JP2022095080A - Method for producing carotenoid having allene structure - Google Patents

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

The present invention relates to a method for producing a carotenoid having an allene structure, which is expected to be useful for human health, using a host.

Carotenoids are yellow-orange-red natural pigments produced by all photosynthetic organisms (plants, algae, etc.) and some bacteria, molds, yeasts, etc. To date, more than 750 species of carotenoids have been isolated and identified from nature. Of these, many carotenoids derived from plants and algae, which are photosynthetic organisms familiar to us, have also been reported.

Leaf carotenoids, which are photosynthetic organs in plants, are usually the same regardless of the plant species (Patent Document 1, Non-Patent Document 1). This biosynthetic pathway is shown in FIG. 1 together with the chemical structure. Among these carotenoids, those with a clear color are carotenoids of the biosynthetic pathway after Lycopene (lycopene; also called lycopene). The proportion of commercially available carotenoids is high among these, Lycopene, β-Carotene (β-carotene), α-Carotene (α-carotene), Lutein (lutein), Zeaxanthin (zeaxanthin), β-Cryptoxanthin. It has been reported that (β-cryptoxanthin) corresponds to it and has a function useful for promoting human health (Non-Patent Document 2).

Not limited to the carotenoids shown in FIG. 1, many carotenoids (hereinafter referred to as “functional carotenoids”) that are expected to have functionality are generally expensive because of their high manufacturing costs. Therefore, in recent years, biotechnology research for inexpensive production of carotenoids using microorganisms, particularly hosts such as Escherichia coli and Saccharomyces cerevisiae , has been actively conducted. Although Escherichia coli and germinated yeast do not originally have the ability to produce carotenoids, they do have the carotenoid precursor farnesyl diphosphate (FPP), so the carotenoid biosynthetic genes downstream from the FPP should be introduced. If so, it is considered possible to produce various functional carotenoids. However, among the leaf-derived carotenoids shown in FIG. 1, those that can be efficiently produced by a microbial host are limited to Lycopene, β-Carotene, and Zeaxanthin. The main reason is that these three carotenoids are produced not only by plants but also by some microorganisms such as Pantoea ananatis , a soil bacterium belonging to the same class as Escherichia coli, so these carotenoid biosynthetic genes This is because a group ( crt gene group) can be used. The functions of the Crt enzyme encoded by these crt genes are shown in parentheses in FIG.

On the other hand, among other leaf-derived carotenoids, those that have not been commercialized include Antheraxanthin, Violaxanthin, and Neoxanthin. Of these, for Antheraxanthin and Violaxanthin, ZEP (zeaxanthin epoxidase) gene ( GlZEP ) derived from the flower of Lindou ( Gentiana lutea ) or ZEP gene ( CaZEP ) derived from red bell pepper (Paplica; Capsicum annuum ). , Zeaxanthin has been reported to be able to be introduced into recombinant Escherichia coli and the like for efficient production to carry out biosynthesis of Antheraxanthin and Violaxanthin (Non-Patent Documents 2 and 3). However, it is known that many ZEP genes derived from various plants or algae do not function (do not express function) even in E. coli. For example, the ZEP gene derived from diatom (diatom; Phaeodactylum tricornutum ) and liverwort ( Marchantia polymorpha ), which are a kind of algae, did not function in Escherichia coli (Non-Patent Documents 3 and 4). Therefore, it is not always easy to efficiently produce Violaxanthin with microorganisms such as Escherichia coli.

On the other hand, regarding Neoxanthin, there are no reports of production in microbial hosts including Escherichia coli and Saccharomyces cerevisiae. Neoxanthin is a carotenoid with an allene (C = C = C) structure in the molecule (see Fig. 1), and it has been found that carotenoids with such a structure have health-friendly functionality such as obesity prevention. There is. Therefore, it has been desired to produce carotenoids having an allene structure such as neoxanthin by microorganisms.

Japanese Unexamined Patent Publication No. 2020-142992

Norihiko Misawa, Biotechnology 93: 403-406, 2015 Akiko Kubo, Takehiko Sahara, Miho Takemura, Norihiko Misawa, Smart Cell Industry-Prospects for Material Production Using Microbial Cells-, Volume 3 Approaches to Industrial Applications, Chapter 8 Microbial Production of Plant-Derived Carotenoids, pp.195- 205, CMC Publishing, 2018. M. Takemura et al, Applied Microbiology and Biotechnology, 103: 9393-9399, 2019. M. Dambek et al, Journal of Experimental Botany, 63: 5607-5612, 2012.

An object of the present invention is to provide a method for producing a carotenoid having an allen structure such as neoxanthin, using a microorganism such as Escherichia coli or budding yeast or algae as a host.

As a result of diligent research to solve the above problems, the present inventors have conducted diatoms together with a gene group encoding a biosynthetic pathway from farnesyl diphosphate to zeaxanthin, a gene encoding zeaxanthin epoxidase derived from a higher plant, and a gene. It was found that by introducing the derived VDL1 gene into a host capable of producing farnesyl diphosphate such as Escherichia coli, neoxanthin is efficiently produced and deepoxyneoxanthin, which is not observed in plant leaves, is produced. , The present invention has been completed.

That is, the present invention
The following genes (1) to (3) or gene clusters;
(1) Genes encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin (2) Genes encoding zeaxanthin epoxidase (3) Farnesyl diphosphate producing ability into which a gene encoding neoxanthin synthase is introduced It is a method for producing a carotenoid having an allen structure by culturing a host having the same and extracting a carotenoid having an allen structure from the host after culturing.
(2) The gene encoding zeaxanthin epoxidase is a gene encoding ZEP derived from higher plants.
(3) A method for producing a carotenoid having an allene structure, wherein the gene encoding neoxanthin synthase is a gene encoding VDL1.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to efficiently mass-produce carotenoids having an allen structure such as neoxanthin and deepoxy neoxanthin having functions such as an obesity preventive effect at low cost.

It is a figure which shows the biosynthetic pathway of carotenoid in the leaf of a plant, and the function of various carotenoid biosynthetic enzymes. It is the result of HPLC analysis and NMR analysis of the carotenoid extracted from the recombinant Escherichia coli (pACHP-Viol + pUC-PtVDL1). (A) is a carotenoid (top) extracted from recombinant Escherichia coli (pAC-Viol + pUC-PtVDL1), neoxanthin (middle) of the standard, deepoxyneoxanthin (bottom) of the standard, and (B) is the chromatogram. As a result of NMR analysis of a peak compound that seems to be Neoxanthin purified from recombinant Escherichia coli (pAC-Viol + pUC-PtVDL1), (C) is Deepoxyneoxanthin purified from recombinant Escherichia coli (pACHP-Viol + pUC-PtVDL1). It is the result of NMR analysis of the peak compound which seems to be. It is a figure which shows the structure of the Escherichia coli transformation vector, pUC18, pACYC184. It is a figure which shows the structure of the plasmid for E. coli transformation pACHP-Viol, pUC-PtVDL1. It is a figure which shows the synthetic pathway in the carotenoid production method of this invention.

In the present invention, (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 into a host. ..

(1) Gene group encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin The gene group encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin includes farnesyl diphosphate (FPP) to geranylgeranyl diphosphate. The gene encoding GGPP synthase (CrtE) that synthesizes (GGPP) (CrtE), the gene encoding phytoensynthase (CrtB) that synthesizes phytoen from GGPP ( CrtB ), and the phytoendesaturase (CrtI) that synthesizes lycopene from phytoen . The gene encoding ( CrtI ), the gene encoding lycopene β-cyclase (CrtY) that synthesizes β-carotene from lycopene ( CrtY ), and the gene encoding β-carotene hydroxylase (CrtZ) that synthesizes zeaxanthin from β-carotene. ( CrtZ ) is included. The origin of the carotenoid biosynthesis gene group is not particularly limited, but those derived from the soil bacterium Pantoea ananatis of the γ-proteobacteria are preferably used.

(2) Gene encoding zeaxanthin epoxidase As a gene encoding zeaxanthin epoxidase, a gene encoding ZEP (zeaxanthin epoxidase) derived from papsicum annuum ( CaZEP ) and a flower of lindo ( Gentiana lutea ) ZEP derived from higher plants such as the ZEP gene derived from GlZEP and the ZEP gene derived from Arabidopsis thaliana (AtZEP ) can be used , but the ZEP gene derived from paprika is preferable because of its excellent production efficiency. Used. Examples of the gene encoding ZEP derived from paprika include a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, and one or more amino acids deleted, substituted or deleted in the amino acid sequence shown in SEQ ID NO: 4. A gene consisting of an added amino acid sequence and encoding a protein having zeoxanthin epoxidation activity that epoxidizes zeaxanthin and synthesizes violaxanthin, 80% or more, preferably 85% or more of the protein consisting of the amino acid sequence shown in SEQ ID NO: 4. It consists of an amino acid sequence having a homology of% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more, and has a zeoxanthin epoxidation activity for epoxidizing zeaxanthin and synthesizing violaxanthin. The gene encoding the protein is preferred.
As such a gene, a gene consisting of the base sequence shown in SEQ ID NO: 3 is preferable. In addition, SEQ ID NO: 3, a gene encoding a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 3 and has zeoxanthine epoxidation activity. Zeoxanthin epoxidation activity that consists 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 the above, and epoxidizes zeaxanthin to synthesize violaxanthin. A gene encoding a protein having the above is preferred.

(3) Gene encoding neoxanthin synthase As a gene encoding neoxanthin synthase, a VDL1 (violaxanthin de-epoxidase-like protein 1) gene derived from diatom (Phaeodactylum tricornutum ) is used. The VDL1 gene is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and 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. 80% or more, preferably 85% or more, more preferably 90% with the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, a gene encoding a protein having neoxanthin synthesizing activity that synthesizes neoxanthin from violaxanthin. As described above, a gene having an amino acid sequence having a homology of 95% or more, more preferably 98% or more, and encoding a protein having a neoxanthin synthesizing activity for synthesizing neoxanthin from violaxanthin is preferable.
As such a gene, a gene consisting of the base sequence shown in SEQ ID NO: 1 is preferable. Further, a protein having a neoxanthin synthesizing activity that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 1 and synthesizes neoxanthin from violaxanthin. Synthesize neoxanthin from violaxanthin, which consists of the encoding gene and the 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. A gene encoding a protein having neoxanthine synthase activity is preferable. Neoxanthin is synthesized from violaxanthin by neoxanthin synthase. Zeaxanthin is converted to antheraxanthin by zeaxanthin epoxidase, and violaxanthin is further synthesized. Neoxanthin synthase synthesizes deepoxyneoxanthin from antheraxanthin.

(4) Isopentenyl diphosphate isomerase gene ( IDI gene)
Isopentenyl diphosphate isomerase converts isopentenyl diphosphate (isopentenyl pyrophosphate: IPP) to dimethylallyl diphosphate (DMAPP) during the FPP production process of Escherichia coli and Saccharomyces cerevisiae. The IDI gene can efficiently produce carotenoids by expressing it together with the crt gene cluster. The origin of the IDI gene is not particularly limited, but for example, IDI (IPP isomerase) derived from the green alga Haematococcus pluvialus is preferably used.
The amount ratio of IPP and dimethylallyl diphosphate (DMAPP) is adjusted by isopentenyl-diphosphate isomerase (Patent Document 1). There are type 1 (type 1) and type 2 (type 2) isopentenyl diphosphate isomerases, which have different structures from each other. Type 2 IDI genes are sometimes present together in the carotenoid biosynthesis gene cluster of carotenoid-producing bacteria belonging to the α-Pseudomonadota or γ-Pseudomonadota (Norihiko Misawa, Oreoscience 9 (9): 385-391, 2009). Regardless of type 1 or type 2, when the IDI gene is introduced into Escherichia coli together with terpene biosynthetic genes such as carotenoids and sesquiterpenes and expressed, the production of terpenes increases compared to the case where the IDI gene is not introduced. Is known (S. Kajiwara, PD Fraser, K. Kondo and N. Misawa, Biochem J. , 324, 421-426. 1997). By introducing and co-expressing the genes encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin, the CaZEP gene and the IDI gene, violaxanthin is efficiently produced, and neoxanthin is produced in high yield using violaxanthin as a substrate. Produced in.

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

When Escherichia coli is used as a host, the method for introducing the exogenous gene is not particularly limited, and the exogenous gene to be introduced is expressed by a known method using an appropriate plasmid and terminator, and the vector for transforming E. coli. A plasmid may be prepared by inserting it into E. coli cells, and the plasmid may be introduced into E. coli cells. When a plurality of genes are introduced, those genes may be introduced by one transformation vector or may be introduced by different transformation vectors.

The Escherichia coli transformation vector may be appropriately selected depending on the number and type of transgenes and the like, but Escherichia coli protein expression vectors and the like are preferable, and examples thereof include pUC18, pACYC184 and RSFDuet-1. Moreover, any promoter may be used as long as it can be expressed in Escherichia coli. For example, T7 promoter, lacZ promoter, tac promoter and the like can be mentioned. Among them, the tac promoter is more preferable because the introduced gene is preferably highly expressed in Escherichia coli. When it is preferable that the introduced gene is transiently expressed in Escherichia coli, it is preferable to use an inducible promoter, and an inducible T7 promoter by IPTG or the like is more preferable. The terminator can be appropriately selected depending on the gene to be introduced and the like, and examples thereof include a T7 terminator and an rrnB terminator.

Various selectable marker genes may be used in order to efficiently select E. coli cells into which the target foreign gene has been introduced by the E. coli transformation vector. The selectable marker gene is not particularly limited, and a known gene can be used. For example, it is preferable to use a gene that imparts drug resistance as a selection marker, and to introduce a plasmid or the like containing the selection marker gene and an exogenous gene into E. coli cells as an E. coli transformation vector. As a result, Escherichia coli cells into which a foreign gene has been introduced can be efficiently selected from the expression of the selectable marker gene. Examples of such a selectable marker gene include various drug resistance genes. More specifically, for example, an ampicillin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene and the like can be mentioned. Further, it is preferable to have a promoter and a terminator for expressing the gene upstream and downstream of the selectable marker gene.

The method for introducing the transformation vector thus prepared into Escherichia coli is not particularly limited, and may be introduced using a known method such as a heat shock method. The expression of the exogenous gene in recombinant Escherichia coli can be confirmed by, for example, methods such as Northern blotting, RT-PCR, and Western blotting. Further, it can be confirmed that neoxanthin, deepoxy neoxanthin and the like are produced in Escherichia coli by, for example, a method such as HPLC analysis.

The method for culturing recombinant Escherichia coli is not particularly limited, and a known culturing method can be used. Since the transgenic Escherichia coli after culturing contains carotenoids having an allen structure such as neoxanthin and deepoxy neoxanthin, these carotenoids can be obtained by a known extraction method.

Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited to these examples.

<Example 1>
(Preparation of plasmid for neoxanthin production)
The DNA fragment of the VDL1 (violaxanthin de-epoxidase-like protein 1) gene ( PtVDL1 gene) of the diatom Phaeodactylum tricornutum was chemically synthesized using a design drawing modified to a base sequence suitable for the codon usage frequency of Escherichia coli. The base sequence is shown in SEQ ID NO: 1. The DNA fragment of the ZEP (zeaxanthin epoxidase) gene ( CaZEP gene) of paprika Capsicum annuum was chemically synthesized using a design drawing modified to a base sequence suitable for the codon usage frequency of E. coli. The base sequence is shown in SEQ ID NO: 3. The amino acid sequences of PtVDL1 and CaZEP (SEQ ID NOs: 2 and 4) can be obtained from DDBJ accession numbers B7G087_PHATC and accession numbers XP_016561102.1, respectively. In addition, the artificial gene fragment of CaZEP has Spe I at the 5'end and the HindIII site at the 3'end, and the artificial gene fragment of PtVDL1 has Eco RI at the 5'end and the Sal I site at the 3'end. Was added.

PACYC184 and pUC18 were used as E. coli transformation vectors (Fig. 3). Spe I- of the zeaxanthin production plasmid pACHP-Zea (M. Takemura et al, Tetrahedron Letters, 56: 6063-6065 , 2015.) prepared by cutting out CaZEP fragments cleaved with Spe I and Hind III and prepared according to a known method. The plasmid pACHP-Viol was prepared by inserting it into the Hin dII site (Fig. 4A). A PtVDL1 gene fragment of diatom cleaved with Eco RI and Sal I was excised and inserted into the Eco RI- Sal I site of vector pUC18 to prepare plasmid pUC-PtVDL1 (Fig. 4B).

NSY gene ( SlNSY gene; F. Bouvier et al, European Journal of Biochemistry, 267: 6346-6352, 2000.) and NXD1 gene ( SlNXD1 gene; H. Neuman et al, The Plant Journal, 78) of tomato ( Solanum lycopersicum ) : 80-93, 2014.) DNA fragments were chemically synthesized using a design drawing modified to a base sequence suitable for the frequency of codon use in E. coli. As in the case of the PtVDL1 gene fragment of diatom, each gene fragment was inserted into the Eco RI- Sal I site of the vector pUC18 to prepare plasmids pUC-SlNSY and pUC-SlNXD1.

<Example 2>
(Transformation of E. coli)
Escherichia 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, it was done as follows.
After culturing in Escherichia coli (JM101 (DE3)), SOB medium (2% bactotrypton, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM DDL ₄ ), TB buffer (10) Treatment with mM PIPES (pH 6.7), 15 mM CaCl ₂ , 250 mM KCl, 55 mM MnCl _{2.4H 2 O} ). Then, the plasmid pACHP-Viol prepared in Example 1 was mixed with the treated E. coli and allowed to stand on ice. After applying a heat shock at 42 ° C, the cells were cultured in SOC medium and sprinkled on a drug-containing medium. After culturing overnight, the drug-resistant colonies that emerged were obtained as transformed Escherichia coli (recombinant Escherichia coli). As a drug (antibiotic), chloramphenicol (30 mg / L) was added to the medium.
The SOB medium (2% bactotrypton, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM) was prepared from the above-prepared Escherichia coli (JM101 (DE3)) having pACHP-Viol and producing violaxanthin. After culturing in MgCl ₂ , 10 mM DDL ₄ ), it was treated with TB buffer (10 mM PIPES (pH 6.7), 15 mM CaCl ₂ , 250 mM KCl, 55 mM MnCl _{2.4H 2 O} ). Then, the plasmid pUC-PtVDL1 (or pUC-SlNSY or pUC-SlNXD1) prepared in Example 1 was mixed with the treated E. coli and allowed to stand on ice. After applying a heat shock at 42 ° C, the cells were cultured in SOC medium and sprinkled on a drug-containing medium. After culturing overnight, the drug-resistant colonies that emerged were obtained as transformed Escherichia coli (recombinant Escherichia coli). As drugs (antibiotics), chloramphenicol (30 mg / L) and ampicillin (40 mg / L) were added to the medium.

<Example 3>
The above recombinant E. coli was cultured in 2 YT (2 x YT) medium (1.6% bactotrypton, 1% yeast extract, 0.5% NaCl) at 37 ° C. At that time, two mediums containing 400 mL in a 2 liter (L) Erlenmeyer flask were used, and the antibiotics chloramphenicol (30 mg / L) and ampicillin (40 mg / L) were added to the medium. .. After culturing until OD ₆₀₀ reached 0.4-0.6, 0.05 mM IPTG was added and the cells were cultured at 20 ° C. for 2 nights. The cultured bacterial solution was centrifuged to obtain bacterial cells. Methanol was added to the cells and suspended well, and then 1 M Tris (pH 7.5) and 1 M NaCl solution were added. Chloroform was further added, the suspension was well suspended, and then centrifugation was performed. The carotenoid extract (chloroform layer) was recovered.

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

HPLC analysis and NMR analysis of carotenoids produced by recombinant E. coli into which two plasmids pACHP-Viol and pUC-PtVDL1 were introduced into E. coli were performed. As a result, the carotenoids produced by recombinant E. coli were Zeaxanthin, Violaxanthin, Neoxanthin, Deepoxyneoxanthin and the like. The result of this HPLC analysis is shown in FIG. 2 (A). From the comparison with the standard, a peak with the same retention time as Neoxanthin and Deepoxyneoxanthin was found. Then, as a result of NMR analysis of these peak compounds, as shown in FIGS. 2 (B) and 2 (C), they were in agreement with the previously reported Neoxanthin and Deepoxyneoxanthin. From the above, it was found that Neoxanthin and Deepoxyneoxanthin are produced in recombinant Escherichia coli into which pACHP-Viol and pUC-PtVDL1 have been introduced.
On the other hand, in the recombinant E. coli into which the plasmids pACHP-Viol and pUC-SlNSY were introduced, and the recombinant E. coli into which the plasmids pACHP-Viol and pUC-SlNXD1 were introduced, no conversion product for Violaxanthin was observed.

Although it was known that the ZEP gene derived from the diatom Phaeodactylum tricornutum does not function in E. coli (Non-Patent Documents 3 and 4), the VDL1 gene derived from diatom efficiently expresses its function in E. coli, and Neoxanthin and Deepoxyneoxanthin are expressed. It was unexpected to be able to produce Neoxanthin and Deepoxyneoxanthin, which had never been reported in microorganisms including Escherichia coli and Saccharomyces cerevisiae. FIG. 5 shows the synthetic route of a carotenoid having an allene structure by the production method of the present invention.
By this method, Deepoxyneoxanthin, which is not observed in the leaves of plants, is produced, and in addition to the synthesis from violaxanthin, neoxanthin is also synthesized from deepoxyneoxanthin, so that neoxanthin can be efficiently produced. It becomes.

INDUSTRIAL APPLICABILITY The present invention is useful as a method for easily producing functional carotenoids at low cost using a normal culture apparatus and stably supplying them.

Claims (11)

The following genes or gene clusters (1) to (3);
(1) Genes encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin (2) Genes encoding zeaxanthin epoxidase (3) Farnesyl diphosphate producing ability into which a gene encoding neoxanthin synthase is introduced It is a method for producing a carotenoid having an allen structure by culturing a host having the same and extracting a carotenoid having an allen structure from the host after culturing.
(2) The gene encoding zeaxanthin epoxidase is a gene encoding ZEP derived from higher plants.
(3) A method for producing a carotenoid having an allene structure, wherein the gene encoding neoxanthin synthase is a gene encoding VDL1 derived from diatom. The method for producing a carotenoid having an allene structure according to claim 1, wherein the carotenoid having an allene structure is neoxanthin. The method for producing a carotenoid having an allene structure according to claim 1, wherein the carotenoid having an allene structure is deepoxy neoxanthin. (1) The genes encoding the biosynthetic pathway from farnesyl diphosphate to zeaxanthin are the gene encoding GGPP synthase (CrtE), the gene encoding phytoene synthase (CrtB), and the gene encoding phytoendesaturase (CrtI). , Production of a carotenoid having an allen structure according to any one of claims 1 to 3, which contains a gene encoding lycopene β-cyclase (CrtY) and a gene encoding β-carotene hydroxylase (CrtZ). Method. The method for producing a carotenoid having an allen structure according to any one of claims 1 to 4, wherein the gene encoding VDL1 is any of the following genes (a) to (f).
(A) A gene consisting of the base sequence shown in SEQ ID NO: 1 (b) Hybridizing under stringent conditions with a polynucleotide having a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 1 and Gene encoding a protein having neoxanthin synthesizing activity (c) Encoding a protein having a base sequence having 90% or more homology with the gene consisting of the base sequence shown in SEQ ID NO: 1 and having neoxanthine synthesizing activity. Gene (d) Gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (e) 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. Gene consisting of and encoding a protein having neoxanthin synthesizing activity (f) It consists of an amino acid sequence having 80% or more homology with the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and has neoxanthine synthesizing activity. Genes that encode proteins The carotenoid having an allen structure according to any one of claims 1 to 5, wherein the gene encoding ZEP derived from a higher plant is a gene encoding one ZEP selected from the group consisting of paprika, white sardine and gentian. Production method. The method for producing a carotenoid having an allen structure according to claim 6, wherein the gene encoding ZEP derived from a higher plant is a gene encoding ZEP derived from paprika. The method for producing a carotenoid having an allen structure according to claim 7, wherein the gene encoding ZEP derived from paprika is any of the following genes (g) to (l).
(G) A gene consisting of the base sequence shown in SEQ ID NO: 3 (h) Hybridizing under stringent conditions with a polynucleotide having a base sequence complementary to the gene consisting of the base sequence shown in SEQ ID NO: 3 and Gene encoding a protein having zeoxanthine epoxidation activity (i) A protein having a base sequence having 90% or more homology with the gene consisting of the base sequence shown in SEQ ID NO: 3 and having zeoxanthine epoxidation activity. Gene encoding (j) Gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (k) In the amino acid sequence shown in SEQ ID NO: 4, one or more amino acids have been deleted, substituted or added. A gene consisting of an amino acid sequence and encoding a protein having zeoxanthin epoxidation activity (l) A gene consisting of an amino acid sequence having 80% or more homology with the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 and zeoxanthin epoxy Gene encoding a protein with chemogenic activity The method for producing a carotenoid having an allen structure according to any one of claims 1 to 8, wherein the host has further introduced (4) a gene encoding isopentenyl diphosphate isomerase. The method for producing a carotenoid having an allen structure according to any one of claims 1 to 9, wherein the host is a microorganism. The method for producing a carotenoid having an allen structure according to claim 10, wherein the microorganism is Escherichia coli.

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