JP7294636B2 - Novel carotenoid and method for producing said caroteroid - Google Patents

Description

The present invention relates to novel carotenoids and methods for producing the caroteroids.

Carotenoids are yellow-orange-red natural pigments produced by all photosynthetic organisms and some bacteria, molds, yeasts, and the like. To date, more than 750 carotenoids have been isolated and identified from nature. Among them, many reports have been made on carotenoids derived from higher plants such as crops and flowers, which are photosynthetic organisms that are most familiar to us. On the other hand, carotenoids common to higher plants are carotenoids in leaves, which are photosynthetic organs (Non-Patent Document 1). This biosynthetic pathway is shown in FIG. 1 together with the chemical structure. Among these carotenoids, those with distinct colors are biosynthetic pathways from Lycopene (also called lycopene) onwards. Among these, the ratio of carotenoids that are commercialized is high, Lycopene, β-Carotene, α-Carotene, Lutein, Zeaxanthin, β-Cryptoxanthin. (β-Cryptoxanthin), which has been reported to have health benefits (functionality) (Non-Patent Document 2). Many carotenoids expected to have functionality (hereinafter referred to as functional carotenoids), not limited to the carotenoids shown in FIG. 1, are generally expensive due to high production costs. In recent years, biotechnological research has been vigorously conducted to produce carotenoids at low cost using microorganisms, particularly hosts such as Escherichia coli and Saccharomyces cerevisiae.

Escherichia coli and budding yeast do not originally have carotenoid-producing ability, but they do have FPP (farnesyl diphosphate), which is a carotenoid precursor. do not have. However, not only among the leaf-derived carotenoids in Fig. 1, but also among the functional carotenoids as a whole, the ones that can be efficiently produced in microbial hosts are currently limited to Lycopene, β-Carotene, and Zeaxanthin. there were. A major reason for this is that these three carotenoids are produced not only by higher plants but also by some microorganisms, including the soil bacterium Pantoea ananatis , which belongs to the same class as E. coli. This is because the gene cluster ( crt gene cluster) can be used. The functions of Crt enzymes encoded by these crt gene clusters are shown in parentheses in FIG.

On the other hand, other leaf-derived carotenoids that have not been commercialized, namely Antheraxanthin, Violaxanthin and Neoxanthin, have been shown to be effective in microbial hosts such as E. coli and Saccharomyces cerevisiae. Production was difficult. Among the microbial hosts, E. coli alone uses a plant-derived ZEP gene such as the Gentiana lutea flower-derived ZEP (zeaxanthin epoxidase) gene ( GlZEP ) to biosynthesize antheraxanthin and violaxanthin. There is a report that it was successful in (Non-Patent Document 3, Non-Patent Document 2). However, it is now known that many ZEP genes derived from various plants do not function (do not express their function) even in E. coli. For example, the ZEP gene of diatom (diatom; Phaeodactylum tricornutum ) was not functionally expressed in Escherichia coli (Non-Patent Document 4).

CYO1 (cotyledon-specific chloroplast biogenesis factor), which is another component of the present invention , is a protein disulfide isomerase ( non Patent document 5). Arabidopsis thaliana-derived CYO1 is hereinafter referred to as AtCYO1, and the gene encoding this is referred to as AtCYO1 .

Patent No. 6348530

Norihiko Misawa, Biotechnology 93: 403-406, 2015 Akiko Kubo, Takehiko Sahara, Miho Takemura, Norihiko Misawa, Smart Cell Industry -Prospects of Substance Production Using Microbial Cells-, Part 3: Approaches to Industrial Applications, Chapter 8: Microbial Production of Plant-Derived Carotenoids, pp.195- 205, CMC Publishing C. Zhu et al, Biochimica et Biophysica Acta (BBA), 1625:305-308, 2003, https://doi.org/10.1016/S0167-4781(03)00017-4 M. Dambek et al, Journal of Experimental Botany, 63:5607-5612, 2012. H. Shimada et al, The PlantCell, 19: 3157–3169, 2007, DOI: https://doi.org/10.1105/tpc.107.051714 M. Takemura et al, TetrahedronLetters, 56:6063-6065, 2015.

The problem to be solved by the present invention is a novel carotenoid and a method for producing the caroteroid.

In order to solve the above problems, the present inventors have conducted intensive research and found that a gene or gene group of an enzyme that synthesizes zeaxanthin from farnesyl diphosphate, a zeaxanthin epoxidation enzyme gene, and cotyledon-specific chloroplast neogenesis The present inventors have found that a novel carotenoid represented by the following formula (1) is produced when a cotton-specific chloroplast biogenesis factor gene is introduced into a host and expressed, and the problem has been solved.

For example, in addition to the crtE , crtB , crtI , crtY and crtZ genes (from Pantoea ananatis , for example), and the ZEP gene (for example, CaZEP ), the CYO1 gene (for example, AtCYO1 ) is introduced into E. coli and expressed to produce novel carotenoids. Furthermore, a gene ( IDI ) encoding IDI {isopentenyl diphosphate (IPP) isomerase} and a gene encoding an electron transfer system protein and/or a gene encoding a NADPH regeneration system protein are introduced together and co-expressed. the novel carotenoids are produced more efficiently.
More specifically, the present inventors analyzed carotenoids extracted from the following recombinant Escherichia coli and found that antheraxanthin, violaxanthin, and novel carotenoids were efficiently produced. That is, the plasmid pACHP-Zea containing the IDI gene derived from Haematococcus pluvilias and the crtE , crtB , crtI , crtY and crtZ genes derived from Pantoea ananatis was introduced into Escherichia coli producing zeaxanthin . By introducing genes encoding zeaxanthin epoxidation enzyme CaZEP, chaperone protein AtCYO1 from Arabidopsis thaliana , electron transport protein NsRF from Nostoc sp . We found that antheraxanthin and violaxanthin can be produced at a level of 20.1% of total carotenoids, respectively, and that new carotenoids can be efficiently produced.
The above results were also surprising and unexpected for the inventors. It was discovered that the gene ( CYO1 gene) encoding the protein disulfide isomerase, which was discovered as a chaperone factor necessary for the formation of chloroplasts in the cotyledons of higher plants, functioned in E. coli and assisted in the production of new carotenoids. be. Furthermore, it was completely unpredictable that this novel carotenoid would have a physiological function such as a strong differentiation-inducing activity for adipocytes.

２．前項１に記載のカロテノイドを含む、抗酸化剤。
３．前項１に記載のカロテノイドを含む、脂質過酸化抑制剤。
４．前項１に記載のカロテノイドを含む、一重項酸素消去剤。
５．前項１に記載のカロテノイドを含む、脂質細胞分化誘導剤。
６．以下の（Ａ）又は（Ｂ）の遺伝子又は遺伝子群を導入した組換え細胞：
（Ａ）
（１）ファルネシル二リン酸からゼアキサンチンを合成する酵素の遺伝子又は遺伝子群、
（２）ゼアキサンチンエポキシ化酵素遺伝子、及び
（３）シャペロンとして働くタンパク質をコードする遺伝子
又は、
（Ｂ）
（１）ファルネシル二リン酸からゼアキサンチンを合成する酵素の遺伝子又は遺伝子群、及び、
（２）ゼアキサンチンエポキシ化酵素遺伝子。
７．さらに、イソペンテニル二リン酸イソメラーゼ遺伝子を導入した、前項６に記載の組換え細胞。
８．さらに、電子伝達系タンパク質をコードする遺伝子及び／又はNADPH再生系タンパク質をコードする遺伝子を導入した、前項６又は７に記載の組換え細胞。
９．前記シャペロンとして働くタンパク質をコードする遺伝子が子葉特異的葉緑体新生因子（cotyledon-specificchloroplast biogenesis factor）遺伝子である、前項６～８のいずれか１に記載の組換え細胞。
１０．前項６～９のいずれか１に記載の組換え細胞を培養して得られる培養物又は菌体からカロテノイドを得ることを特徴とする、カロテノイドの製造方法。
１１．前項６～９のいずれか１に記載の組換え細胞を培養して得られる培養物又は菌体から式（１）又は式（１Ａ’）で表されるカロテノイド、アンテラキサンチン及びビオラキサンチンのいずれか１以上を得る、又は、アンテラキサンチン及びビオラキサンチンのいずれか１以上を得ることを特徴とする、カロテノイドの製造方法。

１２．前記細胞が大腸菌である、前項６～９のいずれか１に記載の組換え細胞。
１３．前記細胞が大腸菌である、前項１０又は１１に記載のカロテノイドの製造方法。 The present invention is as follows.
1. A carotenoid represented by formula (1) or formula (1A').

2. An antioxidant comprising the carotenoid according to 1 above.
3. A lipid peroxidation inhibitor comprising the carotenoid according to the preceding item 1.
4. A singlet oxygen quencher comprising the carotenoid according to the preceding item 1.
5. An agent for inducing adipocyte differentiation, comprising the carotenoid according to 1 above.
6. Recombinant cells into which the gene or gene group of (A) or (B) below has been introduced:
(A)
(1) a gene or gene group of an enzyme that synthesizes zeaxanthin from farnesyl diphosphate;
(2) a zeaxanthin epoxidase gene, and (3) a gene encoding a protein that acts as a chaperone, or
(B)
(1) a gene or gene group of an enzyme that synthesizes zeaxanthin from farnesyl diphosphate, and
(2) zeaxanthin epoxidation enzyme gene;
7. 7. The recombinant cell according to 6 above, further containing an isopentenyl diphosphate isomerase gene.
8. 8. The recombinant cell according to item 6 or 7, further comprising a gene encoding an electron transport system protein and/or a gene encoding a NADPH regeneration system protein.
9. 9. The recombinant cell according to any one of 6 to 8 above, wherein the gene encoding the protein that acts as a chaperone is a cotyledon-specific chloroplast biogenesis factor gene.
10. 10. A method for producing carotenoids, which comprises obtaining carotenoids from a culture or bacterial cells obtained by culturing the recombinant cells according to any one of 6 to 9 above.
11. Any carotenoid, antheraxanthin or violaxanthin represented by formula (1) or formula (1A') from a culture or bacterial cell obtained by culturing the recombinant cell according to any one of the preceding items 6 to 9 A method for producing a carotenoid, characterized by obtaining one or more, or obtaining one or more of antheraxanthin and violaxanthin.

12. 10. The recombinant cell according to any one of 6 to 9 above, wherein the cell is E. coli.
13. 12. The method for producing a carotenoid according to 10 or 11 above, wherein the cells are Escherichia coli.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a production method capable of synthesizing any one or more of caroteroid, antheraxanthin and violaxanthin represented by formula (1).

FIG. 1 shows the carotenoid biosynthetic pathway in plant leaves and the functions of various carotenoid biosynthetic enzymes. Examples of E. coli that biosynthesize antheraxanthin and violaxanthin include GGPP synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (CrtI), and lycopene. Five genes encoding the enzymes β-lycopene β-cyclase (CrtY), β-carotene hydroxylase (CrtZ), and zeaxanthin epoxidase (ZEP) Genetically modified E. coli in which the genes are at least all co-introduced and expressed is required. FIG. 2 shows HPLC analysis results of carotenoids extracted from genetically modified E. coli. (A) Recombinant E. coli A into which pACHP-Zea and pUC-CaZEP were introduced. (B) Recombinant E. coli E introduced with pACHP-Zea, pUC-CaZEP, RSF-NsRF-AtCYO1, and CDF-gdh. (C) Violaxanthin preparation. (D) Purified novel carotenoid 6'-hydroxy-3'-didehydrolutein (standard product) produced and purified by E. coli according to Examples described below and identified by various instrumental analyses. (E) Absorption wavelength of violaxanthin peak of recombinant E. coli E. (F) Peak absorption wavelength of the novel carotenoid 6'-hydroxy-3'-didehydrolutein from recombinant E. coli E. (G) Absorption wavelength of violaxanthin preparation. (H) Absorption wavelength of novel carotenoid 6'-hydroxy-3'-didehydrolutein preparation. Peak 1: violaxanthin, peak 2: novel carotenoid 6'-hydroxy-3'-didehydrolutein. FIG. 1 is a diagram of the putative biosynthetic pathway of the novel carotenoid 6'-hydroxy-3'-didehydrolutein. FIG. 2 shows the structures of E. coli transformation vectors (A) pUC18, (B) RSFDuet-1, and (C) CDFDuet-1. Fig. 2 shows the structures of E. coli transformation plasmids, (A) pUC-CaZEP, (B) RSFD-NsRF-AtCYO1, and (C) CDF-gdh. Fig. 2 shows A: ¹ H-NMR (in CDCl ₃ ), B: ¹³ C-NMR (in CDCl ₃ ) of the novel carotenoid. FIG. 2 shows the novel carotenoids: A: zeaxanthin structural portion, B: structure of β-ionone ring portion, and C: determined overall structure. Rat brain lipid peroxidation inhibitory activity of compound 1. ¹ O ₂ scavenging activity of compound 1. 3T3-L1 cell differentiation-inducing activity of compound 1.

The present invention relates to a method for producing carotenoids, particularly antheraxanthin, violaxanthin and carotenoids represented by formula (1). The production method of the present invention can produce the carotenoid by introducing a specific gene and/or gene group into a microbial host such as Escherichia coli or budding yeast.
The present invention will be described in detail below.

(Gene or gene group of enzyme for synthesizing zeaxanthin from farnesyl diphosphate)
Escherichia coli and budding yeast have the ability to produce carotenoid precursor FPP, but do not have the ability to produce zeaxanthin. Therefore, there is a need to synthesize zeaxanthin from farnesyl diphosphate. Zeaxanthin can be produced from farnesyl diphosphate using the carotenoid biosynthetic gene cluster (crt gene cluster) derived from zeaxanthin-producing microorganisms such as the soil bacteria Pantoea ananatis and Pantoea agglomerans.
The crt gene group in the present invention includes any one or more, two or more, three or more, four or more, or all of crtE, crtB, crtI, crtY and crtZ.

(Isopentenyl diphosphate isomerase gene ( IDI gene))
Isopentenyl diphosphate isomerase converts isopentenyl diphosphate (isopentenyl pyroline: IPP) to dimethylallyl diphosphate (DMAPP) during FPP production in E. coli and budding yeast. The IDI gene can efficiently produce carotenoids by expressing it together with the crt gene group.
Isopentenyl diphosphate isomerase regulates the quantitative ratio of IPP and dimethylallyl diphosphate (DMAPP) (Patent Document 1). There are two types of isopentenyl diphosphate isomerase, type 1 and type 2, which have different structures. Type 2 IDI genes are sometimes present together in the carotenoid biosynthesis gene cluster of carotenoid-producing bacteria belonging to the class α-Proteobacteria or γ-Proteobacteria (Norihiko Misawa, Oreo Science 9 (9): 385-391, 2009). Regardless of type 1 or type 2, when the IDI gene is introduced into Escherichia coli together with the biosynthetic genes for terpenes such as carotenoids and sesquiterpenes and expressed, the amount of terpene produced increases compared to when the IDI gene is not introduced. (S. Kajiwara, PD Fraser, K. Kondo and N. Misawa, Biochem J. , 324, 421-426. 1997).

(Gene encoding zeaxanthin epoxidase)
As for the enzyme gene necessary for synthesizing violaxanthin by epoxidizing zeaxanthin, a gene encoding zeaxanthin epoxidation enzyme is suitable, as described above. As a zeaxanthin epoxidase gene, ZEP ( CaZEP ) isolated from paprika ( Capsicum annuum ) and the like are known. Preferably, at least zeaxanthin epoxidase is used. More preferably, gene sequences encoding ZEP isolated from paprika are used. However, it is not always necessary to use the same gene sequence, and zeaxanthin epoxidases derived from other organisms may be used.
As a gene encoding zeaxanthin epoxidase, ZEP isolated from paprika (the amino acid sequence described in SEQ ID NO: 2 or homology with the amino acid sequence described in SEQ ID NO: 2 in the entire amino acid sequence is 70% or more, preferably is an amino acid sequence that is 80%, more preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more, and has an activity to epoxidize zeaxanthin and synthesize violaxanthin ) is preferred, and such a gene is, for example, DNA containing the base sequence of SEQ ID NO: 1 is more preferred.
In addition, as a gene encoding zeaxanthin epoxidase, it hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and epoxidizes zeaxanthin to synthesize violaxanthin. A DNA that encodes a polypeptide having an activity to do is also preferably used. In addition, such a DNA sequence generally has about 90% or more, preferably about 95%, more preferably about 98% or more sequence identity with the DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and zeaxanthin is an epoxy compound. A DNA encoding a polypeptide having the activity of synthesizing violaxanthin is preferably used.

(gene cluster required for carotenoid synthesis)
Genes or gene groups necessary for the method for producing carotenoids of the present invention are exemplified below, but are not particularly limited.
<Antheraxanthin and Violaxanthin>
(1) A gene or gene group of an enzyme that synthesizes zeaxanthin from farnesyl diphosphate (2) A gene encoding zeaxanthin epoxidase, an enzyme that synthesizes antheraxanthin and further violaxanthin from zeaxanthin (zeaxanthin epoxidase gene )
<Carotenoid Represented by Formula (1)>
(1) a gene or gene group of an enzyme that synthesizes zeaxanthin from farnesyl diphosphate (2) a zeaxanthin epoxidase gene (3) a gene encoding a protein that acts as a chaperone specificchloroplast biogenesis factor) gene, preferably AtCYO1.
Furthermore, in the present invention, an isopentenyl diphosphate isomerase gene and/or a gene encoding an electron transport system protein and a gene encoding a NADPH regeneration system protein are preferably introduced into the host.

(genes encoding chaperone proteins)
As for the protein gene which is highly likely to activate zeaxanthin epoxidase and is necessary for synthesizing the carotenoid represented by formula (1), the CYO1 gene encoding the chaperone protein is preferable as described above. be. AtCYO1 isolated from Arabidopsis thaliana is known as a chaperone protein gene. Preferably, at least chaperone proteins are used. More preferably, gene sequences encoding AtCYO1 isolated from Arabidopsis thaliana are used. However, it is not always necessary to use the same gene sequence, and chaperone proteins derived from other organisms may be used.
As a gene encoding a chaperone protein, AtCYO1 isolated from Arabidopsis thaliana (the amino acid sequence described in SEQ ID NO: 4 or homology with the amino acid sequence described in SEQ ID NO: 4 in the entire amino acid sequence is 70% or more, preferably 80%). %, more preferably 85% or more, still more preferably 90% or more, still more preferably 95% or more, most preferably 98% or more, and having the activity of regulating the activity of zeaxanthin epoxidase ), and as such a gene, for example, a DNA containing the base sequence of SEQ ID NO: 3 is more preferable.
Also, as a gene encoding a chaperone protein, it hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3, and has the activity of regulating the activity of zeaxanthin epoxidase. A DNA encoding a polypeptide having is also preferably used. In addition, such a DNA sequence usually has about 90% or more, preferably about 95%, more preferably about 98% or more sequence identity with the DNA consisting of the base sequence of SEQ ID NO: 3, and has zeaxanthin epoxidation. A DNA encoding a polypeptide having an activity of regulating enzyme activity is preferably used.

(Genes encoding electron transport proteins)
Known genes encoding electron transport proteins include NsRF ( NsRED & NsFER ) isolated from Nostoc sp. strain PCC7120 and camAB ( camA & camB ) isolated from Pseudomonas putida . A gene encoding an electron transport system protein that can be used in the present invention is not particularly limited, and for example, a gene encoding an electron transport system protein derived from another organism may be used.
As genes encoding electron transport proteins, genes encoding NsRF (NsRED and NsFER) isolated from Nostoc sp. strain PCC7120 are preferred.
NsRED (70% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 6 or the amino acid sequence set forth in SEQ ID NO: 6 in the entire amino acid sequence , more preferably 95% or more, and most preferably 98% or more, and having an electron transfer activity). preferable.
NsFER (70% or more, preferably 80%, more preferably 85% or more, more preferably 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 8 or the amino acid sequence set forth in SEQ ID NO: 8 in the entire amino acid sequence , more preferably 95% or more, and most preferably 98% or more, and having an electron transfer activity). preferable.
Also, as a gene encoding an electron transport system protein, it encodes a polypeptide having an electron transfer activity that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of SEQ ID NO:5. A DNA encoding a polypeptide having an electron-transmitting activity that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to a DNA comprising SEQ ID NO: 7 and/or a DNA comprising SEQ ID NO: 7 is also preferably used. be done. In addition, such a DNA sequence usually has a sequence identity of about 90% or more, preferably about 95%, more preferably about 98% or more with the DNA consisting of the base sequence of SEQ ID NO: 5, and can transmit electrons. usually has about 90% or more, preferably about 95%, more preferably about 98% or more sequence identity with the DNA encoding the polypeptide having the activity to and / or the DNA consisting of the nucleotide sequence of SEQ ID NO: 7, A DNA that encodes a polypeptide that also has an electron transfer activity is preferably used.

(Gene encoding NADPH regeneration system protein)
As NADPH regeneration system protein genes, gdh isolated from Bacillus subtilis and zwf isolated from Escherichia coli are known. Preferably, at least a NADPH regeneration system protein is used. More preferably, gene sequences encoding Gdh isolated from Bacillus subtilis or Zwf isolated from E. coli are used. However, it is not always necessary to use the same gene sequence as this, and NADPH regeneration system proteins derived from other organisms may be used.
Examples of the gene encoding the NADPH-regenerating system protein include Gdh isolated from Bacillus subtilis (the amino acid sequence set forth in SEQ ID NO: 10, or having 70% or more homology to the amino acid sequence set forth in SEQ ID NO: 10 in the entire amino acid sequence; preferably 80%, more preferably 85% or more, still more preferably 90% or more, even more preferably 95% or more, most preferably 98% or more, and having an activity to regenerate NADPH). A coding gene is preferred, and such a gene is more preferably, for example, a DNA containing the nucleotide sequence of SEQ ID NO:9.
In addition, as a gene encoding a NADPH regeneration system protein, a polyhybridization under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 9 and having an activity to regenerate NADPH. DNAs encoding peptides are also preferably used. In addition, such a DNA sequence usually has about 90% or more, preferably about 95%, more preferably about 98% or more sequence identity with the DNA consisting of the base sequence of SEQ ID NO: 9, and regenerates NADPH. A DNA that encodes a polypeptide having the activity to do so is preferably used.

(gene)
As used herein, the term "gene (DNA molecule)" is intended to include not only double-stranded DNA, but also single-stranded DNA such as the sense strand and antisense strand that constitute it, and is limited in length. not to be
Therefore, unless otherwise specified, the gene (polynucleotide) encoding the amino acid sequence of the present invention includes double-stranded DNA including genomic DNA, single-stranded DNA including cDNA (sense strand), and complementary to the sense strand. Both single-stranded DNA (antisense strand) and synthetic DNA having similar sequences and fragments thereof are included.

(Carotenoid represented by formula (1))
The present invention provides a carotenoid represented by the following formula (1) (4-hydroxy-4-((1E,3E,5E,7E,9E,11E,13E,15E,17E)-18-(4-hydroxy- 2,6,6-trimethylcyclohex-1-en-1-yl)-3,7,12,16-tetramethyloctadeca-1,3,5,7,9,11,13,15,17-nonaen-1-yl )-3,5,5-trimethylcyclohex-2-enone). The carotenoid represented by formula (1) is obtained by the carotenoid production method of the present invention.
Carotenoids represented by formula (1) include 6'-hydroxy-3'-didehydrolutein. 6'-Hydroxy-3'-didehydrolutein is a carotenoid represented by formula (1A), a carotenoid represented by formula (1A') and a carotenoid represented by formula (1B), or formula (1A) It includes carotenoids represented by and carotenoids represented by formula (1B).

In the present specification, the carotenoid represented by formula (1) is a carotenoid represented by the following formula (1A) ((S)-4-hydroxy-4-((1E,3E,5E,7E,9E, 11E,13E,15E,17E)-18-((R)-4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-3,7,12,16-tetramethyloctadeca-1,3 ,5,7,9,11,13,15,17-nonaen-1-yl)-3,5,5-trimethylcyclohex-2-enone). The carotenoid represented by formula (1A) is obtained by the carotenoid production method of the present invention.

In the present specification, the carotenoid represented by formula (1) is a carotenoid represented by the following formula (1A') (4-hydroxy-4-((1E,3E,5E,7E,9E,11E,13E ,15E,17E)-18-((R)-4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-3,7,12,16-tetramethyloctadeca-1,3,5, 7,9,11,13,15,17-nonaen-1-yl)-3,5,5-trimethylcyclohex-2-enone). The carotenoid represented by formula (1A') is obtained by the carotenoid production method of the present invention.

In the present specification, the carotenoid represented by formula (1) {preferably formula (1B)} is a carotenoid represented by formula (1B) below ((S)-4-hydroxy-4-((1E ,3E,5E,7E,9E,11E,13E,15E,17E)-18-((R)-4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-3,7, 12,16-tetramethyloctadeca-1,3,5,7,9,11,13,15,17-nonaen-1-yl)-3,5,5-trimethylcyclohex-2-enone). The carotenoid represented by formula (1B) is obtained by the carotenoid production method of the present invention.

The present invention also covers the following agents containing carotenoids represented by formula (1):
An antioxidant containing a carotenoid represented by formula (1),
A lipid peroxidation inhibitor containing a carotenoid represented by formula (1),
A singlet oxygen quenching agent containing a carotenoid represented by formula (1), and an adipocyte differentiation inducer containing a carotenoid represented by formula (1).
The carotenoid-containing agent represented by formula (1) can be prepared by using the compound of the present invention alone or pharmacologically according to a method known per se as a method for producing pharmaceutical preparations (e.g., the method described in the Japanese Pharmacopoeia). It can be produced by mixing with an acceptable carrier.
The content in the agent containing the carotenoid represented by formula (1) is 0.01 to 100% by weight of the total agent, for example 0.01 to 0.1% by weight, 0.1 to 1% by weight. , 1-10% by weight, 10-50% by weight, 50-90% by weight, 90-100% by weight.
The dosage of the agent varies depending on the administration subject, administration route, disease, symptoms, etc. For example, in the case of oral administration, 0.01 to 100 mg / day of the carotenoid represented by formula (1) as an active ingredient kg body weight, 0.1 to 50 mg/kg body weight, or 1 to 20 mg/kg body weight. It may be administered in multiple doses per day, for example, in 2 to 3 divided doses.
The carotenoid represented by formula (1) can be food and drink containing the carotenoid represented by formula (1). For example, it can be used as an edible composition (e.g., health food, etc.) with antioxidant effect, lipid peroxidation inhibitory effect, singlet oxygen scavenging effect, and adipocyte differentiation effect by adding or blending it to food and drink. is. The content of the carotenoid represented by the formula (1) in the food and drink and the intake of the food and drink conform to the agent containing the carotenoid represented by the formula (1).

(Method for producing carotenoid of the present invention)
The method for producing carotenoids of the present invention includes a step of culturing recombinant cells into which the following genes or gene groups (1) to (2) or (1) to (3) have been introduced.
(1) a gene or gene group of an enzyme that synthesizes zeaxanthin from farnesyl diphosphate (2) a zeaxanthin epoxidase gene (3) a gene encoding a protein that acts as a chaperone Recombination further introducing an acid isomerase gene and/or a gene encoding an electron transport system protein or a gene encoding a NADPH regeneration system protein, more preferably a gene encoding an electron transport system protein and a gene encoding a NADPH regeneration system protein are cells.
Further, as another aspect of the method for producing carotenoids of the present invention, a cell-free system (wheat germ, etc.) or the like is used to obtain the genes or genes of (1) to (3) or (1) to (2) above. In addition to the group, preferably the genes or gene groups of (1) to (3) or (1) to (2) above, an isopentenyl diphosphate isomerase gene and/or a gene encoding an electron transport system protein and/or Alternatively, the step of expressing a gene encoding a NADPH regeneration system protein may be included.

(Recombinant cells of the present invention)
The recombinant cell of the present invention is a recombinant cell into which a gene or gene group for an enzyme that synthesizes zeaxanthin from farnesyl diphosphate, a gene for zeaxanthin epoxidase, and a cotyledon-specific chloroplast gene have been introduced, Preferably, an isopentenyl diphosphate isomerase gene, a gene encoding an electron transport system protein, or a gene encoding a NADPH regeneration system protein (more preferably, a gene encoding an electron transport system protein and a gene encoding a NADPH regeneration system protein) It is a recombinant cell that has been further introduced.
The recombinant cells of the present invention can produce any one or more of antheraxanthin, violaxanthin and carotenoid represented by formula (1).
The recombinant cells of the present invention are preferably capable of producing antheraxanthin, violaxanthin and carotenoids of formula (1).
Recombinant cells that produce antheraxanthin, violaxanthin and/or the carotenoid represented by formula (1) are specifically exemplified below, but are not particularly limited.
(1) Gene encoding zeaxanthin epoxidation enzyme ( ZEP gene), gene or gene group of enzyme that synthesizes zeaxanthin from farnesyl diphosphate, isopentenyl diphosphate isomerase gene ( IDI gene), gene encoding chaperone protein , a recombinant cell into which a gene encoding an electron transport system protein and a gene encoding a NADPH regeneration system protein have been introduced.
(2) Gene encoding zeaxanthin epoxidation enzyme ( ZEP gene), gene or gene group of enzyme that synthesizes zeaxanthin from farnesyl diphosphate, isopentenyl diphosphate isomerase gene ( IDI gene), gene encoding chaperone protein and recombinant cells into which a gene encoding an electron transport system protein has been introduced.
(3) Gene encoding zeaxanthin epoxidation enzyme ( ZEP gene), gene or gene group of enzyme that synthesizes zeaxanthin from farnesyl diphosphate, isopentenyl diphosphate isomerase gene ( IDI gene), gene encoding chaperone protein and recombinant cells into which a gene encoding a NADPH regeneration system protein has been introduced.
(4) A gene encoding zeaxanthin epoxidation enzyme ( ZEP gene), a gene or gene group of an enzyme that synthesizes zeaxanthin from farnesyl diphosphate, an isopentenyl diphosphate isomerase gene ( IDI gene), and a gene encoding a chaperone protein Recombinant cells into which is introduced.
Recombinant cells having the gene or gene group of an enzyme that synthesizes zeaxanthin from farnesyl diphosphate and the IDI gene include, for example, recombinant cells having zeaxanthin-producing ability. Examples of recombinant cells having zeaxanthin-producing ability include: For example, recombinant cells harboring the plasmid pACHP-Zea.
Cells (hosts) that can be used include E. coli cells, insect cells, yeast cells, plant cell cells, and the like.
Preferred combinations of synthase genes to be introduced for each cell are as follows.
E. coli: IDI , crtE , crtB, crtI, crtY, crtZ, ZEP, AtCYO1, NsRF, gdh
Yeast system: IDI , crtE , crtB, crtI, crtY, crtZ, ZEP, AtCYO1, NsRF, gdh
Insect system: IDI , crtE , crtB, crtI, crtY, crtZ, ZEP, AtCYO1, NsRF, gdh
Plant system: IDI , ZEP, AtCYO1, NsRF, gdh
In addition, when a foreign gene is directly introduced into the chloroplast by particle gun transformation and expressed, isopentenyl diphosphate (IPP) isomerase (type 1 and / or type 2) gene It is believed that co-expression can improve the production of the desired carotenoid.
Among these, particularly preferred are recombinant E. coli into which idi , crtE , crtB, crtI, crtY, crtZ, ZEP, AtCYO1, NsRF and gdh have been introduced.

(E. coli transformation vector)
The enzyme gene for synthesizing the antheraxanthin, violaxanthin and/or carotenoid represented by formula (1) is introduced into, for example, Escherichia coli by a known method. The method of introducing the exogenous gene into Escherichia coli is not particularly limited. For example, the exogenous gene to be introduced is inserted into an Escherichia coli transformation vector in a form in which it is expressed by using an appropriate promoter and terminator by a known method. A plasmid may be prepared by the method described above, and the plasmid may be introduced into Escherichia coli cells. When introducing a plurality of genes, those genes may be introduced by one transformation vector, or may be introduced by different transformation vectors.
E. coli transformation vectors may be appropriately selected according to the number and type of transgenes, but E. coli protein expression vectors are preferred, such as pUC18 and RSFDuet-1 (FIG. 4). Any promoter may be used as long as it can be expressed in E. coli. Examples include T7 promoter, lacZ promoter, tac promoter and the like. Among them, the tac promoter is more preferable because the introduced gene is highly expressed in E. coli. When it is preferable that the introduced gene be transiently expressed in E. coli, it is preferable to use an inducible promoter, more preferably an inducible T7 promoter by IPTG or the like. The terminator can be appropriately selected depending on the gene to be introduced, and examples thereof include T7 terminator, rrnB terminator and the like.
In addition, various selection marker genes may be used to efficiently select E. coli cells into which a target exogenous gene (eg, a gene encoding Idi) has been introduced by an E. coli transformation vector. The selectable marker gene is not particularly limited, and one known per se may be used. For example, it is preferable to use a gene conferring drug resistance as a selectable marker, and introduce a plasmid or the like containing this selectable marker gene and the gene involved in the biosynthesis of violaxanthin into E. coli cells as an E. coli transformation vector. Thus, Escherichia coli cells into which exogenous genes have been introduced can be efficiently selected from the expression of selectable marker genes. Examples of such selectable marker genes include various drug resistance genes. More specific examples include ampicillin resistance gene, kanamycin resistance gene, tetracycline resistance gene and the like. Moreover, it is preferable to have a promoter and terminator for expressing the gene upstream and downstream of the selectable marker gene.
A known E. coli transformation vector and E. coli transformation method using the vector can be used. As an example, FIG. 3 shows an E. coli transformation vector used by the inventors to introduce an enzyme gene for synthesizing violaxanthin into E. coli. A foreign gene to be expressed may be introduced into the cloning site of an E. coli transformation vector. Promoters and terminators that function in E. coli may be used, and examples include T7 promoter and T7 terminator. Furthermore, it is preferable to use a drug resistance gene surrounded by a promoter and a terminator together as a marker gene for transformation.

(Transformation of E. coli and acquisition of genetically modified E. coli)
Transformation of E. coli can be performed by a known method. For example, a DNA fragment is prepared in which a foreign gene to be introduced or a selection marker gene (such as a drug resistance gene) is expressed by using an appropriate promoter and terminator, and the DNA fragment is introduced by a heat shock method or the like. Escherichia coli into which the gene has been introduced is obtained by selection with a drug.
Expression of exogenous genes introduced into E. coli can be confirmed by methods such as Northern blotting, RT-PCR, and Western blotting, for example. Moreover, the production of violaxanthin in the E. coli can be confirmed by, for example, a method such as HPLC analysis.

(Use of genetically modified Escherichia coli to produce antheraxanthin, violaxanthin and/or carotenoid represented by formula (1))
The present invention also includes use for producing antheraxanthin, violaxanthin and/or carotenoid represented by formula (1) from the genetically modified E. coli. Preferred embodiments of the genetically modified Escherichia coli and the method for producing the same are as described above. , or a part of the gene group that encodes zeaxanthin-producing Escherichia coli (isopentenyl diphosphate isomerase, GGPP synthase, phytoene synthase, lycopene synthase, lycopene β-cyclase, β-carotene hydroxylase) ) is preferred.
Each gene and its preferred embodiment are as described above.
Violaxanthin and/or the carotenoid represented by formula (1) are produced in the E. coli by culturing the produced genetically modified E. coli. A method for culturing the genetically modified E. coli is not particularly limited.
For example, genetically modified E. coli contains antheraxanthin, violaxanthin and/or carotenoid represented by formula (1). Therefore, antheraxanthin, violaxanthin and/or carotenoid represented by formula (1) can be obtained by a known extraction method.

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

(Preparation of carotenoid-producing plasmids represented by antheraxanthin, violaxanthin and formula (1))
Antheraxanthin, violaxanthin, and carotenoid-producing plasmids represented by formula (1) were prepared. Details are as follows.
The paprika-derived ZEP gene ( CaZEP ) sequence was prepared by modifying the base sequence to match the codon usage frequency of E. coli. The prepared sequence is shown in SEQ ID NO:1. The sequence of AtCYO1 gene was amplified from Arabidopsis cDNA. The amplified sequence is shown in SEQ ID NO:3. The sequences of the genes ( NsRF ) encoding the electron transfer proteins NsRED and NsFER from Nostoc sp. PCC7120 were amplified from cDNA of Nostoc sp . PCC7120. The amplified sequence is shown in SEQ ID NO:5. The gdh gene sequence encoding Bacillus subtilis Gdh was amplified from Bacillus subtilus cDNA. The amplified sequence is shown in SEQ ID NO:9. The nucleotide sequences of AtCYO1 , NsRF and gdh (SEQ ID NOs: 1, 3, 5 and 9) can be obtained from DDBJ accession numbers SCO2_ARATH, FENR_NOSS1 and FER1_NOSS1, G4EZZ9_BACIU, respectively.
pUC18 (Fig. 4A), RSFDuet-1 (Fig. 4B) and CDFDuet-1 (Fig. 4C) were used as E. coli transformation vectors. The CaZEP fragment cleaved with Eco RI and Bam HI was excised and inserted into the Eco RI- Bam HI site of the vector pUC18 to construct the plasmid pUC-CaZEP (Fig. 5A). The NsRF fragment of Nostoc sp. PCC7120 cleaved with Nco I and Bam HI was excised and inserted into the Nco I- Bam HI site of vector RSFDuet-1 to generate plasmid RSFD-NsRF. Furthermore, the Arabidopsis thaliana AtCYO1 fragment cleaved with Nde I and Kpn I was excised and inserted into the Nde I- Kpn I site of RSFD-camAB to construct the plasmid RSFD-NsRF-AtCYO1 (FIG. 5B). The Gdh fragment cut with Nco I and Sal I was inserted into the Nco I- Sal I site of vector CDFDuet-1 to generate plasmid CDFD-Gdh (FIG. 5C).

Table 1 shows the primers used for PCR amplification. CaZEPF and CaZEPR were used for CaZEP, NsRFF and NsRFRR for NsRF, gdhF and gdhR for gdh, and AtCYO1F and AtCYO1R for AtCYO1.

(Transformation of E. coli and carotenoid production by recombinant E. coli)
Using the plasmids pUC-CaZEP (Fig. 5A), RSFD-NsRF-AtCYO1 (Fig. 5B) and CDFD-gdh (Fig. 5C) prepared in Example 1, carotenoid-producing E. coli represented by zeaxanthin and formula (1) Transformation was performed by a known method. Details are as follows.
Zeaxanthin Escherichia coli {JM101(DE3) with pACHP-Zea} was cultured in SOB medium (2% bactotryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM _MgCl2 , 10 mM _MgSO4 ). After that, it was treated with TB buffer (10 mM PIPES (pH 6.7), 15 mM _CaCl2 , 250 mM KCl, ₅₅ mM _MnCl2.4H2O ). Then, the plasmids prepared in Example 1 were combined, mixed with the treated E. coli, and allowed to stand on ice. After being heat-shocked at 42°C, they were cultured in SOC medium and plated on drug-containing medium. After overnight culture, drug-resistant colonies that appeared were obtained as transformed E. coli (genetically modified E. coli). As drugs (antibiotics), chloramphenicol (33 mg/L), spectinomycin (100 mg/L), ampicillin (40 mg/L) and kanamycin (40 mg/L) were added to the medium.

(Carotenoid production by recombinant E. coli)
crtE (GGPP synthase), crtB (phytoene synthase), crtI (phytoene desaturase), crtY (lycopene β-cyclase), crtZ (β-carotene hydroxylase) genes derived from the soil bacterium Pantoea ananatis , and the green alga Haematococcus pluvialis -derived IDI (IPP isomerase) gene (sometimes referred to as idi ) expression plasmid pACHP-Zea (Non-Patent Document 6), in addition to paprika (red pepper; Capsicum annuum )-derived ZEP ( Escherichia coli A into which a plasmid for expression of a gene encoding a zeaxanthinepoxidase ( CaZEP ) enzyme was simultaneously introduced was prepared. The main carotenoid produced by this (genetically) recombinant E. coli A is zeaxanthin, and it was confirmed that antheraxanthin and violaxanthin were produced, albeit in very small amounts (Table 2).
On the other hand, Escherichia coli E into which expression plasmids for genes encoding AtCYO1 (chaperone) derived from Arabidopsis thaliana, electron transfer system protein (NsRF) derived from Nostoc sp. PCC7120, and NADPH regeneration system protein (Gdh) derived from Bacillus subtilis were introduced was made. In this recombinant Escherichia coli E, violaxanthin and antheraxanthin were detected, and the production of both of them accounted for 20.1% of the total carotenoids.
Violaxanthin and antheraxanthin were also detected in E.coli G and H, which had the AtCYO1 gene and the electron transport system or NADPH regeneration system protein gene introduced, but their production was less than half that of E.coli. On the other hand, trace amounts of violaxanthin and antheraxanthin were detected in Escherichia coli F, B, and C into which AtCYO1 , electron transport system, or NADPH regeneration system genes were introduced. Violaxanthin and antheraxanthin were also detected in trace amounts in E. coli D, which did not contain AtCYO1 and had both the electron transfer system and the NADPH regeneration system.
Table 2 shows a comparison of carotenoid compositions produced by transgenic E. coli. The novel carotenoid 6'-hydroxy-3'-didehydrolutein was detected in E. coli E, F, G, and H, but not in E. coli A, B, C, and D. We confirmed that the function of AtCYO1 is necessary. In Table 2, only simple quantification is performed by the analysis method shown in Non-Patent Document 6, but the function of AtCYO1 for producing novel carotenoids is determined by coexistence with electron transport system and/or NADPH regeneration system proteins. , confirmed that its function is efficiently exhibited. In particular, in addition to the AtCYO1 gene, the novel carotenoid produced by coexisting recombinant E. coli E with the electron transport system and NADPH regeneration system protein genes has a high production of 6'-hydroxy-3'-didehydrolutein, accounting for 16.8% of the total carotenoids. Met.

(Extraction of carotenoids from recombinant E. coli and HPLC analysis)
The genetically modified E. coli obtained in Example 2 was cultured at 37° C. in 2YT (2×YT) medium (1.6% bactotryptone, 1% yeast extract, 0.5% NaCl). At that time, one medium was put in a test tube of 10 mL, and the antibiotics were chloramphenicol (33 mg/L), spectinomycin (100 mg/L), and ampicillin (40 mg/L). Kanamycin (40 mg/L) was added to the medium. After culturing until OD ₆₀₀ reached 0.4-0.6, 0.05 mM IPTG was added and cultured at 20° C. for 2 nights. The cultured bacterial solution was centrifuged to obtain bacterial cells. After adding methanol to the cells and suspending them well, 1 M Tris (pH 7.5) and 1 M NaCl solution were added. Further, chloroform was added, and after well suspended, centrifugation was performed. A carotenoid extract (chloroform layer) was collected.
After the extract solution was concentrated to dryness using an evaporator, it was dissolved in ethyl acetate, and each carotenoid component was separated and purified by HPLC.
HPLC conditions:
HPLC column TSKgel ODS-80Ts 4.6×150mm, 5μm
Mobile phase 0-5 min methanol/water (95:5), 5-10 min gradient from methanol/water (95:5) to methanol/tetrahydrofuran (7:3), 10-18 min methanol/tetrahydrofuran (7: 3), after 18 minutes methanol/water (95:5), flow rate 1.0 ml/min, detection maxplot (280-550 nm)
The composition of each carotenoid was obtained from the peak area of HPLC. In addition, each peak compound was identified by comparison with the retention time of a standard and comparison of absorption curves.
By transgenic E. coli E introduced with three plasmids pUC-CaZEP, pRSF-NsRF-AtCYO1 and CDF-gdh into E. coli (zeaxanthin-producing E. coli) introduced with plasmid pACHP-Zea (Non-Patent Document 6) to synthesize zeaxanthin HPLC analysis of the carotenoids produced was performed. The results are shown in FIG.

(Identification of carotenoids produced by recombinant E. coli)
Carotenoids produced by recombinant E. coli E were cultured by the method described in Non-Patent Document 6 or the following method, separated and purified for each carotenoid, and subjected to UV-VIS and FAB MS analysis. ¹ H-NMR, ¹³ C-NMR and CD analyzes were performed according to. Various carotenoids were identified by these instrumental analyses (spectral data will be described later).

(Method of culturing recombinant E. coli E for identification of novel carotenoids)
Recombinant E. coli E was cultured by the following culture method.
(1) Recombinant E. coli E was inoculated into 100 mL of 2-YT (Amp: 100 μg/mL, Cm: 100 μg/mL, km: 100 μg/mL, Sp: 100 μg/mL). , pre-incubated overnight at 30°C.
(2) 100 mL of preculture medium containing 2-YT (Amp: 100 μg/mL, Cm: 100 μg/mL, km: 100 μg/mL, Sp: 100 μg/mL, IPTG: 50 μg/mL ) and cultured at 20°C for two nights.

(Analysis of carotenoids produced by recombinant E. coli E)
Since the carotenoids produced were not present in the culture supernatant and were contained in the recombinant E. coli E cells, the cultured recombinant E. coli culture (2 L) was centrifuged (7000 rpm, 15 min). , the cells were collected. 50 ml of a solution of CH ₂ Cl ₂ -MeOH (1:1) was added to each centrifuge tube, and extraction was performed for 5 minutes at room temperature while ultrasonically disrupting the cells (× 3 times). (All of the yellow pigment contained in this extraction was extracted.) The extracts were filtered, combined, and once concentrated to dryness. It was then dissolved in 200 ml of EtOAc and shaken in a separatory funnel with an equal volume of H ₂ O to fractionate. The EtOAc layer containing the yellow pigment _was concentrated to dryness ( _0.14 g) and analyzed by silica gel TLC. Yellow spots (=carotenoids) were detected. Therefore, it was developed with a silica gel column {CHROMATOREX FL60D, 20 mm × 200 mm, solvent CH ₂ Cl ₂ :diethylether (6:1)}, and the eluted carotenoids were collected and used as a crude fraction (45.3 mg). This was then developed by silica gel HPLC {Cosmosil 5SL 10 mm×250 mm, 10 mm×250 mm, solvent CH ₂ Cl ₂ :diethylether (4:1), flow rate 3.0 ml/min}.

(Identification of novel carotenoids produced by recombinant E. coli E)
Regarding the retention time of 12 min in silica gel HPLC, there was no corresponding carotenoid library established in our laboratory, so HPLC of the peak with a retention time of 12 min (hereinafter referred to as compound 1) under the same conditions After fractionation, 0.06 mg of pure product was obtained.
Compound 1 was dissolved in MeOH and HRESI-MS was measured. As a result, _a (M+Na) ⁺ ion peak was observed at m/z 605.40498, and the molecular formula _{of 1} was determined to be _C40H54O3 {calcd. for _605.40570 ( _C40H54O3Na )}. Next, ¹ H-NMR (FIG. 6A) and ¹³ C-NMR (FIG. 6B) of 1 were measured in CDCl ₃ in which compound 1 is well soluble and convenient for comparison with other carotenoids.
First, 52 ¹ H, 40 ¹³ C signals were observed in ¹ H, ¹³ C-NMR, respectively, which revealed that the compounds were asymmetric carotenoids.
Next, the ¹ H- ¹ H DQF COZY spectrum, HMQC spectrum, and HMBC spectrum of 1 were measured, and the partial structure existing in compound 1 was analyzed in detail.
First, when ^{the 1} H-NMR, ¹³ C-NMR, ¹ H- ¹ H DQF COZY, HMQC, and HMBC spectra are analyzed in detail, as shown in FIG. 7A, the asymmetric structure from C-1 to C-20 Half of them were confirmed to be zeaxanthin structures. The reported ¹ H, ¹³ C-NMR chemical shift values of zeaxanthin were in good agreement with the C-1 to C-20 peak observed in 1.

Next, we proceeded with the analysis of the measured spectra for the remaining half, C-1' to C-20'. As a result, it was found that the (all E) structure was maintained for the remaining structural olefins (C-7' to C-15') and the methyl groups (C-19', C-20') bonded thereto. , was determined from the J _HH values of olefinic methines and ^{the 13} C chemical shift values of methyl nobles. On the other hand, it became clear that the structure of the β-ionone ring portion was different from that of zeaxanthin, so we proceeded with the structural analysis of the β-ionone ring portion.
In the _HMBC spectrum of 1, C- ₁ ' (d ¹ H- ¹³ C distant spin coupling was observed for _C 41.6), C-2' (d _C 49.8), C-6' (d _C 79.8) (Fig. 7B).

_In addition, from _H -16' (d _H 1.91) bound to C-5', ¹ H- ¹³ C distant spin coupling was observed. Furthermore, ¹ H- ¹³ C distant spin coupling was observed from H-2' (d _H 2.23, d _H 2.48) to ketones at C-1', C-2', C-4' and d _C 198.1. rice field. As a result, it was found that the other terminal structure of 1 also has a six-membered ring structure consisting of C-1' to C-6', and C-3' is a ketone.
In the HMBC spectrum, ¹ H- ¹³ C distant spin bonds were also observed from H-7' (d _H 5.72) to C-6' of the olefin structure (Fig. 7B). was found to be bound at C-6'&#8211;C-7'.
At this point, the only thing that remains unknown in the structure of 1 is the substituent X) attached to C-6' in FIG. 7B, but C Considering the number of , H, and O, it was established that X=OH, and as a result, the structure of compound 1 was uniquely determined as shown in FIG. 7C.
Although the steric structure of the OH group bound to C-6' cannot be discussed in the NMR spectrum analysis so far, since 1 is a compound produced by genetic recombination, the biosynthetic intermediate of 1 is zeaxanthin epoxidase. Since an OH group should have been introduced to C-6' by the action of (ZEP), it is judged to be oriented on the back side of the paper. Compound 1 was a novel compound not registered in the CAS database. Applying the rules of carotenoid nomenclature, the name of Compound 1 is 6'-hydroxy-3'-didehydro-lutein.
For reference, FIG. 3 shows a putative metabolic pathway from the novel carotenoid antheraxanthin.
For reference, the assigned ¹ H, ¹³ C NMR chemical shifts of Compound 1 are shown in Table 3.

(Functional test of novel carotenoid)
Various functions of Compound 1 isolated in Example 4 were analyzed.

(Antioxidant activity of compound 1)
Antioxidant activity of Compound 1 was examined using rat brain lipid peroxidation inhibitory activity and singlet oxygen scavenging activity.

(rat brain lipid peroxidation inhibitory activity)
The amount of malondialdehyde (MDA) produced by the progress of lipid oxidation was measured, and the lipid peroxidation inhibitory activity of compound 1 was evaluated from the degree of progress of lipid oxidation.
The assay was basically performed in disposable culture tubes (φ13 mm×100 mm).
(1) In 0.6 ml of 100 mM phosphate buffer (pH 7.4), 0.05 ml of a methanol solution of the test sample (compound 1 isolated in Example 4), 0.1 ml of 1 mM ascorbic acid (final concentration 100 μM) , and 0.05 ml of _H2O were added. As a control, 0.05 ml of a methanol solution was added instead of the methanol solution of the test sample.
(2) After pre-incubation at 37°C for 5 minutes, the reaction was initiated by adding 0.2 ml of 2.5% (w&#8725;v) rat brain homogenate and incubated at 37°C for 1 hour with shaking. did
(3) Then, 1 ml of a mixture containing 20% (w&#8725;v) trichloroacetic acid and 0.5% (w&#8725;v) 2-thiobarbituric acid (TBA) in 0.2N hydrochloric acid was added to the above reaction solution. The reaction was stopped by adding to
(4) This was boiled at 100°C for 30 minutes to develop color, cooled, and centrifuged at 3000 rpm for 10 minutes.
(5) The absorbance (Abs ₅₃₂ ) at 532 nm, where the reaction product of MDA and TBA has maximum absorption, was measured for the centrifugation supernatant to determine the amount of MDA.
The rat brain homogenate was prepared by homogenizing commercially available frozen rat brain in 67 mM phosphate buffer (pH 7.4) under ice-cooling with a Teflon (registered trademark) homogenizer for about 5 minutes. After diluting this with the same buffer to 10% (w/v), aliquots of 2 ml were dispensed into 15 ml falcon tubes and stored frozen at -80°C until use. At the time of use, it was thawed in a refrigerator at 5°C, and 6 ml of phosphate buffer (pH 7.4) was added to prepare a 2.5% (w/v) rat brain homogenate.
(6) The lipid peroxidation suppression rate (lipid peroxidation suppression rate) was obtained according to the following formula.
(Lipid peroxide production inhibition rate (%)) = {1-(T-B)&#8725;(C-B)}×100
T: A ₅₃₂ in the drug-treated group
C: A ₅₃₂ in the control group (methanol-treated group)
B: A ₅₃₂ in the non-response group (blank group)
From the lipid peroxidation inhibition rate of the compound at various concentrations, the concentration of the compound required to inhibit lipid peroxidation by 50% was calculated as the _IC50 value, which was expressed as rat brain lipid peroxidation inhibition activity.
In this example, a blank without rat brain lipid was used, and its absorbance at 532 nm was set to 0.00. In this assay system, the average absorbance (OD) at 532 nm of the control (drug-free condition) was about 0.37.

(result)
FIG. 8 shows the rat brain lipid peroxidation inhibitory activity of Compound 1. FIG. The _IC50 value for compound 1 was 2.5 μM. Therefore, it was revealed that the tested compound 1 has biolipid peroxidation inhibitory activity. β-carotene used as a positive control was 72 μM. Moreover, astaxanthin did not show inhibitory activity even at 100 µM.

(Singlet oxygen scavenging activity)
The amount of oxidized linoleic acid produced by singlet oxygen was measured.
(1) 80 μl of 0.025 mM methylene blue solution, 100 μl of 0.24 M linoleic acid solution, 40 μl of sample solution (all EtOH solutions), and 180 μl of EtOH were added to a disposable culture tube (400 μl in total) and stirred well.
(2) Singlet oxygen was generated by light irradiation under a fluorescent lamp for 3 hours with a marble placed on the tube to suppress the evaporation of EtOH. The experiment was conducted in a polystyrene foam box so that the amount of light irradiation was constant, and the distance between the fluorescent lamp stand and the sample was set to about 17 cm. The experiment was performed in triplicate, and the average value was used as data.
(3) After 3 hours, 120 μl of the reaction solution was transferred to another tube, and 3,480 μl of EtOH was added to the diluted solution (= 30-fold dilution), and the absorbance at λ ₂₃₅ nm (Abs ₂₃₅ ) was measured, The amount of oxidized linoleic acid produced by singlet oxygen was determined.
(4) The ¹ O ₂ scavenging rate of the sample was obtained by calculating the formula {100−(S−B2)/(C−B1)}×100.
Here, S (sample) is A ₂₃₅ with light irradiation and sample addition, C (Control) is A ₂₃₅ with light irradiation and no sample addition, and B1 (Blank 1) is without light irradiation and no sample addition. A ₂₃₅ is shown, and B2 (Blank 2) shows A ₂₃₅ with no light irradiation and sample addition. The control absorbance was 1.2, and the blank absorbance was about 0.3.
(5) Evaluation of each compound was performed in singles for C, B1, and B2, and in triplicate for each concentration for S. Using the data obtained (average absorbance obtained for S), _a regression line was determined with % inhibition on the vertical axis and compound concentration on the ^horizontal _axis . % inhibitory concentration) was determined.
Experiments were carried out on 1 μM, 10 μM, and 100 μM solutions (final concentrations) adjusted based on the amount of substance in each sample calculated from the absorbance at the absorption maximum wavelength and the molecular extinction coefficient (ε, mM ^-1 cm ^-1 ) It was performed in triplicate. β-carotene and astaxanthin, which are carotenoids widely distributed in nature, were used as positive controls. _IC50 was calculated using the generated regression line.

(result)
FIG. 9 shows the results of ^{the 1} O ₂ scavenging activity of Compound 1. The _IC50 values were 3.2 μM for compound 1, 12 μM for β-carotene and 1.2 μM for astaxanthin, respectively. Compound 1 exhibited only weak activity compared to the positive control astaxanthin, but exhibited strong activity compared to β-carotene.

(Adipocyte differentiation-inducing activity of compound 1)
Compounds that induce adipocyte differentiation (eg, PPARg agonists) are expected to have the effect of lowering blood sugar levels and improving diabetes. Therefore, whether or not compound 1 has such an effect was examined using mouse 3T3-L1 cell differentiation induction experiments. Oil red staining was used to quantify the degree of differentiation. All experiments were performed in triplicate for significance testing.

(cell culture)
(1) 3T3-L1 cells at passage number 10 or less were seeded in a 12-well plate (DMEM + 10% Bovine Calf Serm, BCS) at 4.0 x ¹⁰⁶ cells/well.
(2) The medium was exchanged according to the experimental schedule shown in Table 4, and compound 1 isolated in Example 4 (final concentrations 2.5 mM, 5.0 mM, 10 mM) or rosiglitazone (final concentrations 10 nM, 1000 nM) as a positive control ) was dissolved in DMSO, and 1 μL of the sample was added to 1 mL of the medium and used for Oil red O staining. Each concentration was performed in triplicate.
Rosiglitazone is known as a representative PPARg agonist and is also used as a diabetes drug.
The composition of differentiation-inducing medium I is DMEM + 10% Fetalcalf serum (FBS), insulin (6 μg/mL), 0.6 μM dexamthazone, 0.5 mM IBMX, and the composition of differentiation-inducing medium II is DMEM + 10% FBS, insulin (6 μg/mL).

(Oil red O staining)
(1) 30 mg of OilRed O (Wako, for pathological research, Code No. 154-02072) and 10 mL of 100% isopropanol were placed in a 50 mL Falcon tube and vortexed for 30 seconds. 9 mL of it was transferred to another falcon tube, 6 mL of MilliQ water was added, centrifuged, and allowed to stand to precipitate the insoluble Oil Red O powder. It was then filtered through a 0.45 μm filter. If the liquid after filtration is exposed to the air for a long time (about 2 hours), crystals may precipitate, so it was prepared on the day.
(2) The medium of the cultured 3T3-L1 cells was removed by aspiration, and the cells were washed twice with PBS(-) (1 mL/well).
(3) 1 mL of 4% Paraformaldehyde Phosphate Buffer Solution (PFA) (Wako, for tissue fixation, Code No. 163-20145) was added to each well and allowed to stand at room temperature for 45 minutes.
(4) 4% PFA was removed by aspiration and washed twice with PBS(-) (1 mL/well). After that, 60% isopropanol (1 mL/well) was added and allowed to stand at room temperature for 5 minutes.
(5) 1 mL of the previously prepared Oil red O staining solution was added to each tube and allowed to stand at room temperature for 10 minutes for staining. The staining solution was removed by suction, washed once with 60% isopropanol (1 mL/well), and removed by suction.
(6) Further washed twice with PBS(-) (1 mL/well) and observed under a microscope in PBS(-).
(7) After observation, PBS(-) was removed by aspiration, 100% isopropanol (1 mL/well) was added, and after standing for about 30 minutes, pipetting was performed to dissolve the staining solution. From here, 200 μL was transferred to a 96-well plate, and the absorbance at 492 nm was measured using a microplate reader.
The control value (C) is the average absorbance value of the group to which only DMSO was added without the sample, and the value obtained by subtracting C from the average absorbance value (T) of each concentration of the measured sample and dividing it by C ((TC) /C) was defined as the rate of increase in absorbance, and those with a significantly greater rate of increase than 1 were determined to have differentiation-inducing activity (in FIG. 10, those marked with ** indicate significant differences). For statistical processing, one-way analysis of variance and multiple comparisons were performed for all data. Statistical significance was indicated at P < 0.05.

(result)
Experimental results are shown in FIG. Compound 1 showed adipocyte differentiation-inducing activity of 3T3-L1 cells in a concentration-dependent manner in the range of 2.5 &#8211;
In addition, the strength of induction activity at the most effective concentration was comparable to that of rosiglitazone used as a positive control.

The present invention can provide a production method capable of synthesizing any one or more of carotenoid, antheraxanthin and violaxanthin represented by formula (1).

Claims (10)

A carotenoid represented by formula (1) or formula (1A').

An antioxidant containing a carotenoid represented by formula (1).

A lipid peroxidation inhibitor containing a carotenoid represented by formula (1).

A singlet oxygen quencher containing a carotenoid represented by formula (1).

An adipocyte differentiation inducer containing a carotenoid represented by formula (1).

Recombinant cells derived from Escherichia coli into which the following genes (A) or (B) have been introduced:
(A)
(1) crtE gene, crtB gene, crtI gene, crtY gene and crtZ gene derived from Pantoea ananatis ,
(2) CaZEP gene derived from Capsicum annuum , and (3) AtCYO1 gene derived from Arabidopsis thaliana , or
(B)
(1) crtE gene, crtB gene, crtI gene, crtY gene and crtZ gene derived from Pantoea ananatis , and
(2) CaZEP gene derived from Capsicum annuum .
7. The recombinant cell derived from Escherichia coli according to claim 6, further into which an IDI gene derived from Haematococcus pluvialis has been introduced.
8. The recombinant cell derived from Escherichia coli according to claim 6 or 7, further into which the NsRF gene derived from Nostoc sp. PCC7120 and/or the gdh gene derived from Bacillus subtilis has been introduced.
A method for producing carotenoids, which comprises obtaining carotenoids from a culture or bacterial cells obtained by culturing the E. coli-derived recombinant cells according to any one of claims 6 to 8.
Carotenoids, antheraxanthins and viola represented by formula (1) or formula (1A') from cultures or cells obtained by culturing the E. coli -derived recombinant cells according to any one of claims 6 to 8 A method for producing a carotenoid, characterized by obtaining one or more of xanthines, or obtaining one or more of antheraxanthins and violaxanthins.

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