efficiently produce betaxanthin, which is a group of betalaine dyes, from inexpensive raw materials such as glucose and amino acids, and it is also possible to supply stably.
Moreover, the betaxanthin obtained by this invention can be used suitably for various uses, such as a dye sensitizer of a dye-sensitized solar cell, and an antioxidant.

In Example 1, E.I. The LC / MS analysis result obtained when using E. coli (BL21) / BMTYR / MjDOD is shown. In Example 1, E.I. The LC / MS analysis result obtained when using E. coli (BL21) / BTTYR / MjDOD is shown. In Example 1, E.I. The LC / MS analysis result obtained when using E. coli (BL21) / RSTYR / MjDOD is shown. The LC / MS analysis result obtained in Example 2 (L-valine) is shown. In Example 2 (L-valine), the change of the production amount up to 24 hours after the start of the reaction is shown. The LC / MS analysis result obtained in Example 9 ((S)-(alpha) -propargylalanine) is shown. In Example 9 ((S)-(alpha) -propargylalanine), the change of the production amount to 24 hours after a reaction start is shown. In Example 19, E.I. The LC / MS analysis results obtained using E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / BMTYR + MjDOD are shown. In Example 19, E.I. The LC / MS analysis result obtained when using E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / RSTYR + MjDOD is shown. The LC / MS analysis result obtained when YPH500 / BMTYR / MjDOD was used in Example 20 is shown. The LC / MS analysis result obtained when YPH500 / RSTYR / MjDOD was used in Example 20 is shown. An example of the metabolic pathway which the microorganisms of this invention have is shown.

The present invention relates to a method for producing betaxanthin, which is a group of betalaine pigments, and a microorganism having hydroxylase at the 3-position of the phenol ring of tyrosine and DOPA4,5-dioxygenase activity, or a processed product thereof In the presence of, it has the process (conversion process) which converts a raw material into betaxanthin in an aqueous medium.
Here, DOPA 4,5-dioxygenase is 3,4-dihydroxy-L-phenylalanine: oxygen 4,5-oxidoreductase.
Here, betaxanthin is a general term for a compound group in which an amino acid or an amine is bonded to betaramic acid, and the structure thereof is represented by the following formula (1) or formula (2).

In the formulas (1) and (2), R ₁ , R ₂ and R ₃ are selected from the group consisting of a hydrogen atom, a linear or branched chain having 1 to 40 carbon atoms, or a cyclic saturated or unsaturated hydrocarbon group. One type. However, some or all of the hydrogen atoms of the hydrocarbon group are halogen atoms, acyl groups, amide groups, amino groups, alkoxy groups, aldehyde groups, imidazole groups, indole groups, oxo groups, carboxyl groups, guanidino groups, cyano groups. May be substituted with at least one selected from the group consisting of a sulfo group, a thiol group, a nitro group, a hydroxyl group and a phenyl group, and the hydrocarbon group forms a ring in a form containing nitrogen of iminium. It may be. Furthermore, some or all of the aromatic rings of the imidazole group, the indole group and the phenyl group may be substituted with at least one selected from the same substituent group as described above. Further, some of the carbon atoms of the hydrocarbon group may be substituted with at least one selected from a sulfur atom, an oxygen atom, a nitrogen atom, and a phosphorus atom.

For example, a compound in which R ₁ is a hydrogen atom, R ₂ is a straight-chain hydrocarbon group having 3 carbon atoms and forms a ring in a form containing iminium nitrogen is proline-betaxanthin. A compound in which R ₁ is a hydrogen atom, R ₂ is a straight-chain hydrocarbon group having 3 carbon atoms, and one of the terminal hydrogen atoms of the hydrocarbon group is substituted with a guanidino group is arginine-betaxanthin. . A compound in which R ₁ is a hydrogen atom, R ₂ is a hydrocarbon group having 1 carbon atom, and _one of the hydrogen atoms of the hydrocarbon group is substituted with an imidazole group is histidine-betaxanthin. A compound in which R ₁ is a hydrogen atom, R ₂ is a straight-chain hydrocarbon group having 4 carbon atoms, and the second carbon atom from the end of the hydrocarbon group is substituted with a sulfur atom is methionine-betaxanthin. A compound in which R ₁ is a hydrogen atom, R ₂ is a hydrocarbon group having 1 carbon atom, and _one of the hydrogen atoms of the hydrocarbon group is substituted with an indole group is tryptophan-betaxanthin. R ₁ is a hydrogen atom, R ₂ is a hydrocarbon group having 1 carbon atom, _one of the hydrogen atoms of the hydrocarbon group is substituted with a phenyl group, and the hydrogen atom at the para position of the phenyl group is substituted with a hydroxyl group The compound obtained is tyrosine-betaxanthin.

[Microorganism used in the present invention or processed product thereof]
The microorganism used in the present invention (hereinafter sometimes referred to as “the microorganism of the present invention”) has an enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine and a DOPA4,5-dioxygenase activity.

  The microorganism of the present invention is not particularly limited as long as it has an enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine and a DOPA4,5-dioxygenase activity, and is essentially a microorganism having these enzyme activities. They may be those that have been imparted with these enzyme activities by breeding. Examples of means for imparting these enzyme activities by breeding include mutation treatment and gene recombination treatment. Enhancing the expression of enzyme genes in the biosynthesis pathway of organic compounds and reducing the expression of enzyme genes in the byproduct biosynthesis pathway Any known method can be employed.

  In the production method of the present invention, for example, betaxanthin can be produced by using a microorganism having a metabolic pathway shown in FIG. The chemical reaction represented as Sp in FIG. 12 is a spontaneous reaction that does not require an enzyme. Therefore, if a microorganism having an enzyme activity that hydroxylates at least the 3-position of the phenol ring of tyrosine and a DOPA4,5-dioxygenase activity is used, betaxanthin can be produced by the route shown in FIG. Here, since the reaction described as Sp is not an enzyme reaction, it does not necessarily have to be performed in the metabolism of microorganisms. For example, it is possible to produce betaxanthin by removing betalamic acid produced by microorganisms outside the cells and mixing it with an amino acid or amine as a reagent in an appropriate solvent.

The microorganisms in the present invention are not limited to living microorganisms, but include those that are dead as living organisms but have enzyme activity.
In addition, as a processed product of microorganisms in the present invention, cells treated with acetone, toluene, etc., freeze-dried cells, disrupted cells, cell-free extract obtained by disrupting cells, purified enzyme (partially purified) Including an enzyme). In addition, an immobilized product obtained by immobilizing a treated product of these microorganisms on a carrier by a conventional method is also included in the treated product.
Hereinafter, the microorganisms used in the present invention will be described.

(Enzyme activity to hydroxylate the 3-position of the phenol ring of tyrosine)
The enzyme activity that hydroxylates the 3-position of the phenol ring of tyrosine is an enzyme activity that can add a hydroxyl group to the 3-position of the phenol ring of tyrosine. Whether or not it has this enzyme activity can be determined by measuring the activity of the expressed protein to hydroxylate the 3-position of tyrosine by a conventional assay method. For example, the enzyme activity can be confirmed by allowing an enzyme to be measured to act on tyrosine and directly measuring the amount of L-DOPA converted from tyrosine.

  Examples of the enzyme that hydroxylates the 3-position of the phenol ring of tyrosine include tyrosinase, cytochrome P450, catechol oxidase, tyrosine 3-monooxygenase, and aromatic ring hydroxylated dioxygenase.

Examples of tyrosinases include Aeromonas media, mushrooms (Agaricus bisporus), Azospirillum sp., Bacillus megaterium, Bacillus thuringiensis, and sugar beet (Beta). vulgaris), cabbage (Brassica oleracea), tea tree (Camellia sinensis), arabica coffee tree (Coffea arabica), acai (Euterpe oleracea), human (Homo sapiens), lettuce (Lactuca sativa), Marinomonas mediterania fungus (Marinomonas medi) (Mus musculus), Neurospora crassa, tobacco (Nicotiana tabacum), nameko (Pholiota nameko), Pseudomonas putida F6, Ralstonia solanacearum, Rhizobium Rhizobium etli, tomato (Solanum lycopersicum), eggplant (Solanum melongena), potato (Solanum tuberosum), Streptomyces antibioticus, Streptomyces castaneoglobisporus ), Streptomyces glauescens, Streptomyces lincolnensis, Streptomyces lividans, Streptomyces REN-
21 (Streptomyces sp. REN-21), Thermomicrobium roseum, Vanilla planifolia, Verrucomicrobium spinosum, Vicia faba, European grape ( And an enzyme derived from Vitis vinifera). Among these, those derived from Bacillus megaterium, Bacillus thuringiensis, and Ralstonia solanacearum are preferred.

As cytochrome P450, Arthrobacter spp., Ascochyta spp., Aspergillus flavus, Aspergillus nidulans, Aspergillus niger (Aspergillus niger) ), Aspergillus oryze, Bacillus megaterium, Caldariomyces fumago, Candida albicans, Candida maltosa, Candida maltosa・ Candida tropicalis 、 Fusarium spp. 、 Fusarium oxysporum 、 Fusarium sporotrichioides 、 Fusarium verticillioides 、 Fusarium verticillioides Ma Micrococcus spp., Mortierella isabellina, Mycobacterium tuberculosis, Nectria haematoccoca, Neurospora crassa , Nocardia spp., Phanerochaete chrisosporium, Pseudomonas putida, Streptomyces spp., Rhizobium spp. , Saccharomyces cerevisiae, Saccharomyces pombe, Streptomyces avermitilis, Streptomyces coelicolor, Sulfolibus solfalus Examples include enzymes derived from fungi (Sulfolobus solfataricus), Yarrowia lipolutica, and the like. Among them, Arthrobacter spp., Ascochyta spp., Fusarium spp., Micrococcus spp., Mortierella isabellina, Those derived from Nocardia spp., Streptomyces spp., Rhizobium spp. And Streptomyces avermitilis are preferred.

Catechol oxidases include mushrooms (Agaricus bisporus), Aspergillus oryze, Aspergillus niger, Aspergillus niger, Bepo (Beta vulgaris), cattle (Bos taurus), chanoki (Camellia) sinensis), Henry Guri (Castanea henryi), Arabica coffee (Caffea arabica), melon (Cucumis melo), loquat (Eriobotrya japonica),
Human (Homo sapiens), Sweet potato (Ipomoea batatas), Fuji bean (Lablab purpureus)
), Morus alba, banana (Musa acuminata), Neurospora crassa, tobacco (Nicotiana tabacum), basil (Ocimum basilicum), oyster mushroom (Pleurotus ostreatus), matsuba button (Portulaca grandiflora), apricot (Prunus armeniaca), Ralstonia solanacearum, tomato (Solanum lycopersicum), eggplant (Solanum melongena), potato (Solanum tuberosum), Streptomyces glaucescens, Streptomyces griucescens (Streptomyces griseus), European grape (Vitis vinifera) and the like. Among them, those derived from Aspergillus oryze, Camellia sinensis, Ralstonia solanacearum, Streptomyces griseus, European grape (Vitis vinifera) and the like are preferable.

Tyrosine 3-monooxygenase includes bovine (Bos taurus), guinea pig (Cavia porcellus), Azumanishiki (Chlamys farreri nipponensis), Drosophila melanogaster, gorilla (Gorilla gorilla), human (Homo sapiens),
Rhesus macaque (Macaca mulatta), Mus musculus, rabbit (Oryctolagus cuniculus), chimpanzee (Pan troqlodytes), pine barn button (Portulaca grandiflora), European frog (Rana esculenta), red rat (Rattus norveribosto) And the like. Among them, those derived from Drosophila melanogaster, human (Homo sapiens), and rat rat (Rattus norvegicus) are preferable.

As aromatic ring hydroxylated dioxygenases, Arthrobacter sp., Acinetobacter calcoaceticus, Barkholderia cepacia, Burkholderia terrae , Comamonas testosteroni, Chromohalobacter sp., Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas fluorescens fluormon , Pseudomonas putida, Pseudomonas resinovorans
And enzymes derived from Rhodococcus opacus, Trichosporon cutaneum, and the like. Among them, those derived from Pseudomonas putida, Acinetobacter calcoaceticus, Mesorhizobium loti, and Barkholderia cepacia are preferable.

Among the above-mentioned enzymes, tyrosinase is preferable as the enzyme that hydroxylates the 3-position of the phenol ring of tyrosine.
Specific examples of tyrosinase are as described above. Among them, an enzyme encoded by the following base sequence (A), (B), or (C) is particularly preferable. SEQ ID NO: 13 is a base sequence derived from Bacillus megaterium, SEQ ID NO: 14 is derived from Ralstonia solanacearum, and SEQ ID NO: 15 is a base sequence derived from Bacillus thuringiensis.

(A) DNA having the base sequence represented by SEQ ID NO: 13, 14, or 15
(B) a nucleotide sequence represented by SEQ ID NO: 13, 14, or 15 comprising a nucleotide sequence in which one or several bases are substituted, deleted, and / or added, and the 3rd position of the phenol ring of tyrosine DNA encoding a polypeptide having an enzymatic activity to hydroxylate
(C) includes a base sequence that hybridizes with a complementary strand of the base sequence represented by SEQ ID NO: 13, 14, or 15 under stringent conditions, and has an enzyme activity that hydroxylates the 3-position of the phenol ring of tyrosine DNA encoding a polypeptide

  Here, “1 or (or) several bases” means, for example, 1 to 300, preferably 1 to 150, more preferably 1 to 60, still more preferably 1 to 30, Particularly preferred are 1 to 15 bases. The same applies hereinafter.

  In addition, the production of a base sequence in which one or several bases are deleted, added, inserted, and / or substituted can be obtained by a usual mutation operation such as a method using a mutagen or a site-specific mutation method. it can. These can be easily performed with a commercially available kit such as Diversity PCR Random Mutagenesis Kit (Clontech) or QuickChange Lightening Site-Directed Mutagenesis Kit (Agilent Technologies).

When the above tyrosinase is defined by an amino acid sequence, a polypeptide having the following amino acid sequence (D), (E), or (F) is preferable, and the following (D), (E), or (F) A polypeptide consisting of the amino acid sequence is more preferred.
SEQ ID NO: 16 is an amino acid sequence derived from Bacillus megaterium, SEQ ID NO: 17 is derived from Ralstonia solanacearum, and SEQ ID NO: 18 is derived from Bacillus thuringiensis.

(D) Amino acid sequence represented by SEQ ID NO: 16, 17, or 18 (E) In the amino acid sequence represented by SEQ ID NO: 16, 17, or 18, one or several amino acids are substituted, deleted, and / or added. (F) 60% or more of the amino acid sequence represented by SEQ ID NO: 16, 17, or 18 in the amino acid sequence of the polypeptide having an enzymatic activity that hydroxylates the 3-position of the phenol ring of tyrosine Amino acid sequence of a polypeptide having an amino acid sequence having identity and having an enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine

  Here, “1 or several amino acids” means, for example, 1 to 100, preferably 1 to 50, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably. 1 to 5 amino acids. The same applies hereinafter.

  Further, as described in (F) above, at least 60% or more of the total amino acid sequence shown in SEQ ID NO: 16, 17, or 18 as long as the enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine is retained, preferably May be a protein having a sequence identity of 80% or more, more preferably 90% or more, and still more preferably 95% or more.

As exemplified above, a base sequence based on the genome information of organisms existing in nature, or a homologue thereof, can be used, but each gene has a base sequence according to the codon usage of the host microorganism as necessary. Can be optimized. Codon optimization is described, for example, in J.
There are a method described in Microbiol. Biotechnol. (2012), 22 (3), 316-325 and a method of using Genscript's OptimumGene ^™ service. Codon optimization is preferable because productivity of betaxanthin is improved.

That is, when Escherichia coli is used as a host microorganism, the following bases (G), (H), or (I) obtained by performing codon optimization on the base sequence of SEQ ID NO: 13, 14 or 15 More preferably, it is an enzyme encoded by a sequence.
(G) DNA having the base sequence represented by SEQ ID NO: 1, 2, or 3
(H) a nucleotide sequence represented by SEQ ID NO: 1, 2, or 3 comprising a nucleotide sequence in which one or several bases are substituted, deleted, and / or added, and the 3rd position of the phenol ring of tyrosine DNA encoding a polypeptide having an enzymatic activity to hydroxylate
(I) includes a base sequence that hybridizes under stringent conditions with a complementary strand of the base sequence represented by SEQ ID NO: 1, 2, or 3, and has an enzyme activity that hydroxylates the 3-position of the phenol ring of tyrosine DNA encoding a polypeptide

When yeast is used as a host microorganism, the following (J), (K), or (L) base sequences obtained by performing codon optimization on the base sequences of SEQ ID NOS: 13 and 14 are used. More preferably it is an encoded enzyme.
(J) DNA having the nucleotide sequence represented by SEQ ID NO: 21 or SEQ ID NO: 22
(K) includes a base sequence in which one or several bases are substituted, deleted, and / or added in the base sequences represented by SEQ ID NO: 21 and SEQ ID NO: 22, and the 3rd position of the phenol ring of tyrosine is water DNA encoding a polypeptide having an enzymatic activity to oxidize
(L) a polynucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions with a complementary strand of the nucleotide sequence represented by SEQ ID NO: 21 or SEQ ID NO: 22 and having an enzyme activity that hydroxylates the 3-position of the phenol ring of tyrosine DNA encoding a peptide.

In addition, when the above tyrosinase is defined by an amino acid sequence, it preferably includes the same amino acid sequence as the above (D), (E), (F), and the amino acid of (D), (E), or (F) More preferably, it consists of a sequence. The preferable range of the sequence identity of the amino acid sequence (F) can be considered in the same manner.
In addition, what codon-optimized for E. coli expression with respect to SEQ ID NOS: 13, 14, and 15 are SEQ ID NOS: 1, 2, and 3, respectively, and codons for yeast expression with respect to SEQ ID NOS: 13 and 14 The optimized ones are SEQ ID NOS: 21 and 22, respectively, but these are the same before and after the optimization as translated amino acid sequences. That is, the amino acid sequence defined by the nucleotide sequences of SEQ ID NOs: 1, 13, and 21 is SEQ ID NO: 16, the amino acid sequence defined by the nucleotide sequences of SEQ ID NOs: 2, 14, and 22 is SEQ ID NO: 17, The amino acid sequence defined by the base sequences of 3 and 15 is SEQ ID NO: 18.

(DOPA 4,5-dioxygenase activity)
DOPA4,5-dioxygenase activity has an activity of catalyzing the extradiol cleavage of L-3,4-dihydroxyphenylalanine (DOPA), and converts L-DOPA into 4,5-seco-DOPA. Furthermore, it is an enzyme that converts the L-DOPA into betaramic acid through a subsequent spontaneous reaction. Whether or not it has this enzyme activity depends on whether the protein to be measured causes extradiol cleavage between the 4th and 5th carbons of the phenol ring of L-DOPA, resulting in the formation of betalamic acid. It can be determined by measuring whether it is possible by a usual assay method. For example, the enzyme activity can be confirmed by allowing the enzyme to be measured to act on L-DOPA and directly measuring the amount of betalamic acid converted from L-DOPA.

The DOPA4,5-dioxygenase includes Amanita muscaria, Amaranthus cruentus, Amaranthus tricolor, Beta vulgaris, Celosia argentea, and Quinopodium quinoa.
), Livingstone Daisy (Dorotheanthus bellidiformis), Oiroirobana (Mirabilis jalapa), Prickly Pear (Opuntia ficus), Sennin Cactus (Opuntia stricta)
), Pinula button (Portulaca grandiflora), pokeweed (Phytolacca americana), red beet, spinach (Spinacia oleracea) and the like are preferred.

Among the above, DOPA4,5-dioxygenase is preferably an enzyme encoded by the following base sequence (M), (N), or (O). SEQ ID NO: 19 is a base sequence derived from Osileubana.
(M) DNA having the base sequence represented by SEQ ID NO: 19
(N) a polypeptide comprising a nucleotide sequence in which one or several bases are substituted, deleted, and / or added in the nucleotide sequence represented by SEQ ID NO: 19 and having DOPA4,5-dioxygenase activity DNA to do
(O) DNA comprising a base sequence that hybridizes with a complementary strand of the base sequence represented by SEQ ID NO: 19 under stringent conditions and encoding a polypeptide having DOPA4,5-dioxygenase activity

  Here, “1 or several bases” means, for example, 1 to 300, preferably 1 to 150, more preferably 1 to 60, still more preferably 1 to 30, particularly preferably. 1 to 15 bases. The same applies hereinafter.

In addition, the production of a base sequence in which one or several bases are deleted, added, inserted, and / or substituted can be obtained by a usual mutation operation such as a method using a mutagen or a site-specific mutation method. it can. These include, for example, Diversify PCR Random Muta
It can be easily carried out with commercially available kits such as genesis Kit (Clontech) and QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies).

Further, when the above-mentioned DOPA4,5-dioxygenase is defined by an amino acid sequence, a polypeptide having the following amino acid sequence (P), (Q), or (R) is preferable, and the following (P), (Q) Or a polypeptide comprising the amino acid sequence of (R) is more preferred. SEQ ID NO: 20 is an amino acid sequence derived from Osileubana.
(P) an amino acid sequence represented by SEQ ID NO: 20 (Q) comprising an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence represented by SEQ ID NO: 20, and DOPA4 Amino acid sequence of a polypeptide having 5,5-dioxygenase activity (R) It has an amino acid sequence having 60% or more identity with the amino acid sequence represented by SEQ ID NO: 20, and has DOPA4,5-dioxygenase activity Amino acid sequence of a polypeptide

  Here, “1 or several amino acids” means, for example, 1 to 100, preferably 1 to 50, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably. 1 to 5 amino acids. The same applies hereinafter.

  In addition, as described in (R) above, as long as the DOPA 4,5-dioxygenase activity is maintained, the amino acid sequence shown in SEQ ID NO: 20 is at least 60% or more, preferably 80% or more, more preferably 90% or more. More preferably, it may be a protein having a sequence identity of 95% or more.

  As exemplified above, a base sequence based on the genome information of organisms existing in nature, or a homologue thereof, can be used, but each gene has a base sequence according to the codon usage of the host microorganism as necessary. Can be optimized.

That is, when Escherichia coli is used as a host microorganism, it is encoded by the following base sequence (S), (T), or (U) obtained by performing codon optimization on the base sequence of SEQ ID NO: 19. More preferably, it is an enzyme.
(S) DNA having the base sequence represented by SEQ ID NO: 10
(T) a polypeptide comprising a base sequence in which one or several bases are substituted, deleted, and / or added in the base sequence represented by SEQ ID NO: 10 and having DOPA4,5-dioxygenase activity DNA to do
(U) DNA comprising a base sequence that hybridizes with a complementary strand of the base sequence represented by SEQ ID NO: 10 under stringent conditions and encoding a polypeptide having DOPA4,5-dioxygenase activity

In addition, when yeast is used as a host microorganism, it is encoded by the following nucleotide sequence (V), (W), or (X) obtained by codon optimization for the nucleotide sequence of SEQ ID NO: 19. More preferably, it is an enzyme.
(V) DNA having the base sequence represented by SEQ ID NO: 27
(W) a polypeptide comprising a base sequence in which one or several bases are substituted, deleted and / or added in the base sequence represented by SEQ ID NO: 27 and having DOPA4,5-dioxygenase activity DNA to do
(X) DNA comprising a base sequence that hybridizes under stringent conditions with a complementary strand of the base sequence represented by SEQ ID NO: 27, and encoding a polypeptide having DOPA4,5-dioxygenase activity

Further, when the above-mentioned DOPA4,5-dioxygenase is defined by an amino acid sequence, it preferably has the same amino acid sequence as the above (P), (Q), (R), (P), (Q), or More preferably, it comprises the amino acid sequence (R). The preferable range of the sequence identity of the amino acid sequence (R) can be similarly considered.
Note that SEQ ID NO: 19 was obtained by codon optimization for E. coli expression, and SEQ ID NO: 10 was obtained by codon optimization for yeast expression, and SEQ ID NO: 27. The sequence is the same before and after optimization, and the amino acid sequence defined by any base sequence is SEQ ID NO: 20.

(Method for producing microorganism used in the present invention)
Hereinafter, a method for producing the microorganism used in the present invention (the microorganism of the present invention) will be described. The type of microorganism used as a host microorganism is not particularly limited, but Escherichia coli, Coryneform bacteria, Pseudomonas bacteria, Bacillus bacteria, Rhizobium bacteria, Lactobacillus bacteria, Succinobacillus bacteria, Anaerobiospirillum bacteria, Actino Examples include bacteria belonging to the genus Bacillus, filamentous fungi, yeast and the like. Among these, Escherichia coli, yeast and coryneform bacteria are preferred, Escherichia coli and yeast are more preferred, and Escherichia coli is particularly preferred.

The microorganism of the present invention comprises a DNA encoding an enzyme that hydroxylates the 3-position of the phenol ring of tyrosine (for example, (A), (B), (C), (G), (H), (I) , (J), (K), (L), etc.), and DNA encoding DOPA4,5-dioxygenase (for example, the above (M), (N), (O), (S), (T) , (U), (V), (W), (X), etc.) can be obtained by introducing a plasmid vector or a homologous recombination method into the microorganism.
When introducing the above-described DNA into a host microorganism, the DNA may be introduced on a plasmid vector or the chromosome (genome). When the host microorganism has a gene encoding the enzyme on the chromosome, the microorganism used in the present invention can also be obtained by replacing the promoter of the gene with a strong promoter. When the enzyme gene is not present on the chromosome of the host microorganism, the enzyme gene can be introduced into the chromosome by homologous recombination, or the enzyme gene can be introduced by a plasmid vector.

  When introducing the DNA into a microorganism, an appropriate promoter is preferably incorporated upstream of the 5′-side of the gene, and a terminator is preferably incorporated downstream of the 3′-side of the gene. The promoter and terminator are not particularly limited as long as they are known to function in microorganisms used as hosts, and may be promoters and terminators of the enzyme gene itself or other promoters. And a terminator. Vectors, promoters and terminators that can be used in each of these microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Publishing”.

  In addition, DNA cleavage, ligation, and other methods such as chromosomal DNA preparation, PCR, plasmid DNA preparation, transformation, setting of oligonucleotides used as primers, etc. adopt ordinary methods well known to those skilled in the art. can do. These methods are described in Sambrook, J., Fritsch, EF, and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989) and the like.

  Hereinafter, a method for obtaining a microorganism provided with the activity of the enzyme and / or the enzyme reaction system for each type of microorganism will be described.

(Escherichia coli)
When Escherichia coli is used as a host microorganism, it is preferable to use, for example, K12 strain or B strain as well as these derivatives as the parent strain of Escherichia coli. Examples of parent strains that can be used in the present invention include HB101, DH5α, JM109, JM110, BL21 (DE3), TH2, TOP10, and improved strains thereof, and these strains are commercially available. In addition, for example, the BW25113 strain used in the KEIO collection, which is a single gene disruption strain, can be used, and these can be distributed from the National BioResource Project.

  As a recombination method of E. coli, a recombinant plasmid vector capable of enhancing the expression of the enzyme gene in E. coli is obtained by introducing a DNA fragment containing the target gene or sequence into the plasmid vector. And the like, and the like.

  The plasmid vector into which the target gene or sequence can be introduced into E. coli is not particularly limited as long as it contains at least a gene that controls replication and replication in E. coli. As a plasmid vector, for example, those having a replication origin such as P15A, ColE1 (pBR322, pUC), RSF1030, pSC101, CloDF13 can be preferably used.

  Further, the promoter may be any promoter as long as it functions in E. coli, and may be the promoter of the gene itself to be introduced. For example, trp promoter, trc promoter, PL promoter, T5 promoter, T7 promoter, lac promoter, tac promoter, λPL promoter and the like can be used.

  Plasmid vectors that can be used in the present invention include, for example, pET series (Novagen), Duet Vector series (Novagen), pDUAL series (Stratagen), pMAL series (NEB), pGEX series (GE Healthcare), pKK223 (GE Healthcare) pTrc series (Life Technologies), pUC18, pUC19, pUC118, pUC119 (Takara Bio) and the like.

  Further, in place of or in addition to the above-described recombination method, a part or all of the aforementioned foreign and endogenous genes may be introduced or replaced by a known homologous recombination method. A high expression strain can also be obtained by amplification, in addition, by introducing a mutation into the promoter of the gene introduced onto the chromosome, or by replacing it with a stronger promoter.

(Coryneform bacteria)
When a coryneform bacterium is used as the host microorganism, examples of the coryneform bacterium include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, and bacteria belonging to the genus Arthrobacter. Among them, those belonging to the genus Corynebacterium or Brevibacterium are preferable, and Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes or Brevibacterium Bacteria belonging to Brevibacterium lactofermentum are more preferred.

As parent strains that can be used in the present invention, Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-
1498), Brevibacterium ammoniagenes ATCC6872, Corynebacterium glutamicum ATCC13032, Cocorynebacterium glutamicum AT
CC31831, Brevibacterium lactofermentum ATCC 13869, etc. are mentioned.

Brevibacterium flavum is currently sometimes classified as Corynebacterium glutamicum (Lielbl, W. et al. International Journal of Systematic Bacteriology, 1991, vol. 41, p255-260) In the present invention, Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 are the same strains as Corynebacterium glutamicum MJ-233 and MJ-233 AB-41, respectively. Suppose that In addition, Brevibacterium flavum MJ-233 was released on April 28, 1975 at the Institute of Microbial Industrial Technology, Ministry of International Trade and Industry (currently National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) Deposited as FERMP-3068 at Tsukuba City, Ibaraki Prefecture, Japan, 1st Central, 6), and transferred to the International Deposit under the Budapest Treaty on May 1, 1981, under the accession number of FERM BP-1497 It has been deposited.

  The above microorganisms used as a parent strain in the method of the present invention include not only wild strains but also mutant strains obtained by ordinary mutation treatment such as UV irradiation and NTG treatment, genetic fusion such as cell fusion or gene recombination methods. Any strain such as a recombinant strain induced by a technique may be used.

The target gene or sequence can be introduced into coryneform bacteria by the method shown below.
Specific examples of plasmid vectors that can be used when introducing the gene into a coryneform bacterium using a plasmid vector include, for example, the plasmid pCRY30 described in JP-A-3-210184; JP-A-2-72876 and US Pat. The plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX described in Japanese Patent No. 185,262; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; PHM1519 described in JP-A-58-77895; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; -35197 No. described in Japanese pCG2; those based on pCG4 described in JP 57-183799 publications and pCG11; Plasmid vol.36 (1) p62-66 (1996)
PC2 or a modified plasmid thereof described in 1. above.

  Further, in place of or in addition to the above-described recombination method, a part or all of the aforementioned foreign gene may be introduced, replaced, or amplified by a known homologous recombination method. In addition, a high expression strain can also be obtained by introducing a mutation into the promoter of the gene introduced onto the chromosome, or replacing it with a stronger promoter.

(yeast)
When yeast is used as the host microorganism, the type of yeast used as the parent strain is not particularly limited, but Saccharomyces, Debaryomyces, Schizosaccharomyces, Candida, Yarrowia, Rhodotorula, Lipomyces, Kluyveromyces It is preferable to use yeast belonging to the genus Rhodosporidium, Trichoderma, or Tolropsis, Pichia, etc. as the parent strain. For example, Saccharomyces cerevisiae, Saccharomyces bayanus, Debaryomyces nilssonii, Debaryomyces hansenii, pomacchi glabrata), Candida tropicalis, Candida utilis, Candida Boydny
ida boidinii), Yarrowia lipolytica, Rhodotorula glutinis, Lipomyces lipoferus, Kluyveromyces lactis, Rhodospori, toruloides toruloides・ Resei (Trichoderma
reesei), Torulopsis colliculosa, Pichia farinosa and the like. Of these, the genus Saccharomyces is most preferable because metabolic engineering techniques are easy to use.

  Furthermore, the genus Saccharomyces is preferably an auxotrophic genotype such as his3, leu2, trp1, and ura3. For example, Saccharomyces cerevisiae YPH499 strain (MATa ura3 lys2 ade2 trp1 his3 leu2) (ATCC204679: Americ Cult ect. Alternatively, it can be obtained from STRATAGENE), Saccharomyces cerevisiae FY1679-06c strain (MATα ura3 leu2 trp1 his3) (available from Euroscarf), Saccharomyces cerevisiae BY4741 strain (MATa, his3Δ1, et3Δ0, et. 115-132 (1998), ATCC 2013 8), or the like can also be used.

  The target gene or sequence can be introduced into yeast by the method shown below. As a method for introducing a foreign gene in yeast, a known method can be used, as long as the enzyme targeted for transformation is expressed in the transformant, and the method for introducing the gene is not limited. Examples of the plasmid vector to be used include the three plasmid vectors described later. (I) A multi-copy YEp-type vector using a 2 μm plasmid replication sequence resident in yeast can be used. Specific examples of plasmids that can be used to introduce genes into yeast in the present invention include 2 μm plasmid-derived pAUR112 vector (Takara Bio), pESC vector (Stratagene), pYES vector (Life Technologies), and the like. It is done. These vectors are commercially available. (Ii) A low-copy stable YCp type vector using a self-replicating replication sequence (ARS) and a centromere sequence (CEN) can be used. Examples of plasmid vectors that can be used in the present invention include pAUR123 (Takara Bio). (Iii) A YIp-type plasmid that does not autonomously replicate in yeast for the purpose of chromosome integration can be used. This utilizes the high homologous recombination ability of yeast. A sequence homologous to the chromosomal genome is placed at both ends, and the desired gene is inserted between the selectable marker gene, promoter and terminator in the YIp type. By incorporating it into a plasmid and introducing it into yeast, it can be expected that the foreign gene is incorporated into the chromosome and the foreign gene is stably expressed. This can be constructed using a plasmid for general Escherichia coli or yeast, and can also be produced using PCR or synthetic DNA.

The promoter that can be used in the present invention is not particularly limited as long as it can function in host yeast. For example, the promoter of PGK gene (3-phosphoglycerate kinase 1983, Science, 219, 620-625), TDH gene promoter encoding GAPDH (glyceraldehyde phosphate dehydrogenase 1979, J. Biol. Chem., 254, 9839-9845), TEF1 gene (elongation factor 1 1985, J. Biol. Chem., 260, 3090-3096) ) Promoter, promoter of ADH gene encoding alcohol dehydrogenase, promoter of regulatable CYC1 gene repressed in the presence of glucose (1981, Proc. Natl. Acad. Sci., USA, 78, 2199-2203), PH that can be regulated by thiamine
Examples include the promoter of the O5 gene (1982, EMBO J., 1, 675-680), the promoter of the GAL1 or GAL10 gene induced by galactose in the absence of glucose, and the like.

  In addition, in addition to or in addition to the above-described recombination method, a part or all of a foreign or endogenous gene may be introduced, replaced, or amplified by a known homologous recombination method. In addition, a high expression strain can also be obtained by introducing a mutation into the promoter of the gene introduced onto the chromosome, or replacing it with a stronger promoter.

Note that yeast transformation can be performed by, for example, electroporation (Fromm ME, et al. Nature 1986, 319 (6056): 791-793), spheroplast method, lithium acetate method (Ito, H. et al, (1983) J. Bacteriol. 153 (1), 163-168), and two or more kinds of plasmids can be introduced. In addition, gene expression in yeast can be enhanced by making multiple copies of the gene on the host genome by a known homologous recombination method. For example, a method using a vector in which multiple copies of a genome integration plasmid are integrated into the genome (Bio / Technol. 1991, 9, 1382-1385) can be mentioned. The place where the expression unit containing each gene on the genome is inserted may be any place as long as expression of crotyl alcohol or its isomer synthesis-related gene or growth-related gene is not inhibited.

[Production method of the present invention]
The production method of the present invention comprises an enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine, and a microorganism having the activity of DOPA4,5-dioxygenase (microorganism of the present invention) or a processed product thereof in an aqueous medium. It has the process (conversion process) which converts a raw material into betaxanthin.

It is preferable to have a step (cultivation step) of culturing the microorganism of the present invention in an aqueous medium before the conversion step, and to have a step of recovering betaxanthin from the aqueous medium after the conversion step. Is preferred.
Hereinafter, the manufacturing method of this invention is demonstrated for every process.

(material)
As the raw material, tyrosine and / or a carbohydrate raw material can be used. Here, as tyrosine, a commercially available product having a purity of 90% or more can be suitably used.
The carbohydrate raw materials include carbohydrates such as glucose, galactose, fructose, xylose, sucrose, maltose, lactose, raffinose, saccharose, starch and cellulose, and fermentable carbohydrates such as polyalcohols such as glycerin, mannitol, ribitol and xylitol. Among them, glucose is preferable.

When the metabolic flux for synthesizing tyrosine from a carbohydrate raw material by the microorganism used in the production method of the present invention is not sufficiently strong, it is preferable to use a carbohydrate raw material and tyrosine as raw materials, and particularly preferably glucose and tyrosine.
On the other hand, when the microorganism used in the production method of the present invention has a strong metabolic flux for synthesizing tyrosine from a carbohydrate raw material, only the carbohydrate raw material can be used as the raw material. In order to reduce the cost of the production process of target betaxanthin, it is preferable to use only a saccharide raw material that is a cheaper raw material.

Moreover, in addition to the raw material mentioned above, amino acids other than tyrosine and amines can be added according to the structure of betaxanthin to be produced.
When it is desired to produce the above-mentioned betaxanthin of the formula (1), it is preferable to add an amino acid having the desired R ₁ and R ₂ , and when it is desired to produce the betaxanthin of the formula (2), the desired R ₃ It is preferred to add an amine having

The amino acid other than tyrosine may be a natural amino acid or a non-natural amino acid.
Examples of natural amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. When these are added as amino acids other than tyrosine, as betaxanthine, alanine-betaxanthin, arginine-betaxanthin, asparagine-betaxanthin, aspartic acid-betaxanthin, cysteine-betaxanthine, glutamine-betaxanthine, glutamic acid-beta Xanthine, glycine-betaxanthin, histidine-betaxanthin, isoleucine-betaxanthine, leucine-betaxanthine, Down - solid xanthine, methionine - solid xanthine, phenylalanine - solid xanthine, proline - solid xanthine, serine - solid xanthine, threonine - solid xanthine, tryptophan - solid xanthine, tyrosine - solid xanthine, valine - solid xanthine is manufactured.

  Examples of non-natural amino acids include m-tyrosine, methoxyphenylalanine, methylcysteine, allylalanine, propargylalanine, allylglycine, propargylglycine, ethylalanine, methylaspartic acid, methylcysteine, methylleucine, methylphenylalanine, methyltryptophan, Methyltyrosine, methylvaline, α-methyl-2-bromophenylalanine, α-methyl-3-bromophenylalanine, α-methyl-4-bromophenylalanine, α-methyl-2-iodophenylalanine, α-methyl-3-iodophenylalanine, α-methyl-4-iodophenylalanine, α-methyl-2-nitrophenylalanine, α-methyl-3-nitrophenylalanine, α-methyl-4-nitrophenylalani , Alpha-methyl-.beta.-(4-bisphenyl) alanine and the like. When these are added as amino acids other than tyrosine, m-tyrosine-betaxanthin, methoxyphenylalanine-betaxanthin, methylcysteine-betaxanthin, allylalanine-betaxanthine, propargylalanine-betaxanthin, allylglycine- Betaxanthine, propargylglycine-betaxanthin, ethylalanine-betaxanthine, methylaspartate-betaxanthine, methylcysteine-betaxanthine, methylleucine-betaxanthine, methylphenylalanine-betaxanthine, methyltryptophan-betaxanthine, methyltyrosine-beta Xanthine, methyl valine-betaxanthin, α-methyl-2-bromophenylalanine-betaxanthine, α-methyl -3-Bromophenylalanine-betaxanthine, α-methyl-4-bromophenylalanine-betaxanthine, α-methyl-2-iodophenylalanine-betaxanthine, α-methyl-3-iodophenylalanine-betaxanthine, α-methyl-4 -Iodophenylalanine-betaxanthine, α-methyl-2-nitrophenylalanine-betaxanthine, α-methyl-3-nitrophenylalanine-betaxanthine, α-methyl-4-nitrophenylalanine-betaxanthine, α-methyl-β- ( 4-Bisphenyl) alanine-betaxanthin is produced.

The amine may be a natural amine or a non-natural amine.
Examples of natural amines include methylamine, ethanolamine, piperidine, histamine, dopamine, phenethylamine, tyramine, 3-methoxytyramine, γ-aminobutyric acid, noradrenaline, serotonin, muscimol, spermidine, spermine and the like. When these are added as amines, methylamine-betaxanthin, ethanolamine-betaxanthin, piperidine-betaxanthin, histamine-betaxanthine, dopamine-betaxanthine, phenethylamine-betaxanthine, tyramine-betaxanthine, 3-methoxytyramine-betaxanthin, γ-aminobutyric acid-betaxanthin, noradrenaline-betaxanthin, serotonin-betaxanthin, muscimol-betaxanthin, spermidine-betaxanthin, spermine-betaxanthin are produced.

  Non-natural amines include, for example, ethylamine, piperazine, morpholine, ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethyleneexamine, hexamethylenediamine, 1,3-butadiene-1-amine, 1,3, 5-hexatriene-1-amine, aniline, 4-biphenylamine, amantadine and the like can be mentioned. When these are added as amines, as betaxanthin, ethylamine-betaxanthin, piperazine-betaxanthine, morpholine-betaxanthine, ethylenediamine-betaxanthine, diethylenetriamine-betaxanthine, triethylenetetramine-betaxanthine, tetraethylenepentamine-, respectively. Betaxanthine, pentaethyleneexamine-betaxanthine, hexamethylenediamine-betaxanthine, 1,3-butadiene-1-amine-betaxanthine, 1,3,5-hexatriene-1-amine-betaxanthine, aniline-beta Xanthine, 4-biphenylamine-betaxanthine, amantadine-betaxanthin are produced.

(Culture process)
The culture process of the present invention is a process of culturing the microorganism of the present invention obtained as described above in an aqueous medium. Since this step is performed for culturing and proliferating the microorganism of the present invention or adjusting the state of the microorganism of the present invention, in this step, among the above-mentioned raw materials, the microorganism of the present invention is usually used. Only the raw material necessary for the growth of the plant, specifically, only the saccharide raw material (preferably glucose) is used.
However, when an enzyme necessary for producing betaxanthin is placed under some inducible promoter, it is necessary to add an inducing agent appropriately selected according to the type of the promoter during the culture process.
When a sufficient amount of the microorganism of the present invention is present, this step can be omitted and the next conversion step can be performed.

There is no restriction | limiting in particular in the aqueous medium (medium) used by this invention, Although it can set suitably according to the kind of microorganisms to be used, For example, it can carry out on the following conditions.
As the aqueous medium, a normal medium containing a carbon source, a nitrogen source, an inorganic salt, and other organic micronutrients as necessary can be used. However, a medium containing a carbon source described later, water, phosphate, etc. , Carbonates, acetates, borates, citrates, tris buffers, alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, amides such as acetamide, etc. .

The carbon source is not particularly limited as long as it is a carbon source that can be assimilated by the above-mentioned microorganisms. Fermentable carbohydrates such as polyalcohols such as ribitol are used.
As the nitrogen source, for example, peptone, meat extract, yeast extract, corn steep liquor and the like are used in addition to ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate.

As the inorganic salt, for example, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like are used.
In addition, vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, amino acids, and other factors that promote growth may be added as necessary.

The culture conditions are not particularly limited as long as the microorganisms to be used can be grown. Usually, the culture conditions can be carried out at a culture temperature of 10 ° C. to 45 ° C. for 12 hours to 96 hours. The culture is preferably performed under such a condition that the production of melanin as a by-product is reduced. When the microorganism of the present invention is Escherichia coli, the temperature of the aqueous medium is
It is preferably 16 ° C. or higher, more preferably 20 ° C. or higher, and preferably 29 ° C. or lower, more preferably 25 ° C. or lower.

(Conversion process)
The conversion step of the present invention is a step of converting a raw material into betaxanthin in an aqueous medium in the presence of the microorganism of the present invention or a processed product thereof. The purpose of this step is to convert the raw material into betaxanthin. As the raw material, the above-mentioned carbohydrate raw material and / or tyrosine and, if necessary, an amino acid other than tyrosine are used. .

  Moreover, it is also preferable to use the microbial cell of the microorganism of the present invention cultured in the above-described culturing step as a resting microbial cell and use the quiescent cell as the microorganism of the present invention in this step. Examples of the method of making the microbial cells of the present invention resting microbial cells include a method for recovering microbial cells from the culture solution obtained in the culturing step by centrifugation, etc., water, or a carbon source necessary for the growth of the microorganisms. And a method of recovering bacterial cells by washing with a buffer that does not contain a nitrogen source.

  In addition, when the microorganisms of this invention have all the metabolic pathways which synthesize | combine betaxanthin from saccharide raw materials, you may implement this process continuously, without distinguishing in particular from the above-mentioned culture | cultivation process.

  There is no restriction | limiting in particular in the aqueous medium (medium) to be used, According to the kind of microorganisms to be used, it can set suitably, The thing similar to what was described in the said culture | cultivation process can be used. In this step, the aqueous medium preferably contains at least one selected from the group consisting of ascorbic acid, copper and iron, and more preferably contains ascorbic acid, copper and iron.

  The reaction is not particularly limited as long as the microorganism used can grow, or the enzyme introduced for producing the betalain pigment does not completely lose the enzyme activity. Usually, the reaction temperature is 10 ° C to 45 ° C. The reaction is preferably carried out for 12 hours to 96 hours under such conditions that the enzyme activity is high and the production of melanin as a by-product is reduced. When the microorganism of the present invention is Escherichia coli, the production of betaxanthin is carried out when the temperature of the aqueous medium is preferably 16 ° C or higher, more preferably 20 ° C or higher, and preferably 29 ° C or lower, more preferably 25 ° C or lower. It tends to improve efficiency, which is preferable.

(Recovery process)
It is preferable to have the process of collect | recovering betaxanthins from an aqueous medium as needed after the above-mentioned conversion process.
It can be recovered from an aqueous medium or cells by well-known methods such as extraction using an organic solvent, distillation, column chromatography and the like, and can be further purified as necessary.

[Betaxanthin]
In the production method of the present invention, if any non-natural amino acid is used as a raw material as well as known betaxanthin, betaxanthin to which the non-natural amino acid is added can be produced. Examples of betaxanthin to which such an unnatural amino acid is added include those represented by the following formula (3).

  In Formula (3), R is 1 type selected from the group which consists of a C1-C40 linear or branched or cyclic saturated or unsaturated hydrocarbon group. However, some or all of the hydrogen atoms of the hydrocarbon group are halogen atoms, acyl groups, amide groups, amino groups, alkoxy groups, aldehyde groups, imidazole groups, indole groups, oxo groups, carboxyl groups, guanidino groups, cyano groups. May be substituted with at least one selected from the group consisting of a sulfo group, a thiol group, a nitro group, a hydroxyl group and a phenyl group, and the hydrocarbon group forms a ring in a form containing nitrogen of iminium. It may be. Furthermore, some or all of the aromatic rings of the imidazole group, the indole group, and the phenyl group may be substituted with at least one selected from the same substituent group as described above. Furthermore, some of the carbon atoms of the hydrocarbon group may be substituted with at least one selected from a sulfur atom, an oxygen atom, a nitrogen atom, and a phosphorus atom.

  In Formula (3), when R is a linear or branched alkyl group, the carbon number is preferably 1 to 10, and more preferably 1 to 5. In this case, it is particularly preferable that R is at least one selected from the group consisting of an ethyl group, a propyl group, and an isobutyl group.

  In Formula (3), when R is a linear or branched alkenyl group, the number of carbon atoms is preferably 1 to 10, and more preferably 1 to 5. In this case, it is particularly preferable that R is at least one selected from the group consisting of an allyl group, a 1-butenyl group, and a 2-butenyl group.

  In the formula (3), when R is a linear or branched alkynyl group, the carbon number thereof is preferably 1 to 10, and more preferably 1 to 5. In this case, it is particularly preferable that R is at least one selected from the group consisting of an ethynyl group, a 1-butynyl group, and a 2-butynyl group.

  In Formula (3), when R is a linear or branched aryl group, the carbon number thereof is preferably 1-20, and more preferably 1-10. In this case, R is particularly preferably at least one selected from the group consisting of a benzyl group, a tolyl group, and a phenesyl group.

The compound represented by the formula (3) can be suitably used as an antioxidant or the like by taking advantage of its strong antioxidant activity.
Moreover, among the compounds represented by the formula (3), for example, a compound represented by the following formula (4) is preferable.

  Similarly, by using a non-natural amine as a raw material, betaxanthin to which a non-natural amine is added can be produced. As such a betaxanthin to which an unnatural amine is added, those having a plurality of conjugated structures in the amine skeleton are preferable. For example, those represented by the following formulas (5) and (6) are used. Can be mentioned.

  In the formula (5), n is preferably 1 to 20, and more preferably 1 to 10.

  In Formula (6), n is preferably 1 to 5, and more preferably 1 to 3.

  The compounds represented by the above formulas (5) and (6) can be suitably used as an antioxidant or the like by taking advantage of their strong antioxidant activity.

  EXAMPLES Next, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded.

[Production Example 1] Production of Escherichia coli having tyrosinase activity and DOPA 4,5-dioxygenase activity (1) Construction of tyrosinase expression plasmid for E. coli
The gene sequence information of three types of tyrosinases derived from Bacillus megaterium DSM 319, Ralstonia solanacearum GMI1000, and Bacillus thuringiensis BMB171 (hereinafter, these three types of tyrosinases are referred to as BMTYR, RSTYR, and BTTYR, respectively). NCBI) and optimized for E. coli codon frequency by gene synthesis. The entire gene sequences of the obtained optimized gene fragments are shown in SEQ ID NOs: 1 to 3, respectively. A plasmid was prepared by inserting the gene fragments of SEQ ID NOs: 1 to 3 into the EcoRV site of the pUC57 vector (GenScript). Using the resulting plasmid as a template, each gene fragment was obtained by PCR using the following primers with a restriction enzyme NdeI site added to the 5 ′ end and a restriction enzyme KpnI site added to the 3 ′ end.
BMTYR gene amplification primers: SEQ ID NOs: 4, 5
RSTYR gene amplification primers: SEQ ID NOS: 6 and 7
BTTYR gene amplification primer: SEQ ID NOS: 8 and 9
PCR reaction solution composition: template ^{DNA 2μl, PrimeSTAR (R) HS} DNA
Polymerase (manufactured by Takara Bio Inc.) 0.5 μl, 5 × PrimeStar Buffer 10 μl, 2.5 mM dNTP Mixture 4 μl, 2 μM each primer 5 μl, sterilized water 24 μl (the total volume was 50 μl)
Reaction temperature conditions: Cycle consisting of TaKaRa PCR Thermal Cycler Dice Touch (type TP350), 10 seconds at 98 ° C, 5 seconds at 55 ° C, 1 minute at 72 ° C (BMTYR and BTTYR), 1 minute 30 seconds (RSTYR) Was repeated 30 times. However, the heat retention at 98 ° C. in the first cycle was 1 minute and 10 seconds.

Confirmation of PCR amplification products is performed by separation by gel electrophoresis with 1% agarose (Agarose-RE: Nacalai Tesque), followed by visualization by ethidium bromide staining. 0.5 kb, BTTYR detected a 0.8 kb fragment. Recovery of the target fragment from the gel is performed by Wizard ^(R) SV Gel and
This was performed using PCR Clean-UP System (manufactured by Promega). The PCR fragment obtained tyrosinase, respectively, after the connection to the ^{pCR TM -Blunt II-TOPO (R} ) vector (manufactured by Invitorgen) using ^{Zero Blunt (R) TOPO (R} ) PCR Cloning Kit ( manufactured by Invitorge), Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml kanamycin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 50 μg / ml kanamycin, and then a plasmid was obtained from the obtained bacterial cells using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNAs were named BMTYR / pCR-bluntII, RSTYR / pCR-bluntII, and BTTYR / pCR-bluntII, respectively.

Next, the above-mentioned BMTYR / pCR-bluntII, RSTYR / pCR-bluntII, BTTYR / pCR-bluntII were each treated with NdeI and KpnI, followed by 1% agarose (Agarose-RE: Nacalai Tesque) gel Separated by electrophoresis. The isolated about 0.9 kb gene fragment derived from BMTYR, about 1.5 kb gene fragment derived from RSTYR, and about 0.8 kb gene fragment derived from BTTYR
Zard ^(R) SV Gel and PCR was collected using a Clean-UP System (Promega).
The expression vector pETDuet-1 (manufactured by Novagen) for E. coli was also treated with restriction enzyme and separated in the same manner, and a gene fragment of about 5.4 kb was recovered.

Next, the respective tyrosinase gene fragments obtained above and the E. coli expression vector pETDuet-1 were ligated to High. 2 (manufactured by TOYOBO). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA.
The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 100 μg / ml ampicillin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 100 μg / ml ampicillin, and then the resulting cells were used to generate a plasmid using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNAs were named BMTYR / pETDuet-1, RSTYR / pETDuet-1, and BTTYR / pETDuet-1, respectively.

(2) Construction of DOPA 4,5-dioxygenase (DOD) expression plasmid for E. coli DOPA 4,5-dioxygenase (hereinafter referred to as “MjDOD”) derived from Mirabilis jalapa One type of gene sequence information is NCBI. And optimized to the codon frequency of E. coli by gene synthesis. The entire gene sequence of the optimized MjDOD is shown in SEQ ID NO: 10. The sequence described in SEQ ID NO: 10 was inserted into the EcoRV site of the pUC57 vector (GenScript). Using the resulting plasmid as a template, a gene fragment was obtained by PCR using a primer (SEQ ID NO: 11 and 12) with a restriction enzyme NdeI site added to the 5 ′ end and a restriction enzyme KpnI site added to the 3 ′ end.
PCR reaction solution composition: template ^{DNA 2μl, PrimeSTAR (R) HS} DNA
Polymerase (manufactured by Takara Bio Inc.) 0.5 μl, 5 × PrimeStar Buffer 10 μl, 2.5 mM dNTP Mixture 4 μl, 2 μM each primer 5 μl, sterilized water 24 μl (the total volume was 50 μl)
Reaction temperature conditions: TaKaRa PCR Thermal Cycler Dice Touch (type TP350) was used, and a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 98 ° C. in the first cycle was 1 minute and 10 seconds.

Confirmation of the amplification product and recovery of the target fragment from the gel are carried out in the same manner as described in “(1) Construction of Tyrosinase Expression Plasmid for Escherichia coli”, and an approximately 0.8 kb MjDOD-derived gene fragment is detected. Recovered.
The obtained MjDOD PCR fragment was ligated to pCR ^™ -Blunt II-TOPO ^(R) vector (Invitrogen ⁾ using Zero Blunt ^(R) TOPO ^(R) PCR Cloning Kit (Invitrogen). Escherichia coli (DH5α strain) was transformed with the plasmid DNA.
The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml kanamycin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 50 μg / ml kanamycin, and then a plasmid was obtained from the obtained bacterial cells using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The plasmid DNA obtained here was named MjDOD / pCR-bluntII.

Next, the MjDOD / pCR-bluntII was treated with restriction enzymes with NdeI and KpnI, and then separated by 1% agarose gel electrophoresis. The approximately 0.8 kb gene fragment was obtained from Wizard ^(R) SV Gel and PCR Clean-UP. System
It was collected using (manufactured by Promega).
pRSFDuet-1 (manufactured by Novagen), an Escherichia coli expression vector that can coexist with the pETDuet-1 vector, was also treated with NdeI and KpnI and then separated by the same method, and a gene fragment of about 3.8 kb was recovered. .
Ligation high Ver. 2 (manufactured by TOYOBO) was used to link the MjDOD gene fragment and the E. coli expression vector pRSFDuet-1. Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA.

The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml kanamycin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 50 μg / ml kanamycin, and then a plasmid was obtained from the obtained bacterial cells using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNA was designated as MjDOD / pRSFDuet-1.

(3) Preparation of tyrosinase and DOPA 4,5-dioxygenase co-expression Escherichia coli strains BMTYR / pETDuet-1 and MjDOD / pRSFDuet-1, RSTYR / pETDuet-1 and MjDOD / prepared in (1) and (2) above Escherichia coli (BL21 Star ^™ (DE3) strain) was transformed with mixed plasmids of pRSFDuet-1, BTTYR / pETDuet-1 and MjDOD / pRSFDuet-1, respectively. The obtained recombinant Escherichia coli was smeared on an LB agar medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin. Each of the clones that formed colonies on this medium was E. coli. E. coli (BL21) / BMTYR / MjDOD, E. coli. E. coli (BL21) / RSTYR / MjDOD, E. coli. E. coli (BL21) / BTTYR / MjDOD and used as a betaxanthin producing strain in the following betaxanthin production test.

[Example 1]
First, the microorganism prepared above was cultured by the following method.
(Pre-culture process)
The three co-expression strains E. E. coli (BL21) / BMTYR / MjDOD, E. coli. E. coli (BL21) / RSTYR / MjDOD, E. coli. E. coli (BL21) / BTTYR / MjDOD were added to the preculture medium [5 × M9 Minimal Salts, respectively.
400 μl, 1 M CaCl ₂ 0.2 μl, 1 M MgSO _{4 4} μl, 100 g / L Casamino acid 200 μl, 1 M Glucose 44 μl, 1%
Thiamine ₄ μl, 50 mM FeSO ₄ 2 μl, 40 mg / ml CuSO ₄ 2 μl, 50 mg / ml Kanamicin 2 μl, and 100 mg / ml
Carbicillin 2 μl dissolved in sterilized water to a total volume of 2 ml] was inoculated into 2 ml and cultured at a culture temperature of 32 ° C. and 180 rpm for 18 hours.

(Main culture process)
500 μl of the preculture solution obtained above was added to the main culture medium [5 × M9 Minimal Salts 10 ml, 1 M CaCl ₂ 5 μl, 1 M MgSO ₄ 100 μl, 40 g / L NH ₄ Cl 3.75 ml, 100 g / L Casamino acid 5 ml, 1 _{% Thiamine 100μl, 50mM FeSO 4 50μl} , 40mg / ml CuSO 4 50μl, 50mg / ml Kanamycin 50μl, 100mg / ml Carbenicillin 50μl, Overnight Express TM Autoinduction Systems1 OnEx Solution
1 1 ml, OnEx Solution 2 2.5 ml, and OnEx Solution 3 50 μl dissolved in sterilized water to a total volume of 50 ml] were inoculated into 50 ml, and cultured at 20 ° C. and 200 rpm for 24 hours. Expression was induced.

(Conversion process)
To the main culture obtained above, L-tyrosine (final concentration: 1 mM) and 500 μl of 1M ascorbic acid were added to start the production reaction of betaxanthin, followed by culturing at a reaction temperature of 20 ° C. and 200 rpm for 24 hours. Xanthine was produced. The supernatant obtained by centrifugation (12000 rpm, 1 minute) from 1 ml of the obtained culture solution was passed through a 0.45 μm Cosmo spin filter (manufactured by Japan Millipore Corporation), and the filtrate was used as an analysis sample.

(analysis)
Analysis was performed using Agilent Technologies 6460 Triple Quad LC / MS. Column is SunFire ^™ C18 3.5μm
A 2.1 x 150 mm Column (Waters) was used. For elution, 1% formic acid (Solvent A) and 80% acetonitrile (Solvent B) were used, and the mixing ratio of Solvent B was 0% (0 min) → 0% (2 min) → 20% (20 min) → 100% (24 min) → 100% (26 minutes) Increase over time (flow rate 0.3 ml / min, temperature 40 ° C.). Betaxanthin was detected by monitoring m / z and UV absorption at 470 nm.

(Analysis result)
The obtained LC / MS analysis results (total ion chromatogram (TIC)) are shown in FIG. 1 (E. coli (BL21) / BMTYR / MjDOD) and FIG. 2 (E. coli (BL21) / BTTYR /) for each co-expression strain. MjDOD) and shown in FIG. 3 (E. coli (BL21) / RSTYR / MjDOD). In addition, the time-dependent changes in the signal area value of the molecular ion peak of the produced betaxanthin are shown in Table 1 (E. coli (BL21) / BMTYR / MjDOD) and Table 2 (E. coli (BL21) / BTTYR /) for each co-expression strain. MjDOD), shown in Table 3 (E. coli (BL21) / RSTYR / MjDOD).

As a result of the analysis, E.I. E. coli (BL21) / BMTYR / MjDOD, E. coli. E. coli (BL21) / RSTYR / MjDOD, E. coli. In any of the co-expression strains of E. coli (BL21) / BTTYR / MjDOD, the production of several types of betaxanthins and betalamic acid which is a biosynthetic precursor was detected. In any of the co-expression strains, the production amount of valine-betaxanthin was the highest, followed by isoleucine-betaxanthin, leucine-betaxanthin, and phenylalanine-betaxanthin in that order.
Among the above three co-expression strains, E. coli which is a tyrosinase expression strain derived from Ralstonia solanacearum GMI1000, which is known to have low oxidation activity from L-DOPA to dopaquinone. In E. coli (BL21) / RSTYR / MjDOD, the production amount of betaxanthines was the highest, and yellow coloration derived from the present pigment was strongly confirmed.
From the above, it was shown that betaxanthin can be efficiently produced by introducing tyrosinase and DOPA 4,5-dioxygenase into a microorganism.

[Examples 2 to 8]
Examples of producing betaxanthin added with natural amino acids using resting cells are shown below.
(Pre-culture process)
The E.M. produced in Production Example 1 E. coli (BL21) / RSTYR / MjDOD was inoculated into 2 ml of a preculture medium (the composition was the same as in Example 1), and cultured at a culture temperature of 32 ° C. and 180 rpm for 18 hours.

(Main culture process)
The obtained preculture solution (500 μl) was inoculated into 50 ml of a main culture medium (the composition was the same as in Example 1), and the protein expression was induced by culturing at 200 rpm for 24 hours at a culture temperature of 20 ° C. .

(Conversion process)
The cells obtained by centrifugation (6000 rpm, 5 minutes) of 10 ml of the main culture obtained by the above method were once washed with 2 ml of 50 mM potassium phosphate buffer (pH 7.0), and then centrifuged again. The cells were collected. The collected bacterial cells were treated with a reaction buffer [potassium phosphate buffer (pH 7.0) containing 0.1 mM IPTG, 50 μM FeSO ₄ , 4 μg / ml CuSO ₄ , 10 mM ascorbic acid, 50 μg / ml Kanamicin, 100 μg / ml Carbenicillin]. Suspended in 2 ml.
In addition to L-tyrosine serving as a substrate for betaramic acid, L-valine (Example 2), L-phenylalanine (Example 3), L-tryptophan (Example 4), L-leucine were added to the resulting suspension. (Example 5), L-isoleucine (Example 6), L-histidine (Example 7), and L-methionine (Example 8) were added. The final concentration of these natural amino acids in the suspension was 1 mM. Then, it was made to react for 24 hours on the conditions of reaction temperature 20 degreeC and 200 rpm. The supernatant obtained by centrifugation (12000 rpm, 1 minute) from 1 ml of the obtained reaction solution was passed through a 0.45 μm Cosmo spin filter (manufactured by Nihon Millipore), and the filtrate was used as an analysis sample. The analysis was performed in the same manner as in Example 1.

(Analysis result)
FIG. 4 shows the LC / MS analysis results of the reaction solution obtained in Example 2 (L-valine). In FIG. 4, when valine was added in addition to tyrosine, which is a synthetic substrate for betaramic acid, a molecular ion peak of valine-betaxanthin ([M + H] ⁺ = 311) having UV absorption at 470 nm derived from betaxanthin was detected. .

Further, from the graph of the change over time in the production amount up to 24 hours after the start of the reaction (FIG. 5), it can be seen that the production amount of valine-betaxanthin increases with time. In Example 2, the amount of amino acid-derived betaxanthin other than the added valine was very small. Therefore, if resting cells are used, it is thought that single production of betaxanthin to which the target amino acid is added is possible.
Further, the reaction solutions obtained in Examples 3 to 8 were also analyzed by the same method as in Example 2, and the results are shown in the following table.

From Table 4, it can be seen that when L-valine, L-phenylalanine, L-tryptophan, L-leucine, L-isoleucine, L-histidine, and L-methionine are added, betaxanthin added with the added amino acid can be produced. .
The amino acids added in Examples 2 to 8 have relatively high hydrophobicity. It was suggested that hydrophobic amino acids tend to condense due to betalamic acid and imine bonds, and tend to produce betaxanthin.
In addition, although not listed in Table 4, the production | generation of betaxanthin is confirmed also about L-lysine and L-proline, According to this invention, the production of these betaxanthins is also possible. From these Examples 2 to 8, it can be seen that by adding a single amino acid in addition to L-tyrosine during the resting cell reaction, it is possible to produce almost single betaxanthin added by the added amino acid. .

[Examples 9 to 18]
An example in which betaxanthin added with an unnatural amino acid was produced using resting cells is shown below.
(Pre-culture process, main culture process, betaxanthin production process)
In the (conversion step) of Examples 2 to 8, instead of natural amino acids, (S) -α-propargylalanine (Example 9) and (S) -α- Methylvaline (Example 10), (S) -α-allylalanine (Example 11), (S) -α-methyltryptophan (Example 12), (S) -α-methyl-3-iodophenylalanine (Example) 13), (S) -α-methyl-2-bromophenylalanine (Example 14), (S) -α-ethylalanine (Example 15), (S) -α-methylleucine (Example 16), S) -α-methyl-2-nitrophenylalanine (Example 17) and (S) -α-methylphenylalanine (Example 18) were added under the same conditions as in Example 2 except that the preculture step, Performs main culture process and conversion process became. The non-natural amino acid used here was manufactured by Nagase Sangyo Co., Ltd., and had a purity of ≧ 98% and ≧ 98% ee.

(Analysis result)
FIG. 6 shows the LC / MS analysis results of the reaction solution obtained in Example 9 ((S) -α-propargylalanine). In FIG. 6, when a non-natural amino acid is added in addition to tyrosine, which is a synthetic substrate for betaramic acid, a molecular ion peak derived from propargylalanine-betaxanthin having a UV absorption at 470 nm derived from betaxanthine ([M + H] ⁺ = 321) was detected. This confirmed the production of the desired non-natural betaxanthin pigment. Moreover, it was confirmed from the graph (FIG. 7) of the time course of the production amount up to 24 hours of reaction that the production amount of propargylalanine-betaxanthin increased with time.
Further, Examples 10 to 18 were also analyzed in the same manner, and the results are shown in the following table.

In Example 10, (S) -α-methylvaline-betaxanthin, in Example 11, (S) -α-allylalanine-betaxanthin, in Example 12, (S) -α-methyltryptophan- Betaxanthin, (S) -α-methyl-3-iodophenylalanine-betaxanthin in Example 13, and (S) -α-methyl-2-bromophenylalanine-betaxanthin in Example 14, Example 15 (S) -α-ethylalanine-betaxanthin in Example 16, (S) -α-methylleucine-betaxanthin in Example 16, and (S) -α-methyl-2-nitro in Example 17. Phenylalanine-betaxanthine, (Example 18) produced (S) -α-methylphenylalanine-betaxanthin. And it can be seen. It can be seen from Examples 9 to 18 that a novel non-natural betaxanthin can be produced by adding a single non-natural amino acid in addition to L-tyrosine during the resting cell reaction.

[Production Example 2] Production of tyrosine-producing Escherichia coli having tyrosinase activity and DOPA 4,5-dioxygenase activity (1) Production of tyrosine-producing Escherichia coli Tyrosine-producing Escherichia coli is a transcriptional regulatory factor for genes involved in aromatic amino acid biosynthesis. The TyrR gene was disrupted, and chorismate mutase / prephenate dehydrogenase (TyrA ^fbr ) resistant to tyrosine feedback inhibition, and DAHP synthase (AroG ^fbr ) resistant to phenylalanine feedback inhibition were bred.

According to the protocol of Zhang et al., “Improved RecT or RecET cloning and subcloning method (2001)”, a tyrR gene-deficient strain of E. coli (BL21 Star ^™ (DE3) strain) was obtained. This strain is hereinafter referred to as E. coli. E. coli BL21 Star ^™ (DE3) ΔtyrR :: Kan.
Feedback resistant chorismate mutase / prephenate dehydrogenase (TyrA ^fbr ) obtained the gene sequence information of wild-type enzyme TyrA derived from E. coli from National Center for Biotechnology Information (NCBI), and the methionine of the 53rd amino acid sequence is isoleucine and 354th. Was obtained by gene synthesis in the form of alanine converted to valine (SEQ ID NO: 30). A plasmid was prepared by inserting the gene fragment described in SEQ ID NO: 30 into the EcoRV site of the pUC57 vector (GenScript). Using the resulting plasmid as a template, a gene fragment was obtained by PCR using the following primers with a restriction enzyme NdeI site added to the 5 ′ end and a restriction enzyme KpnI site added to the 3 ′ end.

TyrA ^fbr gene amplification primer: SEQ ID NOS: 31 and 32
The feedback resistant DAHP synthase (AroG ^fbr ) is obtained by obtaining the gene sequence information of the wild-type enzyme AroG derived from E. coli from National Center for Biotechnology Information (NCBI) and converting the aspartic acid at the 146th amino acid sequence into asparagine. Obtained by synthesis (SEQ ID NO: 33). A plasmid inserted into the EcoRV site of the pUC57 vector (GenScript) was prepared. Using the resulting plasmid as a template, a gene fragment was obtained by PCR using the following primers with a restriction enzyme EcoRI site added to the 5 ′ end and a restriction enzyme HindIII added to the 3 ′ end.

AroG ^fbr gene amplification primer: SEQ ID NOs: 34 and 35
PCR reaction solution composition: template ^{DNA 2μl, PrimeSTAR (R) HS} DNA Polymerase ( Takara Bio Inc.) 0.5μl, 5 × PrimeSTAR Buffer 10μl , 2.5mM dNTP Mixture 4μl, 2μM each primer 5 [mu] l, sterile water 24 [mu] l (these To make a total volume of 50 μl.)
Reaction temperature conditions: Using a TaKaRa PCR Thermal Cycler Dice Touch (type TP350), a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 98 ° C. in the first cycle was 1 minute and 10 seconds.

Confirmation of PCR amplification products was performed by separating by gel electrophoresis with 1% agarose (Agarose-RE: Nacalai Tesque) and then visualized by ethidium bromide staining. Both TyrA ^fb and AroG ^fbr were about 1.0 kb. Fragments were detected. Recovery of the desired fragment from the gel was performed using ^{Wizard (R) SV Gel and PCR} Clean-UP System ( manufactured by Promega).
The obtained PCR fragments of TyrA ^fbr and AroG ^fbr were respectively obtained as Zero.
After ligation to pCR ^™ -Blunt II-TOPO ^(R) vector (manufactured by Invitrogen ⁾ using Blunt ^(R) TOPO ^(R) PCR Cloning Kit (manufactured by Invitrogen), E. coli (DH5α strain) was ligated with the obtained plasmid DNA. Transformed. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml kanamycin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 50 μg / ml kanamycin, and then the resulting cells were used to generate a plasmid using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNAs were named TyrA ^fbr / pCR-blunt ^II and AroG ^fbr / pCR-blunt ^II , respectively.

Next, the TyrA ^fbr / pCR- ^bluntII was subjected to restriction enzyme treatment with NdeI and KpnI, respectively, and then separated by 1% agarose (Agarose-RE: Nacalai Tesque) gel electrophoresis. Was recovered about 1.0kb gene fragment from isolated ^{TyrA fbr} using ^{Wizard (R) SV Gel and PCR} Clean-UP System ( manufactured by Promega).
The E. coli expression vector pCDFDuet-1 (manufactured by Novagen) was subjected to restriction enzyme treatment and separation in the same manner, and a gene fragment of about 3.8 kb was recovered.

Next, the TyrA ^fbr gene fragment obtained above and the E. coli expression vector pCDFDuet-1 were ligated to High. 2 (manufactured by TOYOBO). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA.
The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml streptomycin. Clones that formed colonies on this medium were 50 μg / ml.
After liquid culture using an LB liquid medium containing streptomycin, plasmid DNA was extracted from the obtained cells using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). The obtained plasmid DNA was named TyrA ^fbr / pCDFDuet-1.

Further, the AroG ^fbr / pCR- ^bluntII was subjected to restriction enzyme treatment with EcoRI and HindIII, respectively, and then separated by 1% agarose (Agarose-RE: Nacalai Tesque) gel electrophoresis. The isolated 1.0 kb gene fragment derived from AroG ^fbr was recovered ^using Wizard ^(R) SV Gel and PCR Clean-UP System (Promega).
TyrA ^fbr / pCDFDuet-1 was also treated with restriction enzymes and separated in the same manner, and a gene fragment of about 4.8 kb was recovered.

The obtained AroG ^fbr gene fragment and TyrA ^fbr / pCDFDuet-1 were ligated to Ligation high Ver. 2 (manufactured by TOYOBO). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA.
The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml streptomycin. Clones that formed colonies on this medium were 50 μg / ml.
After liquid culture using an LB liquid medium containing streptomycin, plasmid DNA was extracted from the obtained cells using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). The obtained plasmid DNA was named TyrA ^fbr + AroG ^fbr / pCDFDuet-1.

Using the obtained TyrA ^fbr + AroG ^fbr / pCDFDuet-1, the tyrR gene-deficient strain E. E. coli BL21 Star ^™ (DE3) ΔtyrR :: Kan was transformed. The obtained recombinant Escherichia coli was smeared on an LB agar medium containing 50 μg / ml streptomycin and 50 μg / ml kanamycin. Clones that formed colonies on this medium were E. coli. E. coli BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr was designated as tyrosine-producing Escherichia coli.

(2) Construction of tyrosinase for E. coli and DOPA 4,5-dioxygenase (DOD) co-expression plasmid Codon-optimized MjDOD gene fragment for E. coli inserted into pUC57 described above as a template and restriction enzyme EcoRI site at the 5 'end A gene fragment was obtained by PCR using the following primers with a restriction enzyme HindIII site added to the 3 ′ end.
Primers for amplification of MjDOD gene: SEQ ID NOs: 36 and 37
The PCR reaction solution composition and reaction temperature conditions are the same as described above. The obtained gene fragment was ligated into ^{pCR TM -Blunt II-TOPO (R} ) vector (manufactured by Invitorgen), Escherichia coli with the resulting plasmid DNA (DH5 [alpha strain) was transformed. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml kanamycin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 50 μg / ml kanamycin, and then the resulting cells were used to generate a plasmid using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNA was designated as MjDOD / pCR-bluntII-2.

Next, the above-mentioned MjDOD / pCR-bluntII-2 and BMTYR / pETDuet-1 and RSTYR / pETDuet-1 previously constructed in (1) of Preparation Example 1 were treated with restriction enzymes with EcoRI and HindIII, followed by 1% agarose. (Agarose-RE: Nacalai Tesque, Inc.) Separated by gel electrophoresis. MjDOD-derived about 0.8 kb gene fragment, BMTYR / pETDuet-1-derived about 6.3 kb gene fragment, and RSTYR / pETDuet-1 about 6.9 kb gene fragment were ligated to High. 2 (manufactured by TOYOBO). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 100 μg / ml ampicillin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 100 μg / ml ampicillin, and then the resulting cells were used to generate a plasmid using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNAs were named BMTYR + MjDOD / pETDuet-1 and RSTYR + MjDOD / pETDuet-1, respectively.

(3) Production of tyrosinase and DOPA 4,5-dioxygenase co-expressing tyrosine-producing Escherichia coli strain Tyrosine producing E. coli E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr was transformed. The resulting recombinant E. coli was 100 μg / ml ampicillin and 50 μg / ml.
The LB agar medium containing kanamycin and 50 μg / ml streptomycin was smeared. Each of the clones that formed colonies on this medium was E. coli. E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / BMTYR + MjDOD, E. coli. E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / RSTYR + MjDOD was used as a betaxanthin production strain in the betaxanthin production test using the following saccharides as raw materials.

[Example 19]
First, the microorganism prepared above was cultured by the following method.
(Pre-culture process)
Two types of betaxanthin producing strains E. coli produced in Production Example 2 were prepared. E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / BMTYR + MjDOD, E. coli. E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / RSTYR + MjDOD were added to the preculture medium [5 × M9 Minimal Salts 400 μl, 1 M CaCl ₂ 0.2 μl, 1 M MgSO _{4 4} μl, 100 g / L Cas 100 44 μl, 1% Thiamine ₄ μl, 50 mM FeSO ₄ 2 μl, 40 mg / ml CuSO ₄ 2 μl, 50 mg / ml Kanamicin 2 μl, 100 mg / ml Carbenicillin 2 μl, 50 mg / ml Streptomycin 2 μl in a total volume of 2 ml Inoculated into 2 ml and cultured at 32 ° C. and 180 rpm for 20 hours.

(Main culture, conversion process)
The preculture solution obtained above was added to the main culture medium [5 × M9 Minimal Salts 4 ml, 1 M CaCl ₂ 2 μl, 1 M MgSO ₄ 40 μl, 40 g / L NH ₄ Cl 1.5 ml, 100 g / L Casamino acid, 1% _{Thiamine 40μl, 50mM FeSO 4 20μl,} 40mg / ml CuSO 4 20μl, 50mg / ml Kanamycin 20μl, 100mg / ml Carbenicillin 20μl, 50mg / ml Streptomycin 20μl, Overnight Express TM Autoinduction Systems1 OnEx Solution1 400μl, OnEx Solution2 1ml, and OnEx Solution3 20μl To a total volume of 20 ml Thus, 20 ml was inoculated so that OD600 = 0.3, and cultured at 20 ° C. and 180 rpm for 27 hours to induce protein expression. Thereafter, 200 μl of 1M ascorbic acid alone was added and further cultured for 21 hours to produce betaxanthin using carbohydrate as a raw material. The supernatant obtained by centrifugation (12000 rpm, 1 minute) from 1 ml of the obtained culture solution was passed through a 0.45 μm Cosmo spin filter (manufactured by Japan Millipore Corporation), and the filtrate was used as an analysis sample.
The analysis was performed in the same manner as in Example 1.

(Analysis result)
The obtained LC / MS analysis results (total ion chromatogram (TIC) and UV 470 nm) are shown in FIG. 8 (E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / BMTYR + MjDOD), FIG. ) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / RSTYR + MjDOD). Further, the signal Area values of the molecular ion peak of betaxanthin produced after 48 hours of culture are shown in Table 6 (E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / BMTYR + MjDOD), Table 7 (E. coli). (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / RSTYR + MjDOD).

As a result of the analysis, E.I. E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / BMTYR + MjDOD, E. coli. E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / RSTYR + MjDOD Both of these strains are precursors of several types of betaxanthins and betaxanthins by using tyrosine produced from saccharides in the medium. It was confirmed that some betaramic acid was produced. Both strains produced the largest amount of valine-betaxanthin. E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / BMTYR + MjDOD, followed by leucine-betaxanthin, isoleucine-betaxanthin, proline-betaxanthin, lysine-betaxanthin, phenylalanine-betaxanthin in this order. In E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / RSTYR + MjDOD, L-DOPA-betaxanthin, leucine-betaxanthin, isoleucine-betaxanthine, proline-betaxanthin, phenylalanine-betaxanthin, lysine-betaxanthin Many were produced in order. This accumulation tendency was similar to that in Example 1.

Further, as in Example 1, E. coli using tyrosinase derived from Ralstonia solanacearum GMI1000. In E. coli (BL21) ΔtyrR :: Kan / TyrA ^fbr + AroG ^fbr / RSTYR + MjDOD, the amount of betaxanthins produced was high, and the yellow color derived from this dye was strongly confirmed.
From the above, it was shown that by introducing tyrosinase and DOPA 4,5-dioxygenase into tyrosine-producing Escherichia coli, it is possible to efficiently produce betaxanthin using carbohydrate as a raw material without adding tyrosine.

[Production Example 3] Production of yeast having tyrosinase activity and DOPA 4,5-dioxygenase activity (1) Construction of tyrosinase expression plasmid for yeast
The gene sequence information of two types of tyrosinases derived from Bacillus megaterium DSM 319 and Ralstonia solanacearum GMI1000 (hereinafter referred to as BMTYR and RSTYR, respectively) were obtained from National Center for Biotechnology Information (NCBI), Synthesis was optimized for yeast codon frequency. The entire gene sequences (yBMTYR and yRSTYR) of the obtained optimized gene fragments are shown in SEQ ID NOs: 21 and 22, respectively. A plasmid was prepared by inserting the gene fragments of SEQ ID NOs: 21 and 22 into the EcoRV site of the pUC57 vector (GenScript). Using the obtained plasmid as a template, each gene fragment was obtained by PCR using the following primers with a restriction enzyme BamHI site added to the 5 ′ end and a restriction enzyme HindIII site added to the 3 ′ end.

Primer for yBMTYR gene amplification: SEQ ID NOS: 23 and 24
Primer for yRSTYR gene amplification: SEQ ID NOS: 25 and 26
PCR reaction solution composition: template ^{DNA 2μl, PrimeSTAR (R) HS} DNA
Polymerase (manufactured by Takara Bio Inc.) 0.5 μl, 5 × PrimeSTAR Buffer 10 μl, 2.5 mM dNTP Mixture 4 μl, 2 μM each primer 5 μl, sterilized water 24 μl (mixed to make 50 μl in total) Reaction temperature conditions: TaKaRa Using PCR Thermal Cycler Dice Touch (type TP350), a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, 72 ° C. for 1 minute (BMTYR), 1 minute 30 seconds (RSTYR) was repeated 30 times. However, the heat retention at 98 ° C. in the first cycle was 1 minute and 10 seconds.

Confirmation of PCR amplification products is performed by separating by gel electrophoresis with 1% agarose (Agarose-RE: Nacalai Tesque), followed by visualization with ethidium bromide staining. YBMTYR is about 0.9 kb, yRSTYR is about 1 A .5 kb fragment was detected. Recovery of the desired fragment from the gel was performed using ^{Wizard (R) SV Gel and PCR} Clean-UP System ( manufactured by Promega).
PCR fragment obtained tyrosinase, respectively, by using the ^{Zero Blunt (R) TOPO (R} ) PCR Cloning Kit ( manufactured by Invitorgen) pC
After linked to ^{R TM -Blunt II-TOPO (R} ) vector (manufactured by Invitorgen), Escherichia coli with the resulting plasmid DNA (DH5 [alpha strain) was transformed. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml kanamycin. Clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 50 μg / ml kanamycin, and then GenElute ^™ HP was obtained from the resulting cells.
Plasmid DNA was extracted using Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). The obtained plasmid DNAs were named yBMTYR / pCR-bluntII and yRSTYR / pCR-bluntII, respectively.

Next, the yBMTYR / pCR-bluntII and yRSTYR / pCR-bluntII were subjected to restriction enzyme treatment with BamHI and HindIII, respectively, and then separated by 1% agarose (Agarose-RE: Nacalai Tesque) gel electrophoresis. The isolated about 0.9 kb gene fragment derived from yBMTYR and the about 1.5 kb gene fragment derived from yRSTYR were subjected to Wizard ^(R) SV Gel and PCR Clean-UP.
It recovered using System (made by Promega).
The yeast expression vector pESC-LEU (manufactured by Stratagene) was subjected to restriction enzyme treatment and separation in the same manner, and a gene fragment of about 7.8 kb was recovered.

Subsequently, each tyrosinase gene fragment obtained above and the yeast expression vector pESC-LEU were combined with Ligation high Ver. 2 (manufactured by TOYOBO). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA.
The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 100 μg / ml ampicillin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 100 μg / ml ampicillin, and then the resulting cells were used to generate a plasmid using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNAs were named yBMTYR / pESC-LEU and yRSTYR / pESC-LEU, respectively.

(2) Construction of DOPA 4,5-dioxygenase (DOD) expression plasmid for yeast DOPA 4,5-dioxygenase (hereinafter referred to as “MjDOD”) derived from Mirabilis jalapa One type of gene sequence information is NCBI And optimized for yeast codon frequency by gene synthesis. The entire gene sequence (yMjDOD) of the optimized MjDOD is shown in SEQ ID NO: 27. The sequence described in SEQ ID NO: 27 was inserted into the EcoRV site of the pUC57 vector (GenScript). Using the resulting plasmid as a template, a gene fragment was obtained by PCR using primers (SEQ ID NOs: 28 and 29) in which a restriction enzyme BamHI site was added to the 5 ′ end and a restriction enzyme HindIII site was added to the 3 ′ end.

The reaction solution composition: template ^{DNA 2μl, PrimeSTAR (R) HS} DNA Polymerase ( Takara Bio Inc.) 0.5μl, 5 × PrimeSTAR Buffer 10μl , 2.5mM dNTP Mixture 4μl, 2μM each primer 5 [mu] l, sterile water 24 [mu] l (these Mix to make a total volume of 50 μl.)
Reaction temperature conditions: Using a TaKaRa PCR Thermal Cycler Dice Touch (type TP350), a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 5 seconds and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 98 ° C. in the first cycle was 1 minute and 10 seconds.

Confirmation of the amplification product and recovery of the target fragment from the gel are carried out in the same manner as described in “(1) Construction of a tyrosinase expression plasmid for yeast”, and an about 0.8 kb yMjDOD-derived gene fragment is detected. Recovered.
The obtained yMjDOD PCR fragment was ligated to pCR ^™ -Blunt II-TOPO ^(R) vector (Invitrogen ⁾ using Zero Blunt ^(R) TOPO ^(R) PCR Cloning Kit (Invitrogen). Escherichia coli (DH5α strain) was transformed with the plasmid DNA.

The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / ml kanamycin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 50 μg / ml kanamycin, and then the resulting cells were used to generate a plasmid using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The plasmid DNA obtained here was named yMjDOD / pCR-bluntII.

Next, the above yMjDOD / pCR-bluntII was subjected to restriction enzyme treatment with BamHI and HindIII, and then separated by 1% agarose gel electrophoresis, and the approximately 0.8 kb gene fragment was isolated from Wizard ^(R) SV Gel and PCR Clean-UP. It recovered using System (made by Promega).
pESC-URA (manufactured by Stratagene), a yeast expression vector that can coexist with the pESC-LEU vector, was also treated with restriction enzymes BamHI and HindIII and then separated in the same manner, and a gene fragment of about 6.6 kb was recovered. .

Ligation high Ver. 2 (manufactured by TOYOBO) was used to link the yMjDOD gene fragment and the yeast expression vector pESC-URA. Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA.
The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 100 μg / ml ampicillin. The clones that formed colonies on this medium were subjected to liquid culture using an LB liquid medium containing 100 μg / ml ampicillin, and then the resulting cells were used to generate a plasmid using GenElute ^™ HP Plasmid Miniprep Kit (manufactured by SIGMA-ALDRICH). DNA was extracted. The obtained plasmid DNA was named yMjDOD / pESC-URA.

(3) Preparation of tyrosinase and DOPA 4,5-dioxygenase co-expression yeast strains yBMTYR / pESC-LEU and yMjDOD / pESC-URA, yRSTYR / pESC-LEU and yMjDOD / produced in (1) and (2) above The plasmids of pESC-URA were mixed with Saccharomyces cerevisiae YPH500 strain (MATα ura3 lys2 ade2 trp1 his3 leu2) (Stratagene) by the lithium acetate method (Ito, H. et al, (1983) J. Bacteriol. 153 (1). ), 163-168), or electroporation (Fromm ME, et al., (1986) Nature 319 (6056): 791-793). The obtained recombinant yeast was treated with yeast synthetic drop-out medium supplements without histide, leucine, tryptophan and uracil (SIGMA-ALDRICH) and 20 mg / L histidine containing 0.6 mg Yeast Nitrogen Base w / o Amino Acids (Difco), 2% glucose, 2% agar). The clones that formed colonies on this medium were designated as YPH500 / BMTYR / MjDOD and YPH500 / RSTYR / MjDOD, respectively, and used as the betaxanthin-producing yeast strains in the following betaxanthin production tests.

[Example 20]
Examples of producing betaxanthin using resting cells are shown below.
(Pre-culture process)
YPH500 / BMTYR / MjDOD and YPH500 / RSTYR / MjDOD produced in Production Example 3 were produced by Yeast Synthetic Drop-out Medium with histodin, leucine, triptophan MG L Was inoculated into 2 ml of an SD medium containing, and cultured at 30 ° C. and 180 rpm for 18 hours.

(Main culture process)
Yeast Synthetic so that the obtained pre-cultured solution becomes OD600 = 0.2
Drop-out Medium Supplements without histide, leucine, tryptophan and uracil (SIGMA-ALDRICH) and SR medium containing 20 mg / L tryptophan and 20 mg / L histidine (0.67% Yeast Beast West
The solution was added to 20 ml of Amino Acids (Difco), 2% raffinose) and cultured at a culture temperature of 30 ° C. and 180 rpm. When OD600 = 0.7, galactose (final concentration 2%), CuSO ₄ (final concentration 40 μg / ml), and FeSO ₄ (final concentration 50 μM) were added to induce protein expression. Subsequently, the culture was continued, and galactose (final concentration 1%) and raffinose (final concentration 1%) were obtained 9 hours after the cultivation, and lysine (final concentration 30 mg / L) and tryptophan (final concentration 20 mg / liter) were obtained 24 hours after the cultivation. L), histidine (final concentration 20 mg / L) and adenine (final concentration 20 mg / L) were added, and the culture was carried out until 50 hours.

(Conversion process)
The cells obtained by centrifuging the whole culture solution obtained by the above method (6000 rpm, 5 minutes) were washed once with 2 ml of 50 mM potassium phosphate buffer (pH 7.0), and then centrifuged again. The cells were collected. The collected cells were suspended in 4 ml of a reaction buffer [50 mM FeSO ₄ , 4 μg / ml CuSO ₄ , 50 mM potassium phosphate buffer (pH 7.0) containing 10 mM ascorbic acid]. The reaction was started by adding L-tyrosine as a substrate to the obtained suspension and allowed to react at 30 ° C. and 180 rpm for 24 hours. The supernatant obtained by centrifugation (12000 rpm, 1 minute) from the obtained reaction solution was passed through a 0.45 μm Cosmo spin filter (manufactured by Nihon Millipore), and the filtrate was used as an analysis sample.
The analysis was performed in the same manner as in Example 1.

(Analysis result)
The obtained LC / MS analysis results (extracted ion chromatogram (EIC) and UV 470 nm) are shown in FIG. 10 (YPH500 / BMTYR / MjDOD) and FIG. 11 (YPH500 / RSTYR / MjDOD). In addition, Table 8 (YPH500 / BMTYR / MjDOD) and Table 9 (YPH500 / RSTYR / MjDOD) show changes over time in the signal area value of the molecular ion peak of the produced betaxanthin.

  As a result of the analysis, it was confirmed that both YPH500 / BMTYR / MjDOD and YPH500 / RSTYR / MjDOD strains produced several types of betaxanthins and betalamic acid which is a precursor of betaxanthin. Both strains produced the largest amount of glycine-betaxanthin. In YPH500 / BMTYR / MjDOD, proline-betaxanthin, DOPA-betaxanthin, isoleucine-betaxanthin, valine-betaxanthin, and leucine-betaxanthin in this order. In YPH500 / RSTYR / MjDOD, DOPA-betaxanthin, proline-betaxanthin, isoleucine-betaxanthin, and valine-betaxanthin were produced in this order. From the above, it was shown that betaxanthin can be produced by introducing tyrosinase and DOPA 4,5-dioxygenase into yeast.

Claims (8)

  A step of converting a raw material to betaxanthin in an aqueous medium in the presence of a microorganism having hydroxylation at the 3-position of the phenol ring of tyrosine and DOPA4,5-dioxygenase activity or a processed product thereof (conversion step) A process for producing betaxanthin, comprising: Before the conversion step, the method comprises culturing a microorganism having an enzyme activity for hydroxylating the 3-position of the phenol ring of tyrosine and DOPA 4,5-dioxygenase activity in an aqueous medium (cultivation step). The method for producing betaxanthin according to claim 1.   The method for producing betaxanthin according to claim 1 or 2, further comprising a step (recovery step) of recovering betaxanthin from the aqueous medium after the conversion step.   The method for producing betaxanthin according to any one of claims 1 to 3, wherein the enzyme that hydroxylates the 3-position of the phenol ring of tyrosine is tyrosinase.   The method for producing betaxanthin according to any one of claims 1 to 4, wherein in the conversion step, resting cells are used as the microorganism.   The method for producing betaxanthin according to any one of claims 1 to 5, wherein the raw material is tyrosine and / or a saccharide raw material.   The method for producing betaxanthin according to any one of claims 1 to 6, wherein an amino acid or an amine other than tyrosine is added in addition to the raw material.   In the said conversion process, the said aqueous medium contains at least 1 type chosen from the group which consists of ascorbic acid, copper, and iron, The betaxanthin as described in any one of Claims 1-7 characterized by the above-mentioned. Method.

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