Method for producing carotenoids having allene structure

Abstract

To provide a method for producing carotenoid having an allene structure of neoxanthin or the like while using a microorganism such as coliform bacillus and budding yeast or algae as a host.SOLUTION: A method for producing carotenoid having an allene structure in which a host having farnesyl diphosphate productivity into which (1) a gene group encoding a biosynthetic pathway from farnesyl diphosphate to zeaxanthin, (2) a gene encoding zeaxanthin epoxidase, and (3) a gene encoding neoxanthin synthase are introduced is cultured, and carotenoid having an allene structure is extracted from a host after being cultured. The above described (2) is a gene encoding ZEP derived from a higher plant, and the above described (3) is a gene encoding VDL1 derived from diatoms.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to a method for producing carotenoids having an allene structure, which are expected to be useful for human health, using a host. [Background technology]

[0002] Carotenoids are natural yellow-orange-red pigments produced by all photosynthetic organisms (plants, algae, etc.), as well as some bacteria, fungi, and yeasts. To date, more than 750 types of carotenoids have been isolated and identified in nature. Of these, many carotenoids derived from plants and algae, photosynthetic organisms familiar to us, have been reported.

[0003] The carotenoids in leaves, which are photosynthetic organs in plants, are usually the same regardless of plant species (Patent Document 1, Non-Patent Document 1). This biosynthetic pathway is shown in Figure 1 along with its chemical structure. Among these carotenoids, those with a clear color are those in the biosynthetic pathway after lycopene (also called lycopene). Among these, a high proportion of carotenoids have been commercialized, and these include lycopene, β-carotene, α-carotene, lutein, zeaxanthin, and β-cryptoxanthin, and they have been reported to have functionality useful for promoting human health (Non-Patent Document 2).

[0004] Not only the carotenoids in Figure 1, but many other carotenoids that are expected to have functions (hereinafter referred to as "functional carotenoids") are generally expensive due to the high production costs. Escherichia coli ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae), biotechnology research has been actively conducted to produce carotenoids cheaply using hosts such as Escherichia coli and Saccharomyces cerevisiae. Although Escherichia coli and Saccharomyces cerevisiae do not originally have the ability to produce carotenoids, they do possess the carotenoid precursor farnesyl diphosphate (FPP), and it is therefore believed that it would be possible to produce various functional carotenoids by introducing the carotenoid biosynthesis genes downstream from FPP. However, of the leaf-derived carotenoids shown in Figure 1, only lycopene, β-carotene, and zeaxanthin can be efficiently produced using microbial hosts. The main reason for this is that these three carotenoids can be produced not only in plants but also in soil bacteria that belong to the same class as Escherichia coli. Pantoea ananatis Since carotenoids are also produced in some microorganisms, including crt This is because it is possible to use the gene group. crt The functions of the Crt enzymes encoded by the gene clusters are indicated in brackets in FIG.

[0005] On the other hand, other carotenoids derived from leaves that have not been commercialized include antheraxanthin, violaxanthin, and neoxanthin. Of these, antheraxanthin and violaxanthin are not commercially available. Gentiana lutea ) flower derived ZEP (zeaxanthin epoxidase) gene ( GlZEP ), or red bell pepper (paprika; Capsicum annuum ) derived from ZEP gene( CaZEP It has been reported that the biosynthesis of antheraxanthin and violaxanthin can be carried out by introducing the enzyme into recombinant Escherichia coli that efficiently produces zeaxanthin (Non-Patent Documents 2 and 3). However, many enzymes derived from various plants or algae have not been reported. ZEPIt has been found that the gene does not function (is not expressed) even in E. coli. For example, in the case of diatoms, a type of algae ( Phaeodactylum tricornutum ) and Marchantia polymorpha ( Marchantia polymorpha ) derived from ZEP The gene did not function in E. coli (Non-Patent Documents 3 and 4). Therefore, it is still not easy to efficiently produce Violaxanthin in microorganisms such as E. coli.

[0006] On the other hand, there have been no reports of neoxanthin production in microbial hosts, including Escherichia coli and Saccharomyces cerevisiae. Neoxanthin is a carotenoid that has an allene (C=C=C) structure in its molecule (see Figure 1), and carotenoids with this structure are known to have health benefits, such as preventing obesity. For this reason, there has been a demand for the production of carotenoids with an allene structure, such as neoxanthin, in microorganisms. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2020-142992 A [Non-patent literature]

[0008] [Non-Patent Document 1] Norihiko Misawa, Bioengineering 93: 403-406, 2015 [Non-Patent Document 2] Akiko Kubo, Takehiko Sahara, Miho Takemura, Norihiko Misawa, Smart Cell Industry - Prospects for Material Production Using Microbial Cells -, Part 3: Approaches to Industrial Applications, Chapter 8: Microbial Production of Plant-derived Carotenoids, pp.195-205, CMC Publishing, 2018. [Non-Patent Document 3] M. Takemura et al, Applied Microbiology and Biotechnology, 103: 9393-9399, 2019. [Non-Patent Document 4] M. Dambek et al, Journal of Experimental Botany, 63: 5607-5612, 2012. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a method for producing a carotenoid having an allene structure, such as neoxanthin, using a microorganism such as Escherichia coli or Saccharomyces cerevisiae or an alga as a host. [Means for solving the problem]

[0010] The present inventors have conducted intensive research to achieve the above object, and have found that, in addition to a group of genes encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin and a gene encoding zeaxanthin epoxidase derived from a higher plant, a gene encoding zeaxanthin epoxidase derived from a diatom is also included. VDL1 The inventors discovered that by introducing the gene into a host capable of producing farnesyl diphosphate, such as Escherichia coli, neoxanthin can be produced efficiently, and that de-epoxyneoxanthin, which is not observed in plant leaves, can also be produced, thereby completing the present invention.

[0011] That is, the present invention provides: The following genes or gene groups (1) to (3): (1) Gene cluster encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin (2) The gene encoding zeaxanthin epoxidase (3) A gene encoding neoxanthin synthase a host having an ability to produce farnesyl diphosphate, into which is introduced a compound represented by the formula (I), and a carotenoid having an allene structure is extracted from the host after the culture, (2) the gene encoding zeaxanthin epoxidase is a gene encoding ZEP derived from a higher plant; (3) A method for producing a carotenoid having an allene structure, characterized in that the gene encoding neoxanthin synthase is a gene encoding VDL1. Effect of the Invention

[0012] INDUSTRIAL APPLICABILITY The present invention makes it possible to efficiently and inexpensively mass-produce carotenoids having an allene structure, such as neoxanthin and de-epoxyneoxanthin, which have functional properties such as obesity prevention effects. [Brief description of the drawings]

[0013]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0014] In the present invention, (1) a group of genes encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin, (2) a gene encoding zeaxanthin epoxidase, and (3) a gene encoding neoxanthin synthase are introduced into a host.

[0015] (1) Gene cluster encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin The gene cluster encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin includes the gene encoding GGPP synthase (CrtE), which synthesizes geranylgeranyl diphosphate (GGPP) from farnesyl diphosphate (FPP) ( CrtE ), and the gene encoding phytoene synthase (CrtB) that synthesizes phytoene from GGPP ( CrtB ), a gene encoding phytoene desaturase (CrtI) that synthesizes lycopene from phytoene ( CrtI ), and the gene encoding lycopene β-cyclase (CrtY) that synthesizes β-carotene from lycopene ( CrtY ) and the gene encoding β-carotene hydroxylase (CrtZ), which synthesizes zeaxanthin from β-carotene ( CrtZ The origin of the carotenoid biosynthesis gene cluster is not particularly limited, but it is possible to use the soil bacterium Pantoea ananatis ( Pantoea ananatis ) is preferably used.

[0016] (2) The gene encoding zeaxanthin epoxidase The gene encoding zeoxanthin epoxidase was identified in paprika (red pepper; Capsicum annuum )-derived ZEP (zeaxanthin epoxidase)-encoding gene ( CaZEP ), Gentian ( Gentiana lutea ) flower derived ZEP gene( GlZEP), Arabidopsis thaliana ( Arabidopsis thaliana ) derived from ZEP gene( AtZEP ZEP derived from higher plants such as , can be used, but among these, the ZEP gene derived from paprika is preferably used because of its excellent production efficiency. As the gene encoding ZEP derived from paprika, a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, a gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 and having zeoxanthin epoxidation activity that epoxidizes zeaxanthin to synthesize violaxanthin, and a gene encoding a protein consisting of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more homology with the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 and having zeoxanthin epoxidation activity that epoxidizes zeaxanthin to synthesize violaxanthin are preferred. A preferred example of such a gene is a gene consisting of the base sequence shown in SEQ ID NO: 3. Also preferred is a gene that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 3 and encodes a protein having zeaxanthin epoxidation activity, and a gene consisting of a base sequence having 90% or more, preferably 95% or more, more preferably 98% or more homology with the gene consisting of the base sequence shown in SEQ ID NO: 3 and encodes a protein having zeaxanthin epoxidation activity that epoxidizes zeaxanthin to synthesize violaxanthin.

[0017] (3) A gene encoding neoxanthin synthase The gene encoding neoxanthin synthase was found in the diatom ( Phaeodactylum tricornutum ) derived from VDL1 (violaxanthin de-epoxidase-like protein 1) gene is used. VDL1Preferred genes are genes that encode a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, genes that encode a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and that have neoxanthin synthesis activity to synthesize neoxanthin from violaxanthin, and genes that encode a protein consisting of an amino acid sequence that has a homology of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more to the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 and that have neoxanthin synthesis activity to synthesize neoxanthin from violaxanthin. As such a gene, a gene consisting of the base sequence shown in SEQ ID NO: 1 is preferred. Also, a gene that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 1 and codes for a protein having neoxanthin synthesis activity that synthesizes neoxanthin from violaxanthin, and a gene consisting of a base sequence having 90% or more, preferably 95% or more, more preferably 98% or more homology with the gene consisting of the base sequence shown in SEQ ID NO: 1 and codes for a protein having neoxanthin synthesis activity that synthesizes neoxanthin from violaxanthin are preferred. Neoxanthin synthase synthesizes neoxanthin from violaxanthin. Zeaxanthin is converted to antheraxanthin by zeaxanthin epoxidase, and violaxanthin is further synthesized, while neoxanthin synthase synthesizes de-epoxyneoxanthin from antheraxanthin.

[0018] (4) Isopentenyl diphosphate isomerase gene ( IDI gene) Isopentenyl diphosphate isomerase converts isopentenyl diphosphate (isopentenyl pyrroline: IPP) to dimethylallyl diphosphate (DMAPP) during FPP production in Escherichia coli and Saccharomyces cerevisiae. IDI Genes are crtBy expressing the IDI gene together with a group of genes, it is possible to efficiently produce carotenoids. The origin of the IDI gene is not particularly limited, but for example, Haematococcus pluvialus Origin IDI (IPP isomerase) is preferably used. Isopentenyl diphosphate isomerase adjusts the ratio of IPP to dimethylallyl diphosphate (DMAPP) (Patent Document 1). There are two types of isopentenyl diphosphate isomerase, type 1 and type 2, which are structurally different from each other. IDI The genes are sometimes found together in the carotenoid biosynthesis gene cluster of carotenoid-producing bacteria belonging to the α-proteobacteria or γ-proteobacteria classes (Norihiko Misawa, Oleoscience 9 (9): 385-391, 2009). Regardless of whether it is type 1 or type 2, IDI When the gene was introduced into E. coli together with terpene biosynthetic genes such as carotenoids and sesquiterpenes and expressed, IDI It has been shown that the amount of terpenes produced increases compared to when the gene is not introduced (S. Kajiwara, P.D. Fraser, K. Kondo and N. Misawa, Biochem J ., 324, 421-426, 1997). By introducing and coexpressing the gene group coding for the biosynthetic pathway from farnesyl diphosphate to zeaxanthin, CaZEP gene and IDI gene, violaxanthin is efficiently produced, and neoxanthin is produced in high yields using violaxanthin as a substrate.

[0019] (Transformation) The group of genes encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin, the gene encoding ZEP derived from paprika, and the gene encoding VDL1 can be introduced into a host capable of producing farnesyl diphosphate, such as Escherichia coli, Saccharomyces cerevisiae, algae, etc., by a known method. Hereinafter, a transformation method using Escherichia coli as a host will be described as an example.

[0020] When E. coli is used as a host, the method of introducing the exogenous gene is not particularly limited, and the exogenous gene to be introduced may be made to be expressible by using an appropriate promoter and terminator by a known method, inserted into a vector for transformation of E. coli to prepare a plasmid, and then the plasmid may be introduced into E. coli cells. When multiple genes are introduced, the genes may be introduced using a single transformation vector, or each of the genes may be introduced using a different transformation vector.

[0021] The vector for transforming E. coli may be appropriately selected depending on the number and type of genes to be introduced, but vectors for expressing proteins in E. coli are preferred, such as pUC18, pACYC184, and RSFDuet-1. Any promoter may be used as long as it can be expressed in E. coli. For example, T7 Promoter, lacZ Promoter, tac Among them, it is preferable that the introduced gene is highly expressed in E. coli. tac In addition, when it is desired that the introduced gene is expressed transiently in E. coli, it is preferable to use an inducible promoter, and an inducible promoter using IPTG or the like is preferable. T7 A promoter is more preferable. The terminator can be appropriately selected depending on the gene to be introduced, and examples thereof include T7 terminator, rrnB Terminator, etc.

[0022] In order to efficiently select E. coli cells into which a foreign gene of interest has been introduced by an E. coli transformation vector, various selection marker genes may be used. The selection marker gene is not particularly limited, and any known selection marker gene can be used. For example, it is preferable to use a gene that confers drug resistance as a selection marker, and to introduce a plasmid or the like containing this selection marker gene and a foreign gene into E. coli cells as an E. coli transformation vector. This allows the selection of E. coli cells into which a foreign gene has been introduced efficiently based on the expression of the selection marker gene. Examples of such selection marker genes include various drug resistance genes. More specifically, examples include an ampicillin resistance gene, a kanamycin resistance gene, and a tetracycline resistance gene. In addition, it is preferable that the selection marker gene has a promoter and a terminator for expressing the gene upstream and downstream.

[0023] The method for introducing the transformation vector thus prepared into E. coli is not particularly limited, and may be carried out by a known method such as the heat shock method. Expression of the exogenous gene in the recombinant E. coli can be confirmed by, for example, Northern blotting, RT-PCR, Western blotting, etc. Furthermore, production of neoxanthin, de-epoxyneoxanthin, etc. in E. coli can be confirmed by, for example, HPLC analysis, etc.

[0024] The method for culturing the recombinant E. coli is not particularly limited and any known culturing method can be used. Since the recombinant E. coli after culturing contains carotenoids having an allene structure such as neoxanthin and de-epoxyneoxanthin, these carotenoids can be obtained by known extraction methods. EXAMPLES

[0025] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0026] <Example 1> (Construction of a plasmid for producing neoxanthin) diatom Phaeodactylum tricornutum of VDL1 (violaxanthin de-epoxidase-like protein 1) gene ( PtVDL1 The DNA fragment of the gene was chemically synthesized using a blueprint modified to have a base sequence suitable for the codon usage of E. coli. The base sequence is shown in SEQ ID NO:1. Capsicum annuum of ZEP (zeaxanthin epoxidase) gene ( CaZEP The DNA fragment of PtVDL1 gene was chemically synthesized using a blueprint modified to have a base sequence suitable for the codon usage of E. coli. The base sequence is shown in SEQ ID NO: 3. The amino acid sequences of PtVDL1 and CaZEP (SEQ ID NO: 2 and 4) are available from DDBJ under accession numbers B7G087_PHATC and XP_016561102.1, respectively. CaZEP The artificial gene fragment has a 5' end Spe I and 3' end Hin Addition of a dIII site, PtVDL1 The artificial gene fragment has a 5' end Eco RI and 3' end Sal I site was added.

[0027] pACYC184 and pUC18 were used as vectors for transforming E. coli (Figure 3). Spe I and Hin Digested with dIII CaZEP The fragment was excised and transformed into the plasmid pACHP-Zea for zeaxanthin production (M. Takemura et al., Tetrahedron Letters, 56: 6063-6065, 2015.) prepared according to a known method. Spe I- Hin This was inserted into the dII site to generate the plasmid pACHP-Viol (Fig. 4A ). Eco RI and Sal Diatom cut with I PtVDL1 The gene fragment was excised and inserted into the vector pUC18. Eco RI-Sal I site to generate the plasmid pUC-PtVDL1 (Figure 4B).

[0028] tomato ( Solanum lycopersicum )of NSY gene( SlNSY Genes; F. Bouvier et al, European Journal of Biochemistry, 267: 6346-6352, 2000.) and NXD1 gene( SlNXD1 The DNA fragment of the gene (H. Neuman et al, The Plant Journal, 78: 80-93, 2014.) was chemically synthesized using a blueprint modified to match the codon usage of E. coli. PtVDL1 As with the gene fragments, each gene fragment was inserted into the vector pUC18. Eco RI- Sal I site to generate plasmids pUC-SlNSY and pUC-SlNXD1.

[0029] <Example 2> (Transformation of E. coli) E. coli was transformed by a known method using the plasmids pACHP-Viol and pUC-PtVDL1 (or pUC-SlNSY or pUC-SlNXD1) prepared in Example 1. Specifically, the transformation was carried out as follows. E. coli (JM101(DE3)), SOB medium (2% bactotryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 ), then cultured in TB buffer (10 mM PIPES (pH 6.7), 15 mM CaCl 2 , 250 mM KCl, 55 mM MnCl 2 4H 2The E. coli was treated with 100 mM EDTA (1.0 mM NaCl). The plasmid pACHP-Viol prepared in Example 1 was then mixed with the treated E. coli and allowed to stand on ice. After a heat shock of 42°C, the E. coli was cultured in SOC medium and plated on a drug-containing medium. After overnight culture, drug-resistant colonies were obtained as transformed E. coli (genetically modified E. coli). Chloramphenicol (30 mg / L) was added to the medium as a drug (antibiotic). The E. coli (JM101(DE3)) that had pACHP-Viol and produced violaxanthin was grown in SOB medium (2% bactotryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 ), then cultured in TB buffer (10 mM PIPES (pH 6.7), 15 mM CaCl 2 , 250 mM KCl, 55 mM MnCl 2 4H 2 The E. coli was treated with pUC-PtVDL1 (or pUC-SlNSY or pUC-SlNXD1) plasmid prepared in Example 1 and the treated E. coli was mixed and left to stand on ice. After heat shock at 42°C, the E. coli was cultured in SOC medium and plated on drug-containing medium. After overnight culture, drug-resistant colonies were obtained as transformed E. coli (genetically modified E. coli). Chloramphenicol (30 mg / L) and ampicillin (40 mg / L) were added to the medium as drugs (antibiotics).

[0030] <Example 3> The recombinant E. coli was cultured at 37°C in 2YT (2 x YT) medium (1.6% bactotryptone, 1% yeast extract, 0.5% NaCl). Two 2-liter (L) Erlenmeyer flasks were used, each containing 400 mL of medium, and antibiotics chloramphenicol (30 mg / L) and ampicillin (40 mg / L) were added to the medium. OD 600After culturing until the pH reached 0.4-0.6, 0.05 mM IPTG was added and the culture was continued at 20°C for 2 nights. The cultured bacterial solution was centrifuged to obtain bacterial cells. Methanol was added to the bacterial cells, which were then thoroughly suspended, followed by the addition of 1 M Tris (pH 7.5) and 1 M NaCl solutions. Chloroform was then added, the cells were thoroughly suspended, and the mixture was centrifuged. The carotenoid extract (chloroform layer) was collected.

[0031] The above extract solution was concentrated to dryness using an evaporator, then dissolved in ethyl acetate, and each carotenoid component was separated and purified by HPLC. HPLC conditions: HPLC column: Acquity 1.7 μm BEH UPLC C18 (2.1 id x 100 mm) Mobile phase 0-15 min Solvent CH 3 CN:H 2 O (85:15), flow rate 0.2 ml / min, detection 450 nm NMR was measured using a Varian INOVA 500 MHz CDCL3 solvent.

[0032] Carotenoids produced by recombinant E. coli carrying two plasmids, pACHP-Viol and pUC-PtVDL1, were analyzed by HPLC and NMR. The carotenoids produced by the recombinant E. coli were Zeaxanthin, Violaxanthin, Neoxanthin, Deepoxyneoxanthin, etc. The results of this HPLC analysis are shown in Figure 2(A). Comparison with the authentic samples revealed peaks with the same retention times as Neoxanthin and Deepoxyneoxanthin. The NMR analysis of these peaks, as shown in Figure 2(B)(C), showed that they were consistent with the previously reported Neoxanthin and Deepoxyneoxanthin. From the above, it was found that Neoxanthin and Deepoxyneoxanthin were produced in the recombinant E. coli carrying pACHP-Viol and pUC-PtVDL1. On the other hand, no conversion products were observed for Violaxanthin in the recombinant E. coli carrying the plasmids pACHP-Viol and pUC-SlNSY, and in the recombinant E. coli carrying the plasmids pACHP-Viol and pUC-SlNXD1.

[0033] diatom Phaeodactylum tricornutum Origin ZEP Although it was known that the gene did not function in E. coli (Non-Patent Documents 3 and 4), VDL1 It was unexpected that the genes could be efficiently expressed in E. coli to produce neoxanthin and deepoxyneoxanthin, and this enabled the production of neoxanthin and deepoxyneoxanthin, the production of which had never been reported in microorganisms including E. coli and Saccharomyces cerevisiae. The synthetic pathway of carotenoids having an allene structure according to the production method of the present invention is shown in Figure 5. This method allows the production of deepoxyneoxanthin, which is not observed in plant leaves, and since neoxanthin is synthesized not only from violaxanthin but also from deepoxyneoxanthin, it is possible to efficiently produce neoxanthin. [Industrial Applicability]

[0034] The present invention is useful as a method for easily producing functional carotenoids at low cost using a conventional culture apparatus and for providing a stable supply of the same.

Claims

1. The following genes or gene groups (1), (2), and (3): (1) Gene cluster encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin (2) A gene encoding zeaxanthin epoxidase (3) A gene encoding neoxanthin synthase a host having an ability to produce farnesyl diphosphate, into which is introduced a compound represented by the formula (I), and a carotenoid having an allene structure is extracted from the host after the culture, (2) The gene encoding zeaxanthin epoxidase is a gene encoding ZEP derived from a higher plant, The method for producing a carotenoid having an allene structure (3), wherein the gene encoding neoxanthin synthase is a gene encoding VDL1 derived from a diatom.

2. The method for producing a carotenoid having an allene structure according to claim 1, wherein the carotenoid having an allene structure is neoxanthin.

3. The method for producing a carotenoid having an allene structure according to claim 1, wherein the carotenoid having an allene structure is de-epoxyneoxanthin.

4. (1) The method for producing a carotenoid having an allene structure according to any one of claims 1 to 3, wherein the gene group encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin comprises a gene encoding GGPP synthase (CrtE), a gene encoding phytoene synthase (CrtB), a gene encoding phytoene desaturase (CrtI), a gene encoding lycopene β-cyclase (CrtY), and a gene encoding β-carotene hydroxylase (CrtZ).

5. 5. The method for producing a carotenoid having an allene structure according to any one of claims 1 to 4, wherein the gene encoding VDL1 is any one of the following genes (a) to (f): (a) a gene consisting of the base sequence shown in SEQ ID NO: 1 (b) a gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the gene having the base sequence shown in SEQ ID NO:1 and encodes a protein having neoxanthin synthesis activity. (c) a gene having a base sequence having 90% or more identity with the gene having the base sequence shown in SEQ ID NO: 1 and encoding a protein having neoxanthin synthesis activity (d) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:2 (e) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having neoxanthin synthesis activity. (f) a gene encoding a protein having an amino acid sequence having 90% or more identity with the protein having the amino acid sequence shown in SEQ ID NO:2 and having neoxanthin synthesis activity;

6. 6. The method for producing a carotenoid having an allene structure according to any one of claims 1 to 5, wherein the gene encoding a ZEP derived from a higher plant is a gene encoding one type of ZEP selected from the group consisting of paprika, Arabidopsis thaliana, and gentian.

7. 7. The method for producing a carotenoid having an allene structure according to claim 6, wherein the gene encoding ZEP derived from a higher plant is a gene encoding ZEP derived from paprika.

8. 8. The method for producing a carotenoid having an allene structure according to claim 7, wherein the gene encoding ZEP derived from paprika is any one of the following genes (g) to (l): (g) a gene consisting of the base sequence shown in SEQ ID NO:3 (h) a gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the gene having the base sequence shown in SEQ ID NO:3 and encodes a protein having zeoxanthin epoxidation activity. (i) a gene having a base sequence having 90% or more identity with a gene having the base sequence shown in SEQ ID NO: 3 and encoding a protein having zeoxanthin epoxidation activity; (j) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:4 (k) a gene encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 and having zeoxanthin epoxidation activity. (l) a gene encoding a protein having an amino acid sequence having 90% or more identity with the protein having the amino acid sequence shown in SEQ ID NO:4 and having zeoxanthin epoxidation activity;

9. The host further (4) A gene encoding isopentenyl diphosphate isomerase 9. The method for producing a carotenoid having an allene structure according to claim 1, wherein the allene structure is introduced into the carotenoid.

10. 10. The method for producing a carotenoid having an allene structure according to any one of claims 1 to 9, wherein the host is a microorganism.

11. The method for producing a carotenoid having an allene structure according to claim 10, wherein the microorganism is Escherichia coli.

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