JP2014050374A - Method for extracting carotenoid from plant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an efficient method for extracting carotenoid useful as a functional food material or an antioxidant such as astaxanthin fatty acid ester, and also provide a plastid-genome modified plant that produces carotenoid such as astaxanthin fatty acid ester.SOLUTION: Provided is a method for extracting carotenoid from a plant or its tissue, which comprises the steps of: (I) dehydrating carotenoid-producing plant or its tissue by using a dehydration solvent, (II) bringing in contact the dehydrated plant or its tissue with an extraction solvent to obtain a solution containing carotenoid, and (III) adding water and an organic solvent to the dehydration solvent used in (I) and the carotenoid-containing extract obtained in (II) to collect the organic solvent layer containing carotenoid after phase separation.

Description

  The present invention relates to a method for efficiently extracting carotenoids useful as functional food materials, antioxidants, and the like, for example, carotenoids such as astaxanthin fatty acid esters from plants, plastid genome modified plants, and the like.

  Isoprenoids (also called terpenoids) are the most diverse group of compounds in nature, with over 23,000 species. Carotenoids (tetraterpenes) are a group of isoprenoids consisting of a basic skeleton with 40 carbon atoms and are composed of more than 750 dyes. Astaxanthin (Astaxanthin; the structure is shown in FIG. 1; hereinafter also referred to as Asta) is one of carotenoids contained in crustaceans such as shrimp and crab, red fish such as salmon, trout, and Thailand. Asta contained in these marine animals is often a fatty acid ester. However, salmon, trout muscle and egg Asta are free. The Asta content in sockeye salmon muscle and eggs is 20-38 μg / g wet weight. The crustacean crayfish and Antarctic krill have an Asta content of 1 to 3 μg and 20 to 40 μg per 1 g (wet weight), respectively (weight is calculated as Asta free form). The crab is also a carotenoid that is commonly found in plants and algae (lutein; the structure shown in FIG. 1) is a ketocaronoid (a carotenoid having a keto group at the 4 or / and 4 'position), frithielaxanthin (fritschiellaxanthin) ; The structure is converted into FIG. 1) and accumulated, but it is very small compared to the accumulated amount of Asta (Non-patent Document 1).

alpha-proteobacteria (alpha-Proteobacteria) steel (class) belonging to (authentic) bacteria ((eu) bacteria) some or freshwater green alga Haematococcus pluvialis (Haematococcus pluvialis is a degradation product of ingested nutrients Asta can be biosynthesized from the beginning ( de novo synthesis), while most of the Asta produced by bacteria is free (free), whereas Asta produced by H. pluvialis is a fatty acid ester. Asta fatty acid ester has higher intestinal absorbability when administered orally compared to the free form, and is useful as a functional food material, etc. (Non-patent Document 2) Asta fatty acid produced by H. pluvialis Esters are sold by Fuji Chemical Industry Co., Ltd. as a health food ingredient, and the market size is about 2 billion yen / year at the original level.

An enzyme gene that converts β-carotene (β-carotene; structure shown in FIG. 1) into Asta was first clarified in 1995 (Patent Document 1 and Non-Patent Document 3). It is two enzyme genes isolated from the α-proteobacteria marine bacterium Paracoccus sp. N81106 strain (originally called Agrobacterium aurantiacum ) They were named crtW and crtZ , respectively. The crtW and crtZ genes encoded carotenoid 4,4'-ketolase (also called keto group-introducing enzyme; also called carotenoid 4,4'-oxygenase) and carotenoid 3,3'-hydroxylase (hydroxyl-inducing enzyme), respectively. Document 1 and Non-Patent Document 3). These enzymes, CrtW and CrtZ are both non-heme iron proteins and their catalytic function is shown in FIG. Thereafter, it was found that the crtW and crtZ genes are commonly present in α-proteobacteria that synthesize Asta (Non-patent Document 4). In particular, CrtW and CrtZ derived from the marine bacterium Brevundimonas sp. Strain SD212 were shown to efficiently convert β-carotene to Asta (Patent Document 2). H. pluvialis has three carotenoid 4,4'-ketolase gene of CrtW homology, these genes are called respectively Bkt1, Bkt2, Bkt3 (Non-Patent Document 5). This BKT (CrtW) type enzyme seems to exist widely in green algae that can synthesize ketocarotenoids (Non-patent Document 6). H. pluvialis also has a carotenoid 3,3′-hydroxylase gene that is homologous to CrtZ (Non-patent Document 7). Higher plants also have the same gene with homology to CrtZ and are called Bhy (Non-patent Document 5). BHY functions to convert β-carotene into zeaxanthin in plant leaves (Non-Patent Document 5) (FIG. 1).

Higher plants and other land plants usually do not have an enzyme gene that introduces a keto group at the 4,4′-position of the β ring, so they cannot synthesize ketocarotenoids such as Asta and Frithiellaxanthin. However, with the exception of plants belonging to the genus Adonis of Fukujusou (for example, Adonis aestivalis ), Asta fatty acid esters accumulate in the red petals (Non-patent Document 8). The key gene involved in Asta synthesis in Adonis plants is the carotenoid β-ring 4-dehydrogenase gene (carotenoid β-ring 4-dehydrogenase gene) ( CBFD ) (Non-patent Document 9). CBFD has a completely different structure and function from the CrtW-type enzyme, and is structurally close to higher plant BHY (β-carotene 3,3'-hydroxylase), so the CBFD gene has evolved from the Bhy gene. Can be considered. Today, metabolic engineering (pathway engineering; one technical field of advanced biotechnology) using a keto group-introducing enzyme (carotenoid 4,4'-ketolase) can give Asta-producing ability to various higher plants. it can. As may keto group-introducing enzyme gene used, Paracoccus sp. N81106 strain or Brevundimonas sp. SD212 strain crtW and from, H. pluvialis Bkt1 or Bkt2 the like from (non-patent document 5). The keto group transductase gene was introduced into the chromosomal DNA of various higher plants under the control of appropriate promoters and terminators. Among food crops, potato ( Solanum phureja ) tubers (Non-patent Document 5), carrot ( Daucus carota ) main roots (Non-patent Documents 5 and 10), corn ( Zea mays ) endosperm (Non-patent Document 11), rapeseed ( Asta was produced in a canola ( Brassica napus ) seed (Non-patent Document 12). In particular, carrots expressing the Bkt1 gene under the control of the double CaMV 35S promoter produced a high amount of Asta, producing 91.6 μg (27% of total carotenoids) of Asta per 1 g of main root (wet weight). The total amount of ketocarotenoids was 236 μg (68% of the total carotenoids) (Non-patent Document 10). On the other hand, Hasunuma et al., In the Brevundimonas sp. SD212 strain crtZ and crtW genes (codon usage has been changed to plant type) directly under the control of the rrn promoter, non-food crop tobacco ( Nicotiana tabacum ) chloroplasts Introduced into the genome, chloroplast-genome modified (hereinafter referred to as CGM) tobacco was obtained (Non-patent Document 13). Even though 99% of carotenoids were converted to ketocarotenoids such as Asta (74% of all carotenoids) in CGM tobacco leaves, they were able to grow normally after photosynthesis. Recently, a leaf of lettuce ( Lactuca sativa L. var. Berkeley) with a similar plasmid using the same crtZ and crtW genes (and using the IPP isomerase ( idi ) gene from the marine bacterium Paracoccus sp. N81106) by chloroplast transformed issues create a CGM lettuce with leaves reddish, it was reported that could get a T ₁ seeds (non-Patent documents 14 and 15). CGM lettuce seeds showed a germination rate of 99%, which was higher than the germination rate of non-transformants (93%) (Non-patent Document 15).

Recently, a leaf of lettuce ( Lactuca sativa L. var. Berkeley) with a similar plasmid using the same crtZ and crtW genes (and using the IPP isomerase ( idi ) gene from the marine bacterium Paracoccus sp. N81106) by chloroplast transformed issues create a CGM lettuce with leaves reddish, it was reported that could get a T ₁ seeds (non-Patent documents 14 and 15). CGM lettuce seeds showed a germination rate of 99%, which was higher than the germination rate of non-transformants (93%) (Non-patent Document 15).

As mentioned above, today, advanced biotechnology has made it possible to synthesize Asta in various crops, but in each case, this is an example of the production of free Asta, which is actually a material for health food. The Asta fatty acid ester produced by H. pluvialis sold as is different. For this reason, the technique which can obtain efficiently Asta fatty acid ester etc. useful as a functional food material etc. from plant raw materials etc. is calculated | required.

International publication WO95 / 18220 pamphlet International Publication WO2005 / 118812 Pamphlet (US Patent No. 7,999,151)

Miyuki Tsushima, FFI Journal, 212: 539-549, 2007 Eiji Yamashita, Food and Development, 27 (3): 38-40, 1992 N. Misawa, Y. Satomi, K. Kondo, A. Yokoyama, S. Kajiwara, T. Saito, T. Ohtani, and W. Miki, J. Bacteriol., 177 (22): 6575-6584, 1995 N. Misawa, Marine Drugs, 9 (5): 757-771, 2011 N. Misawa, Plant Biotechnol., 26: 93-99, 2009 Y. J. Zhong, J. C. Huang, J. Liu, Y. Li, Y. Jiang, Z. F. Xu, G. Sandmann, and F. Chen, J. Exp. Botany, 62 (10): 3659-3669, 2011 H. Linden, Biochim. Biophys. Acta., 1446 (3): 203-212, 1999 T. Maoka, T. Etoh, S. Kishimoto, S. Sakata, J. Oleo Sci., 60, 47-52, 2011 F. X. Cunningham, Jr., E. Gantt, Plant Cell, 23, 3055-3069, 2011 J. Jayaraj, R. Devlin, Z. Punja., Transgenic Res., 17: 489-501, 2008 C. Zhu, S. Naqvi, J. Breienbach, G. Sandmann, P. Christou, and T. Capell, PNAS, 105 (47): 18232-18237, 2008 M. Fujisawa, E. Takita, H. Harada, N. Sakurai, H. Suzuki, K. Ohyama, D. Shibata, and N. Misawa, J. Exp. Botany, 60 (4): 1319-1332, 2009 T. Hasunuma, S. Miyazawa, S. Yoshimura, Y. Shinzaki, K. Tomizawa, K. Shindo, S. K. Choi, N. Misawa, and C. Miyake, Plant J., 55: 857-88, 2008 N. Misawa, The 16th International Symposium on Carotenoids, Krakow, Poland, July 17-22, 2011 (Acta Biologica Cracoviensia, 53 (suppl. 1): 83-84, 2011) Naoshi Harada, Miyuki Murakami, Kazuko Arai, Kazutoshi Shindo, Toshihiko Otomatsu, Norihiko Misawa, 2012 Annual Meeting of the Japanese Society for Agricultural Chemistry, 2A31p19, 2012

  The present invention is a method capable of efficiently extracting carotenoids such as astaxanthin fatty acid esters useful as functional food materials, antioxidants, etc. from plants, etc., plastid genome modified plants capable of producing carotenoids such as astaxanthin fatty acid esters, etc. The purpose is to provide.

  As a result of intensive studies to solve the above problems, the present inventors have found that each of carotenoid 3,3′-hydroxyl introducing enzyme, carotenoid 4,4′-keto group introducing enzyme and isopentenyl diphosphate isomerase. When carotenoids are extracted from the leaves of chloroplast genome modified lettuce (CGM lettuce) expressed by introducing an enzyme-encoding gene into the chloroplast genome, (I) CGM lettuce is contacted with a solvent for dehydration. (II) The dehydrated CGM lettuce is extracted with an extraction solvent, and (III) the dehydrating solvent contacted with the CGM lettuce in (I) and the extract obtained in (II) above are mixed with water and an organic solvent. When liquid separation is performed, the organic solvent layer is found to contain a large amount of carotenoids mainly composed of astaxanthin fatty acid esters, and by performing the operations (I) to (III), Carotenoid fat Found that can be extracted carotenoid esters efficiently. Astaxanthin fatty acid ester cannot be extracted from CGM lettuce by a conventional extraction method, and is a carotenoid fatty acid ester extracted from CGM lettuce for the first time by performing the operations (I) to (III).

  The present inventors also analyzed the carotenoid extracted from the CGM lettuce by the extraction method, so that CGM lettuce produced about 2.67 times the total carotenoid of normal lettuce, of which 67.4% was Asta fatty acid ester (49.2%: diester, 18.2%: monoester), fatty acid part in Asta fatty acid ester is myristate or palmitate, other than Asta fatty acid ester Ketocarotenoids include Asta free form (10%), Frithielaxanthin (6%), Canthaxanthin (5.3%), 4-ketoanthaxanthin (4.3%), etc. It was found that chucaxanthin (3.8%) was contained (Table 1).

As described above, it has been reported that Asta is synthesized in various agricultural crops by advanced biotechnology, and in any case, it is an example of production of Asta free form (Non-patent Documents 5 and 10-15). As in the case of CGM lettuce this time, Asta's fatty acid ester was synthesized in large quantities, and more than half of the total carotenoid was included. This is the first finding that has been clarified by studying efficient extraction and purification methods. Currently, those produced by H. pluvialis as fatty acid esters of Asta are actually sold as health food materials, but plants such as CGM lettuce can be easily grown in plant plants for recombinant plants, It is preferably used to efficiently produce fatty acid esters of Asta on an industrial scale. In addition, the extraction method of the present invention can efficiently extract carotenoids useful as functional food materials such as Asta fatty acid esters, antioxidants and the like from the CGM lettuce and the like, so that carotenoids can be stably supplied. become.

  In addition, for example, the CGM lettuce also produces rare carotenoids such as frithielaxanthin, 4-ketoantheraxanthin and lactucaxanthin together with fatty acid esters of Asta, but other organisms that produce carotenoid mixtures of these combinations Since they do not exist, the total carotenoids of CGM lettuce containing these rare carotenoids (also called multi-carotenoids) cannot be realized by other production methods or extraction methods. In addition, the total production of Asta in the CGM lettuce is 180 μg / g wet weight in terms of free form. Not only among the recombinant food crops produced so far, but also Asta-containing foods such as salmon and salmon (for example, The content of Asta in salmon and salmon roe was the highest level compared to 20-38 μg / g wet weight in terms of free form. The plastid genome-modified plant such as CGM lettuce can be easily grown in a plant plant for recombinant plants, and thus is useful as a stable source of other rare ketocarotenoids in addition to Asta fatty acid esters.

  The present invention has been completed based on the above findings, and relates to the following carotenoid extraction method and use of a plastid genome-modified plant for producing an astaxanthin fatty acid ester.

(1) (I) a step of bringing a carotenoid-producing plant or tissue thereof into contact with a dehydrating solvent to dehydrate, (II) extracting the dehydrated plant or tissue thereof with an extraction solvent to obtain a carotenoid-containing extract. Steps (III) and (3) Carotenoids are separated by adding water and an organic solvent to the dehydrating solvent brought into contact with the plant or tissue thereof in Step (I) and the carotenoid-containing extract obtained in Step (II). A method for extracting a carotenoid from a plant or a tissue thereof, which comprises a step of recovering an organic solvent layer containing.
(2) The extraction method according to (1) above, wherein the carotenoid contains a ketocarotenoid.
(3) The extraction method according to (1) or (2) above, wherein the carotenoid contains an astaxanthin fatty acid ester.
(4) The extraction method according to (3), wherein the astaxanthin fatty acid ester is a mixture of a diester and a monoester.
(5) The dehydrating solvent is at least one organic solvent selected from the group consisting of acetone, ethanol, methanol, and 2-propanol. Any one of (1) to (4) above The extraction method according to item.
(6) The extraction method according to any one of (1) to (5), wherein the extraction solvent is a polar organic solvent.
(7) The organic solvent used in step (III) is at least one organic solvent selected from the group consisting of hexane, diethyl ether, petroleum ether, ethyl methyl ether, and dimethyl ether. The extraction method according to any one of the above.
(8) The extraction method according to any one of (1) to (7), wherein in step (I), the minced plant or tissue thereof is contacted with a dehydrating solvent.
(9) The carotenoid-producing plant or tissue thereof is a plastid genome-modified plant or tissue obtained by introducing a gene encoding a carotenoid 4,4′-keto group-introducing enzyme into the plant plastid genome. The extraction method according to any one of (1) to (8), characterized in that:
(10) The gene encoding the carotenoid 4,4′-keto group-introducing enzyme is complementary to (i) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or (ii) DNA comprising the nucleotide sequence of SEQ ID NO: 1 A DNA encoding a polypeptide that hybridizes with a DNA consisting of a base sequence under stringent conditions and has an activity of introducing a keto group into the 4-position and / or 4'-position of a β-ring of a carotenoid having a β-ring The extraction method according to (9), wherein the extraction method is provided.
(11) A pigment obtained by introducing a carotenoid-producing plant or tissue thereof into a plastid genome by further introducing genes encoding carotenoid 3,3′-hydroxyl transferase and isopentenyl diphosphate isomerase enzymes The extraction method according to (9) or (10) above, which is a body genome-modified plant or a tissue thereof.
(12) A gene encoding a carotenoid 3,3′-hydroxyl transferase is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 3 or (ii) a nucleotide sequence complementary to a DNA comprising the nucleotide sequence of SEQ ID NO: 3 DNA encoding a polypeptide having the activity of introducing a hydroxyl group into the 3rd and / or 3'-position carbon of a carotenoid having a β ring, which hybridizes with DNA comprising The extraction method according to (11) above.
(13) The gene encoding isopentenyl diphosphate isomerase is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 5 or (ii) a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 5 And DNA encoding a polypeptide having an isomerase activity that hybridizes under stringent conditions and converts isopentenyl diphosphate to dimethylallyl diphosphate. ) Extraction method.
(14) A plastid genome-modified plant introduces DNA containing the nucleotide sequence of SEQ ID NO: 1, DNA containing the nucleotide sequence of SEQ ID NO: 3, and DNA containing the nucleotide sequence of SEQ ID NO: 5 into the plastid genome of the plant. The extraction method according to any one of (9) to (13), which is obtained by the following.
(15) The plant according to any one of (9) to (14), wherein the plant is lettuce, cauliflower, cabbage, rapeseed, potato, tomato, carrot, rice, soybean, or tobacco. Extraction method.
(16) Use of a plastid genome-modified plant obtained by introducing a gene encoding a carotenoid 4,4′-keto group-introducing enzyme into a plastid genome of a plant to produce an astaxanthin fatty acid ester.
(17) The gene encoding the carotenoid 4,4′-keto group-introducing enzyme is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, or (ii) a base complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 A DNA encoding a polypeptide that hybridizes with a DNA comprising a sequence under stringent conditions and has an activity of introducing a keto group into the 4th and / or 4 'position of the β ring of a carotenoid having a β ring Use as described in said (16) characterized by the above-mentioned.
(18) A plastid genome modification plant obtained by introducing a gene encoding each enzyme of carotenoid 3,3′-hydroxyl transferase and isopentenyl diphosphate isomerase into a plastid genome. The use according to (16) or (17) above, which is a plant.
(19) The gene encoding the carotenoid 3,3′-hydroxyl transferase is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 3 or (ii) a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 3. DNA encoding a polypeptide having the activity of introducing a hydroxyl group into the 3rd and / or 3'-position carbon of a carotenoid having a β ring, which hybridizes with DNA comprising The use according to (18) above.
(20) The gene encoding isopentenyl diphosphate isomerase is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 5 or (ii) a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 5 And a DNA encoding a polypeptide having an isomerase activity that hybridizes under stringent conditions and converts isopentenyl diphosphate to dimethylallyl diphosphate. ) Use as described.
(21) A plastid genome-modified plant introduces a DNA comprising the nucleotide sequence of SEQ ID NO: 1, a DNA comprising the nucleotide sequence of SEQ ID NO: 3, and a DNA comprising the nucleotide sequence of SEQ ID NO: 5 into the plastid genome of the plant. The use according to any one of (16) to (20) above, which is obtained.
(22) The use according to any one of (16) to (21), wherein the astaxanthin fatty acid ester is a mixture of a diester and a monoester.
(23) The use according to any one of (16) to (22), wherein the astaxanthin fatty acid ester is astaxanthin myristic acid ester or palmitic acid ester.
(24) The plant according to any one of (16) to (23), wherein the plant is lettuce, cauliflower, cabbage, rapeseed, potato, tomato, carrot, rice, soybean, or tobacco. use.
(25) The plastid genome-modified plant is a chloroplast genome-modified lettuce obtained by introducing the gene into a chloroplast genome, according to any one of (16) to (24), Use of description.

  According to the present invention, carotenoids such as astaxanthin fatty acid esters that are useful as functional food materials, antioxidants and the like can be efficiently extracted from plants. As a result, carotenoids such as astaxanthin fatty acid esters can be efficiently supplied from plants that are not normally edible, for example.

  Further, according to the present invention, for example, a carotenoid containing an astaxanthin fatty acid ester from a plastid genome-modified plant obtained by introducing a gene encoding a carotenoid 4,4′-keto group-introducing enzyme into a plastid genome such as a chloroplast. Can be obtained. Furthermore, for example, if the plastid genome-modified plant is a chloroplast genome-modified lettuce, the astaxanthin fatty acid ester is the main component, and other rare ketocarotenoids fritschiellaxanthin (fritschiellaxanthin) and 4-ketoanthelaxanthin ( A carotenoid mixture containing 4-ketoantheraxanthin) and lactucaxanthin, a carotenoid specific to lettuce, can be obtained. Thus, according to the present invention, carotenoids such as astaxanthin fatty acid esters can be stably supplied from plants.

FIG. 1 shows carotenoid 3,3′-hydroxyl transferase (CrtZ), carotenoid 4,4′-keto transferase (CrtW) and isopentenyl diphosphate isomerase (CrtZ) as examples of plants that biosynthesize astaxanthin fatty acid esters. Biosynthesis pathways and various carotenoids of various carotenoids in chloroplast genome modified (CGM) lettuce in which three genes (each crtZ , crtW , and idi ) encoding each enzyme of Idi) are introduced and expressed in the chloroplast genome It is a figure which shows the function of a synthase. In FIG. 1, carotenoids and carotenoid fatty acid esters extracted in the examples are indicated by underlining. Of those shown underlined, lactucaxanthin is a carotenoid that does not have a β ring, and all other than these are carotenoids that have a β ring. FIG. 2 is a diagram showing the results of HPLC analysis of carotenoids extracted from CGM lettuce leaves. FIG. 3 is a diagram showing the results of HPLC analysis of carotenoids extracted from control lettuce leaves. FIG. 4 shows the structure of the lettuce chloroplast transformation vector pRL200. FIG. 5 shows the structure of the lettuce chloroplast transformation plasmid pRL-crtZWidi, and the insertion position of the gene into the lettuce chloroplast genome.

The present invention is described in detail below.
In the present specification, the carotenoid includes a carotenoid having a β ring and a carotenoid having no β ring. Carotenoids having a β ring include carotenoid fatty acid esters in addition to free carotenoids.

1. Extraction Method The extraction method of the present invention is a method for extracting carotenoids from a plant or tissue thereof, and includes the following steps (I) to (III) in this order.
(I) A step of dehydrating a plant producing carotenoids or a tissue thereof by contacting with a dehydrating solvent (dehydration step)
(II) A process of extracting a dehydrated plant or tissue thereof with an extraction solvent to obtain a carotenoid-containing extract (extraction process)
(III) Water and an organic solvent are added to the solvent for dehydration brought into contact with the plant or its tissue in step (I) and the carotenoid-containing extract obtained in step (II), and the mixture is separated to contain carotenoids. Process to recover the organic solvent layer (recovery process)

  In the step (I), the plant or the tissue used as a material can be used as it is or after being cut, crushed, shredded (chopped), or powdered into an appropriate size. Preferably, a chopped plant or a tissue thereof is used.

  The dehydrating solvent used in the step (I) is preferably an organic solvent, more preferably a polar organic solvent, and for example, a polar organic solvent such as acetone, methanol, ethanol, 1-propanol, 2-propanol, and methyl ethyl ketone is preferable. These may be used alone or in combination of two or more. Among them, preferably, is at least one organic solvent selected from the group consisting of acetone, ethanol, methanol, and 2-propanol, more preferable dehydrating solvent is acetone, ethanol, or a mixture thereof, More preferred is a polar organic solvent containing acetone, and particularly preferred is acetone.

  The method for bringing the plant or its tissue into contact with the dehydrating solvent is not particularly limited, and examples thereof include a method of immersing the plant or its tissue in the dehydrating solvent. The amount of the solvent for dehydration is, for example, preferably about 1 to 100 mL, more preferably about 1 to 30 mL, and still more preferably about 2 to 10 mL with respect to 1 g of plant (usually wet weight).

  The conditions for dehydration are not particularly limited. For example, the time for contacting the plant with the solvent for dehydration is usually about 10 minutes to 24 hours, preferably about 1 to 12 hours, more preferably about 3 to 5 hours. . The temperature at the time of dehydration is not particularly limited, and is usually about 5 to 30 ° C, preferably about 18 to 24 ° C.

  The plant or tissue dehydrated by contacting with the dehydrating solvent is usually separated from the dehydrating solvent by filtration or the like and used in the next extraction step. The dehydrating solvent brought into contact with the plant or its tissue (used for dehydrating the plant or its tissue) is recovered and used in the subsequent recovery step (III).

In step (II) (extraction step), the dehydrated plant or its tissue is extracted with an extraction solvent to obtain a carotenoid-containing extract.
The extraction solvent is not particularly limited, and is preferably an organic solvent, more preferably a polar organic solvent, and examples thereof include polar organic solvents such as halogen-containing hydrocarbons, lower fatty acid esters, lower alcohols, and ketones. Examples of halogen-containing hydrocarbons include halogen-containing hydrocarbons having 1 to 3 carbon atoms such as chloroform, dichloromethane, dichloroethane, and trichloroethane; examples of lower fatty acid esters include 1 to 6 carbon atoms such as methyl acetate, ethyl acetate, and butyl acetate An ester of a fatty acid of; a lower alcohol such as methanol, ethanol, 1-propanol, 2-propanol, etc .; a C 1-3 alcohol; a ketone such as acetone, methyl ethyl ketone, etc. Is mentioned. These polar organic solvents may be used alone or in combination of two or more. Preferably, it is an extraction solvent such as a halogen-containing hydrocarbon, a lower alcohol, a ketone having 1 to 4 carbon atoms (acetone, methyl ethyl ketone, etc.) or a combination of two or more thereof, more preferably a halogen-containing hydrocarbon. And a mixed solvent of alcohol and lower alcohol. More preferably, the halogen-containing hydrocarbon is chloroform or the like, and the lower alcohol is methanol, ethanol or the like.

  When performing extraction using a mixed solvent of two or more organic solvents as an extraction solvent, the use ratio of each organic solvent may be appropriately set according to the type of the solvent, and is not particularly limited. For example, when a mixed solvent of a halogen-containing hydrocarbon and a lower alcohol is used as the extraction solvent, the mixing ratio of the halogen-containing hydrocarbon to the lower alcohol is, for example, 1:10 halogen-containing hydrocarbon: lower alcohol (volume ratio). ˜5: 1 is preferable, and about 1: 2 to 1: 1 is more preferable.

  The extraction method may be a commonly used method, for example, a method of immersing a dehydrated plant or its tissue in an extraction solvent, a method of performing extraction while heating and stirring at a temperature below the boiling point of the extraction solvent, etc. Can be mentioned. Moreover, in order to raise the efficiency of extraction, you may perform operations, such as chopping a plant or the structure | tissue in an extraction solvent, crushing, and grinding.

  The extraction conditions are not particularly limited. For example, the extraction time is usually about 10 minutes to 24 hours, preferably about 1 to 12 hours, more preferably about 6 to 8 hours. Although the temperature at the time of extraction is not specifically limited, Usually, it is about 5-30 degreeC, Preferably it carries out at about 18-24 degreeC.

  The amount of the extraction solvent used for extraction is usually about 2 to 50 mL, preferably about 5 to 25 mL, with respect to 1 g (usually wet weight) of the plant or its tissue used in the dehydration step.

  Through step (II) (extraction step), carotenoids such as carotenoid fatty acid esters are extracted into the extraction solvent, and a carotenoid-containing extract is obtained. After the extraction step, it is preferable to separate the plant or its tissue from the carotenoid-containing extract by filtration or the like.

  In step (III) (recovery step), water and an organic solvent are added to the dehydrating solvent brought into contact with the plant or its tissue in step (I) and the carotenoid-containing extract obtained in step (II). Liquid is collected and an organic solvent layer containing carotenoid is recovered.

  The organic solvent used in the step (III) may be any organic solvent that can be separated from water such as hydrocarbon, linear or branched hydrocarbon, and ether, such as hexane, petroleum ether, diethyl ether, ethyl methyl ether. , Dimethyl ether, normal pentane, normal heptane, normal octane and the like are preferable. Only one organic solvent may be used, or two or more organic solvents may be used in combination. Among these, the organic solvent is preferably at least one selected from the group consisting of hexane, diethyl ether, petroleum ether, ethyl methyl ether, and dimethyl ether, more preferably hexane, diethyl ether, and the like, and particularly preferably hexane.

  In step (III), the amount of water and organic solvent used is 2 to 10 times the total amount of water and organic solvent in a volume ratio with respect to the total of the dehydrating solvent and the carotenoid-containing extract obtained in step (II). It is preferable to use about 2 to 4 times.

  The use ratio of water and the organic solvent can be appropriately set depending on the type of the organic solvent and the like, and is not particularly limited. For example, the water: organic solvent (volume ratio) is about 3: 1 to 1:10. It is preferably about 2: 1 to 1: 5, and more preferably about 1: 0.8 to 0.8: 1.2.

  In the case of liquid separation, (i) the dehydrating solvent brought into contact with the plant or tissue thereof in step (I) and the carotenoid-containing extract obtained in step (II) are combined, and water and an organic solvent are added to this mixed solution. The liquid may be separated by adding, and (ii) water and an organic solvent are added to each of the dehydrating solvent brought into contact with the plant or its tissue in step (I) and the carotenoid-containing extract obtained in step (II). It may be added to carry out liquid separation. Preferably, liquid separation is performed by the method (i). Separation can be performed by a normal method. In both methods (i) and (ii), carotenoids migrate to the organic solvent layer. Usually, the organic solvent layer is colored orange or red with a carotenoid.

  In the organic solvent layer, carotenoids contained in the plant as a raw material are extracted. The type of carotenoid varies depending on the plant as a raw material, but usually a carotenoid mixture containing two or more types of free carotenoids and / or carotenoid fatty acid esters is obtained.

  According to the method of the present invention, carotenoids contained in plants or tissues thereof, for example, ketocarotenoid free compounds, ketocarotenoid fatty acid esters, and the like can be efficiently extracted from plants or tissues thereof. Among these, the method of the present invention is particularly suitable for extracting astaxanthin fatty acid esters. The astaxanthin fatty acid ester may be a diester, a monoester, or a mixture of a diester and a monoester. When a mixture of a diester body and a monoester body is obtained, the diester body and the monoester body can be separated by further purification and the like. The astaxanthin fatty acid ester is preferably astaxanthin myristic acid ester or palmitic acid ester.

  For example, when a plastid genome-modified plant expressed by introducing a gene encoding a carotenoid 4,4′-keto group-introducing enzyme, which will be described later, into a plant plastid genome or expressed by the extraction method of the present invention, usually, A carotenoid mixture containing astaxanthin fatty acid ester can be obtained. In addition, for example, plastid genomes in which genes encoding carotenoid 3,3′-hydroxyl transferase, carotenoid 4,4′-keto transferase and isopentenyl diphosphate isomerase are introduced and expressed in the chloroplast genome. When a modified plant or tissue thereof is used as a raw material, it is usually possible to obtain a carotenoid mixture containing astaxanthin fatty acid ester as a main component and further containing free carotenoids such as astaxanthin, frithielaxanthin, 4-ketoanthelaxanthin. it can. The present invention is suitable as a method for extracting a carotenoid mixture containing such a carotenoid fatty acid ester.

  In particular, if the plastid genome-modified plant is a chloroplast genome-modified lettuce, the organic solvent layer usually contains an astaxanthin fatty acid ester, an astaxanthin free form, frityra xanthine, 4-ketoanthaxanthin, lactucaxanthin. Etc. are included. In the case of chloroplast genome modified lettuce, astaxanthin fatty acid ester is usually contained in an amount of about 50 to 90% by weight, preferably about 50 to 80% by weight, in all carotenoids extracted by the extraction method. Astaxanthin fatty acid ester is usually a mixture of monoester and diester. The ester is usually an astaxanthin myristic acid ester or palmitic acid ester.

  The method of the present invention may include steps other than the above (I) to (III) as long as the effects of the present invention are exhibited. For example, you may perform the process of cut | disconnecting, crushing, shredding | pulverizing, etc. to the suitable magnitude | size the plant or its structure | tissue used at process (I). Moreover, you may dry the plant which is a raw material, or its processed material before process (I). Moreover, you may perform the process of further concentrating and refine | purifying the organic-solvent layer containing the collect | recovered carotenoid. Each carotenoid can be isolated by further purifying the organic solvent layer containing the carotenoid. Concentration, purification, etc. can be performed by known methods.

2. Plant producing carotenoid or tissue thereof Plant or tissue producing carotenoid used as a raw material in the present invention is a plant having plastids such as chloroplast, amyloplast, elaioplast, or colored body, or tissue thereof. If it is. Among them, a plant having a plastid such as a chloroplast or a colored body or a tissue thereof is preferable. In the present invention, the plant includes not only plant cells, tissues and organs but also individual plants (plants). Examples of plant tissues having plastids are leaves, stems, tubers, roots, rhizomes, flowers, florets, seeds, fruits, and the like. Plant cells such as protoplasts and callus are also included in the plant of the present invention. The plant may be a natural plant or a genome-modified plant (genetically modified plant) into which an exogenous gene has been introduced.

It does not specifically limit as a plant, Any of a seed plant, a fern plant, a moss plant, and algae may be sufficient. The seed plant may be a gymnosperm or an angiosperm. In the case of angiosperms, either monocotyledonous plants or dicotyledonous plants may be used, and there is no particular limitation. Examples of seed plants include cereals such as rice, barley, corn, and wheat; vegetables such as lettuce, cauliflower, cabbage, rapeseed, potato, tomato, carrot, basil, radish, spinach, turnip, turnip, pumpkin, and pepper; Green manure plants such as alfalfa, clover, and lotus; cosmos, torenia, chrysanthemum, gerbera, pansies, orchids, peonies, tulips and other flowering plants; soybeans, azuki beans, green beans, peanuts, broad beans, peas, and other beans; Other examples include: Arabidopsis thaliana, periwinkle and tobacco. Among them, lettuce, cauliflower, cabbage, oilseed rape, potatoes, tomatoes, carrots, rice, soybean, tobacco, Adonis (Adonis) Genus like. As Ferns, for example, the mainspring, bracken, field horsetail, and examples bryophyte, for example, liverwort, Tsunogoke, polytrichum juniperinum, sphagnum, and examples algae, Haematococcus pluvialis (Haematococcus pluvialis), Chlamydomonas (Chlamydomonas sp), Euglena ( Euglena algae (genus Euglena )) and the like. As a raw material in the present invention, a tissue having such a plant plastid is preferably used.

The plant or tissue thereof in the present invention is preferably a plant that produces a ketocarotenoid, more preferably a plant that produces a ketocarotenoid fatty acid ester, and even more preferably a plant that produces an astaxanthin fatty acid ester. Examples of plants that produce astaxanthin fatty acid esters include green algae such as the plants belonging to the genus Adonis (for example, Adonis aestivalis ), Haematococcus pluvialis, and the like. Further, for example, even if a plant or tissue thereof in which production of ketocarotenoid fatty acid ester has not been confirmed, if ketocarotenoid fatty acid ester is actually produced, the ketocarotenoid fatty acid ester can be obtained by subjecting it to the method of the present invention. Is extracted.

3. Plastid genome modified plant In the present invention, as a carotenoid-producing plant or tissue thereof, a genome-modified plant (genetically modified plant) or tissue obtained by introducing a gene of an enzyme involved in carotenoid biosynthesis into the genome. It can be used suitably. The genome modified plant is preferably a plastid genome modified plant. Since carotenoids are usually biosynthesized in plastids such as chloroplasts and colored bodies, plants or tissues that efficiently produce carotenoids by introducing genes for enzymes involved in carotenoid biosynthesis directly into the plastids. Can be obtained. The plastid genome-modified plant is preferably a plant that produces astaxanthin fatty acid ester or a tissue thereof.

  Plants that produce astaxanthin fatty acid esters or tissues thereof include, for example, genes encoding enzymes that synthesize astaxanthin from β-carotene (convert β-carotene to astaxanthin) as genes for enzymes involved in carotenoid biosynthesis. Or it can obtain by introduce | transducing into the structure | tissue. A plastid genome-modified plant or tissue obtained by introducing a gene encoding an enzyme that synthesizes astaxanthin from β-carotene into the plastid genome can be suitably used as a raw material in the present invention.

A plastid genome-modified plant obtained by introducing an enzyme gene that synthesizes astaxanthin from β-carotene into the plastid genome will be described below.
3.1 Enzyme gene that synthesizes astaxanthin from β-carotene As described above, the enzyme gene required to synthesize Asta from β-carotene typically has two enzyme genes, namely carotenoids 4,4. Genes encoding a '-keto group introducing enzyme (keto group introducing enzyme) and a carotenoid 3,3'-hydroxyl introducing enzyme (hydroxyl introducing enzyme) are preferred. Keto group-introducing enzyme gene, alpha-proteobacteria from belonging to bacteria leash bacteria crtW gene or isolated, Bkt gene isolated from H. pluvialis (Bkt1, Bkt2, Bkt3 ) and the like are known (the non Patent Documents 4 and 5). CrtW (meaning the protein encoded by the crtW gene) and BKT (meaning the protein encoded by the Bkt gene) have homology with each other. Known hydroxyl-transducing enzyme genes include the crtZ gene isolated from bacteria belonging to the α-proteobacteria class or the γ-proteobacteria class, the Bhy gene isolated from higher plants, and the like (Non-Patent Document 4). ). CrtZ and BHY have homology with each other. For example, any of these known genes may be used for production of a plastid genome-modified plant that produces astaxanthin fatty acid ester. Preferably, at least carotenoid 4,4′-keto group introducing enzyme (keto group introducing enzyme) is used. More preferably, a carotenoid 4,4′-keto group-introducing enzyme (CrtW) derived from Brevundimonas sp. Strain SD212, which is a combination of genes that synthesize Asta most efficiently from β-carotene, and Gene sequences encoding carotenoid 3,3′-hydroxyl transferase (CrtZ) are used (patent document 3; sequence information is described in DDBJ accession number AB377271 and accession number AB377272, respectively). The gene sequence (DDBJ accession number AB377271) encoding the carotenoid 4,4′-keto group-introducing enzyme (CrtW) derived from the Brevundimonas sp. Strain SD212 ( Brevundimonas sp. SD212 strain) is represented by SEQ ID NO: 1. The gene sequence (DDBJ accession number AB377272) encoding the carotenoid 3,3′-hydroxyl transferase (CrtZ) derived from the Bandimonas sp. Strain SD212 is shown in SEQ ID NO: 3, respectively. In addition, the amino acid sequence of carotenoid 4,4'-keto group-introducing enzyme (keto group-introducing enzyme) (CrtW) derived from Brevundimonas sp. Strain SD212 is represented by SEQ ID NO: 2, and carotenoid 3,3'-hydroxyl introducing enzyme (CrtZ ) Are shown in SEQ ID NO: 4, respectively. For example, by introducing these two genes into a lettuce chloroplast genome, a chloroplast genome modified plant such as a chloroplast genome modified (CGM) lettuce that produces Asta ester as a main component can be obtained. However, it is not always necessary to use the same crtW and crtZ gene sequences, and keto group-introducing enzymes and hydroxyl-introducing enzyme genes derived from other organisms may be used, and the latter equivalent gene ( Bhy ) must be in the plant. Therefore, only the former keto group-introducing enzyme gene may be used. It is also possible to use a conversion enzyme gene (CBFD and HBFD; Non-patent Document 9) from β-carotene to Asta derived from an Adonis plant. In addition, the enzyme gene which makes Asta fatty acid diester from Asta (free body) through Asta fatty acid monoester is not isolated. For example, in the case of CGM lettuce obtained by introducing an enzyme gene that synthesizes astaxanthin from β-carotene, it is thought that Asta (free form) produced in lettuce chloroplasts was esterified by endogenous enzymes in chloroplasts. .

3.2 Carotenoid biosynthetic pathway in higher plants In the plastids of plants, not the mevalonate pathway but the non-mevalonate pathway (2-C-methyl-D-erythritol 4-phosphate (hereinafter sometimes referred to as MEP) The first isoprenoid substrate, isopentenyl diphosphate (also called isopentenyl pyrophosphate; may be referred to as IPP hereinafter), is made. IPP is converted to dimethylallyl diphosphate (hereinafter sometimes referred to as DMAPP) by IPP isomerase (IPP isomerase; hereinafter sometimes referred to as IDI or Idi). DMAPP is a 10-carbon geranyl diphosphate (hereinafter referred to as GPP) by condensing with IPP sequentially by geranylgeranyl diphosphate (hereinafter sometimes referred to as GGPP) synthase (GGPS). A farnesyl diphosphate having 15 carbon atoms (hereinafter may be referred to as FPP) and GGPP having 20 carbon atoms. Branching from this GGPP, carotenoids (tetraterpenes) are synthesized (FIG. 1).

Two molecules of GGPP are condensed by phytoene synthase (PSY) to synthesize colorless phytoene (Phytoene) which is the first carotenoid (FIG. 1). In plant plastids, phytoene is converted to lycopene through a dehydrogenation reaction (unsaturation reaction) and an isomerization reaction. Details of this reaction are as follows. First, 9,15,9'-tri- cis- ζ-Carotene is synthesized from phytoene by phytoene desaturase (PDS), and then ζ-carotene unsaturation. Lycopene is synthesized by zymogenase (ζ-carotene desaturase; ZDS) and two types of carotene isomerase (Z-ISO and CRTISO) (Fig. 1) (Q. Yu, S. Ghisla, J. Hirschberg, V Mann, P. Beyer, J. Biol. Chem. 286: 8666-8676, 2011). This step is not yet fully understood.

  For example, in the case of a chloroplast of a general higher plant, two types of cyclized carotene are synthesized from lycopene. When only lycopene β-cyclase (LCYb) acts on lycopene, it is converted into β-carotene having two β rings (FIG. 1). β-carotene undergoes hydroxylation at the 3,3 ′ position by BHY and is converted to Zeaxanthin via β-Cryptoxanthin. Zeaxanthin is converted into Violaxanthin via Antheraxanthin by zeaxanthin epoxidase (ZEP). Violaxanthin is thought to be converted to neoxanthine, one of the final carotenoid forms in the leaves of higher plants, by neoxanthin synthase (NSY). When lycopene ε-cyclase (LCYe) and LCYb act on lycopene, it is converted into α-carotene (α-Carotene) having an ε ring and a β ring (FIG. 1). α-Carotene undergoes hydroxylation at the 3 ′ position by ε-carotene hydroxylase (EHY), a cytochrome P450 of the CYP97C subfamily. α-Cryptoxanthin (β, ε-Caroten -3'-ol), and is thought to be converted to lutein by hydroxylation at position 3 by P450 of the BHY or CYP97A subfamily (Fig. 1) (RF Quinlan, TT Jaradat, ET Wurtzel, Arch Biochem. Biophys. 458: 146-157, 2007).

  Carotenoid biosynthesis in normal higher plant chloroplasts is usually up to here, but in the case of lettuce, for example, another kind of cyclized carotene is produced. That is, only LCYe acts on lycopene and is converted to ε-Carotene having two ε rings (Fig. 1) (FX Jr. Cunningham, E. Gantt, PNAS 98: 2905-2910, 2001) ). It is believed that ε-carotene is converted to Lactucaxanthin by EHY (FIG. 1).

3.3 IPP isomerase gene
The amount ratio of IPP and DMAPP is adjusted by IDI (Idi; IPP isomerase). There are IDI (Idi) types of type 1 (type 1) and type 2 (type 2) that have different structures. Type 2 idi genes are sometimes present together in the carotenoid biosynthesis gene cluster of carotenoid-producing bacteria belonging to the α-proteobacteria or γ-proteobacteria class (Norihiko Misawa, Oreoscience 9 (9): 385-391, 2009). Type 1, irrespective of the type 2, when the idi gene is expressed and then is introduced into E. coli with carotenoid biosynthesis genes, in comparison with the case of not introducing the idi gene, production of carotenoids has been found to be elevated (H Harada, N. Misawa, Appl. Microbiol. Biotechonol. 84: 1021-1031, 2009). The plastid genome-modified plant used in the present invention is obtained by introducing a gene encoding isopentenyl diphosphate isomerase into the plant plastid genome in addition to the enzyme gene that synthesizes astaxanthin from β-carotene. It is preferably a plastid genome-modified plant.

In the following examples, the present inventors used type 2 idi gene as an example. Specifically, a gene sequence encoding Idi derived from Paracoccus sp. Strain N81106 ( Paracoccus sp. Strain N81106) (Non-Patent Document 12; sequence information is described in DDBJ Accession No. AB453829) was used. That is, in the embodiment, the idi gene sequence, Brevundimonas sp. SD212 introduced into lettuce chloroplast genome with the gene sequence encoding a CrtW and CrtZ derived, chloroplast genome to produce a main component Asta ester described above Modified (CGM) lettuce was obtained. The gene sequence (DDBJ accession number AB453829) encoding the IPP isomerase (Idi) derived from the Paracoccus bacterium N81106 strain ( Paracoccus sp. N81106 strain) is represented by SEQ ID No. 5, and the IPP isomerase derived from the Paracoccus bacterium N81106 strain. The amino acid sequence of (Idi) is shown in SEQ ID NO: 6, respectively.

  The plant or tissue thereof that produces carotenoid in the present invention is preferably a plastid genome-modified plant or tissue thereof obtained by introducing a gene encoding a carotenoid 4,4'-keto group-introducing enzyme into the plastid genome. Such a plastid genome-modified plant is preferable because it can produce, for example, an astaxanthin fatty acid ester.

  Examples of the gene encoding the carotenoid 4,4′-keto group-introducing enzyme include the gene encoding carotenoid 4,4′-keto group-introducing enzyme (CrtW) (SEQ ID NO: 2) derived from the serobacter genus SD212 of Brevandymonas sp. Preferably, as such a gene, for example, DNA containing the base sequence of SEQ ID NO: 1 is preferable, and DNA consisting of the base sequence of SEQ ID NO: 1 is more preferable.

  In addition, as a gene encoding a carotenoid 4,4′-keto group-introducing enzyme, it hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of SEQ ID NO: 1 under stringent conditions, and a β-ring A DNA encoding a polypeptide having an activity of introducing a keto group into the 4-position and / or the 4'-position of a β-ring of a carotenoid having β (carotene, zeaxanthin, etc.) is also preferably used. In addition, such a DNA sequence usually has a sequence identity of about 90% or more, preferably about 95% or more, more preferably about 98% or more with the DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and a β ring A DNA encoding a polypeptide having an activity of introducing a keto group into the 4-position and / or the 4′-position of a β-ring of a carotenoid having an amino acid is preferably used.

  In the present specification, “stringent conditions” refer to general conditions such as those described in Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Vol 2, p11.45. Specifically, it refers to the case where hybridization occurs at a temperature 5 to 10 ° C. lower than the melting temperature (Tm) of the complete hybrid.

  More preferably, the plastid genome-modified plant or the tissue thereof contains a carotenoid 3,3′-hydroxyl transferase and an isopentenyl diphosphate isomerase in addition to a gene encoding a carotenoid 4,4′-keto transferase. A plastid genome-modified plant or tissue thereof obtained by introducing a gene encoding each enzyme into the plastid genome. It is preferable to introduce more genes for astaxanthin fatty acid esters by introducing genes encoding carotenoid 3,3′-hydroxyl transferase and isopentenyl diphosphate isomerase.

  As a gene encoding the carotenoid 3,3′-hydroxyl transferase, a gene encoding a carotenoid 3,3′-hydroxyl transferase (CrtZ) (SEQ ID NO: 4) derived from a brendimonous bacterium SD212 strain is preferable. As such a gene, for example, DNA containing the base sequence of SEQ ID NO: 3 is preferable, and DNA consisting of the base sequence of SEQ ID NO: 3 is more preferable.

  In addition, as a gene encoding a carotenoid 3,3′-hydroxyl transferase, it hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of SEQ ID NO: 3, and a β ring. A DNA encoding a polypeptide having an activity of introducing a hydroxyl group into the 3rd and / or 3'-position carbon of a carotenoid possessed (such as β-carotene and canthaxanthin) is also preferably used. Further, as such DNA, for example, it has a sequence identity of usually about 90% or more, preferably about 95% or more, more preferably about 98% or more with DNA consisting of the nucleotide sequence of SEQ ID NO: 3, and β A DNA encoding a polypeptide having an activity of introducing a hydroxyl group into the 3rd and / or 3'position carbon of a carotenoid having a ring is preferably used.

  The gene encoding isopentenyl diphosphate isomerase is preferably a gene encoding isopentenyl diphosphate isomerase (Idi) (SEQ ID NO: 6) derived from the genus Paracoccus bacterium N81106. Examples of such a gene include the sequence DNA containing the base sequence of No. 5 is preferred, and DNA consisting of the base sequence of SEQ ID No. 5 is more preferred.

  Further, as a gene encoding isopentenyl diphosphate isomerase, it hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of SEQ ID NO: 5, and isopentenyl diphosphate is A DNA encoding a polypeptide having an isomerase activity that is converted to dimethylallyl diphosphate is also preferably used. Further, as such DNA, for example, it has a sequence identity of usually about 90% or more, preferably about 95% or more, more preferably about 98% or more with DNA consisting of the base sequence of SEQ ID NO: 5, and A DNA encoding a polypeptide having an isomerase activity for converting pentenyl diphosphate to dimethylallyl diphosphate is preferably used.

  In the present invention, a carotenoid-producing plant or tissue thereof includes genes encoding carotenoid 3,3′-hydroxyl transferase, carotenoid 4,4′-keto transferase and isopentenyl diphosphate isomerase. A plastid genome-modified plant or a tissue thereof introduced and expressed in the plastid genome is preferably used.

  As the plastid genome-modified plant, a dye comprising a DNA comprising the nucleotide sequence of SEQ ID NO: 1, a DNA comprising the nucleotide sequence of SEQ ID NO: 3, and a DNA comprising the nucleotide sequence of SEQ ID NO: 5 introduced into the plant plastid genome Body genome modified plants are particularly preferred. It is preferable to introduce these three genes because more astaxanthin fatty acid esters are produced.

3.4 Plastid Transformation Vector An enzyme gene or the like that synthesizes astaxanthin from β-carotene is introduced into a plant plastid by a known method. The method for introducing an exogenous gene into a chloroplast is not particularly limited. For example, a vector for plastid transformation in which a foreign gene to be introduced is expressed in a form that uses a suitable promoter and terminator by a known method. A plasmid may be prepared by inserting the plasmid into a plant cell, and the plasmid may be introduced into plant cells. When a plurality of genes are introduced, these genes may be introduced by one transformation vector, or may be introduced by different transformation vectors.

The plastid transformation vector may be appropriately selected depending on the type of plant and the like, but a chloroplast transformation vector is preferred. For example, lettuce chloroplast transformation vector pRL200 (H. Kanamoto, A. Yamashita, H. Asao, S. Okumura, H. Takase, M. Hattori, A. Yokota, K. Tomizawa, Transgenic Research 15: 205-217, 2006), tobacco chloroplast transformation vector pLD200 (supra Non-patent document 13), vector pASCC201 for cabbage chloroplast transformation (C.-W. Liu, C.-C. Lin, JJW Chen, M.-J. Tseng, Plant Cell Reports 26 (10): 1733- 1744, 2007), carrot chloroplast transformation vector pDD-Dc-aadA / badh (S. Kumar, A. Dhingra, and H. Daniell, Plant Physiology 136: 2843-2854, 2004), oilseed rape chloroplast. Transformation vector pNRAB (B.-K. Hou, Y.-H. Zhou, L.-H. Wan, Z.-L. Zhang, G.-F. Shen, ZM Hu Transgenic research 12 (1): 111-114, 2003). Any promoter may be used as long as it can be expressed in the plastids of plants. Examples include rrn promoter, psbA promoter, clpP promoter, psbD promoter, rbcL promoter, rpoC1 promoter, atpI promoter, rpl23 promoter, pabD promoter and the like. Among them, since it is preferable that the introduced gene is constantly expressed in plastids such as chloroplasts, a constitutive promoter is preferably used, and an rrn promoter 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 psbA terminator, rps16 terminator, rbcL terminator, rps7 terminator, psbB terminator and the like.

  In addition, various selection marker genes may be used in order to efficiently select plant cells into which a desired foreign gene (for example, a gene encoding CrtZ) has been introduced by a plastid transformation vector. The selection marker gene is not particularly limited, and a known marker gene may be used. For example, it is preferable to use a gene conferring drug resistance as a selection marker and introduce a plasmid or the like containing the selection marker gene and the gene involved in the biosynthesis of the carotenoid into a plant cell as a plastid transformation vector. As a result, plant cells in which a foreign gene has been introduced into the plastid genome can be efficiently selected from the expression of the selection marker gene. Examples of such selection marker genes include various drug resistance genes. More specifically, for example, herbicide chlorsulfuron resistance gene, streptomycin resistance gene, spectinomycin resistance gene and the like. Moreover, it is preferable to have a promoter and a terminator for expressing the gene upstream and downstream of the selection marker gene.

  As an example, a chloroplast transformation vector for introducing the enzyme gene that synthesizes astaxanthin from β-carotene into the chloroplast genome of lettuce will be described. A plastid genome-modified plant can be obtained by preparing a vector for transformation and introducing the vector into cells.

A vector for transforming lettuce chloroplasts and a method for transforming lettuce chloroplasts using the same were reported by Kanamoto et al. (H. Kanamoto, A. Yamashita, H. Asao, S. Okumura, H Takase, M. Hattori, A. Yokota, K. Tomizawa, Transgenic Research 15: 205-217, 2006). Lettuce chloroplast transformation vector pRL200 is a vector in which a cloning site of Not I- Nhe I- Sal I is inserted between rbcL and accD genes derived from a lettuce chloroplast genome (FIG. 4). What is necessary is just to introduce into this cloning site what inserted the exogenous gene to express in lettuce chloroplast between promoter and terminator. As the promoter and terminator, those that function in lettuce chloroplasts may be used. As an example, the former may be a promoter of rrn or psbA , and the latter may be a terminator of psbA or rps16 . These promoter and terminator sequences are preferably derived from the lettuce chloroplast genome, but those derived from other plants such as those derived from tobacco chloroplasts can also be used. For example, in the examples, a promoter of rrn derived from tobacco chloroplasts and a terminator of psbA or rps16 were used. Furthermore, it is preferable to use a drug resistance gene surrounded by a promoter and terminator together as a marker gene for transformation. Examples of drug resistance genes include Streptomycin / Spectinomycin Adenyltransferase ( aadA ) gene, which confers resistance to streptomycin and spectinomycin to the host (H. Kanamoto, A. Yamashita , H. Asao, S. Okumura, H. Takase, M. Hattori, A. Yokota, K. Tomizawa, Transgenic Research 15: 205-217, 2006). For example, chloroplast transformants can be selected by adding 50 mg / L spectinomycin dihydrochloride to the medium. The aadA gene is also used for selection of transformation of various bacteria including E. coli (NC Clark, O. Olsvik, JM Swenson, CA Spiegel, FC Tenover, Antimicrobial Agent and Chemotherapy 43: 157-160). 1999).

3.5 Transformation of plant plastids and acquisition of a plastid genome-modified plant Plant plastids can be transformed by known methods. For example, a DNA fragment is prepared in which a foreign gene to be introduced (for example, an enzyme gene that synthesizes astaxanthin from β-carotene) or a selection marker gene (such as a drug resistance gene) is expressed by using an appropriate promoter and terminator, A plant comprising the DNA fragment inserted into a vector having sequences of two plastid genes adjacent to each other, for example, and using a particle gun or the like, preferably a plant containing chloroplasts such as plant leaves and stems Just put it in the cells of your tissue. “A vector having sequences of two plastid genes next to each other” is a vector containing two genes adjacent to each other in the natural plant plastid genome, and a gene to be introduced is inserted between the two genes. It is preferable to do. A plastid genome-modified plant is obtained by cultivating or cultivating a plant into which a gene has been introduced or a tissue thereof and redifferentiating it. Carotenoids are produced in plastids in plastid genome-modified plants or tissues thereof. A method for cultivating or culturing a plant into which a gene has been introduced or a tissue thereof, a method for redifferentiation, etc. are not particularly limited, and may be appropriately set according to the type of plant.

For example, plastid transformation of food crops has been reported in cauliflower, cabbage, rapeseed (canola), potato (potato), tomato, carrot, rice, and soybean other than lettuce (D. Verma, H. Daniell, Plant Physiology 145: 1129-1143, 2007). Therefore, in the Examples, transformation was performed using lettuce, but the same thing can be performed using these crops and the like. The transformation method of lettuce chloroplasts has been reported by Kanamoto et al. (H. Kanamoto, A. Yamashita, H. Asao, S. Okumura, H. Takase, M. Hattori, A. Yokota, K. Tomizawa , Transgenic Research 15: 205-217, 2006). Although this method may be used, the method for introducing a foreign gene is the same as the ordinary particle gun method. For example, in the case of lettuce, a foreign gene (eg, crtW , crtZ , idi , and aadA ) to be introduced into a vector for transforming lettuce chloroplasts (eg, pRL200 described above) is expressed by using the promoter and terminator described above. Then, the inserted plasmid is prepared, and it is injected into lettuce leaves with a particle gun. For example, when the aadA gene is used, it is placed on a lettuce redifferentiation medium containing spectinomycin (an example of the medium composition is shown in Test Example 2) and cultured to form adventitious buds and leaves. Chloroplast transformation lettuce can be selected. For example, when the vector pRL200 is used, foreign genes (for example, crtW , crtZ , idi , aadA, etc. ) can be introduced between the rbcL and accD genes that are adjacent in the chloroplast genome. When the aadA gene is used, the chloroplast genome is mostly recombined by introducing a foreign gene into the lettuce regeneration medium containing spectinomycin. become. The lettuce thus obtained is referred to as Chloroplast-Genome Modified (CGM) lettuce.

  The plant into which the gene encoding an enzyme that synthesizes astaxanthin from β-carotene is introduced is not particularly limited, and for example, the above-described seed plants are preferable. Among them, lettuce, cauliflower, cabbage, rapeseed, potato, tomato, carrot, rice, soybean, tobacco and the like are preferable, and lettuce is more preferable.

  A plastid that introduces a gene that encodes an enzyme that synthesizes astaxanthin from β-carotene (such as the carotenoid 4,4′-keto group-introducing enzyme), that is, a plastid that is transformed into a plant type, tissue, etc. For example, chloroplasts, amyloplasts, elaioplasts, colored bodies and the like may be selected. For example, in the case of plants such as lettuce, cabbage and tobacco, the gene can be introduced into chloroplasts to make a chloroplast genome modified plant. For example, in the case of a plant having amyloplasts such as cauliflower, potato (potato), rice, soybean, rapeseed (canola), etc., the gene can be introduced into amyloplast or the like to obtain an amyloplast genome-modified plant. For example, in the case of a plant having a colored body such as tomato and carrot, the gene can be introduced into the colored body to obtain a colored body genome-modified plant. When the introduced gene is expressed in the plastid, carotenoids such as astaxanthin and astaxanthin fatty acid ester are efficiently produced in the plastid.

  For example, in lettuce, cabbage, tobacco, etc., in which the gene is introduced into the chloroplast and the chloroplast genome is modified, astaxanthin fatty acid ester and the like are usually produced in the leaf. In plants such as cauliflower, potato (potato), rice, soybean, rapeseed (canola), in which the gene is introduced into amyloplast and the amyloplast genome has been modified, tissues in which amyloplast is present, for example, cauliflower flower bud; potato tuber; Rice, rapeseed seeds; Astaxanthin fatty acid esters and the like are produced in soybean cotyledons and the like. In tomatoes, carrots and the like in which the gene is introduced into the colored body and the colored body genome is modified, astaxanthin fatty acid esters and the like are produced in tissues where the colored body exists, for example, tomato fruits; carrot roots. Astaxanthin fatty acid esters and the like produced in the plastid usually accumulate in the plastid.

  The expression of a foreign gene introduced into a plastid in a plastid genome-modified plant or its tissue can be confirmed, for example, by methods such as Northern blotting, RT-PCR, and Western blotting. Moreover, it can confirm that carotenoid is produced in this plant or its structure | tissue by methods, such as HPLC analysis and LC-MS analysis, for example. In addition, a gene encoding an enzyme that synthesizes astaxanthin from β-carotene was introduced into the plastid, and the expressed plastid genome-modified plant or its tissue was normally produced compared to the wild-type plant or its tissue. It exhibits yellow to red color derived from carotenoids. For example, in the case of a chloroplast genome-modified plant, a green-colored leaf or the like in a wild-type plant is yellowish to reddish. For this reason, the production of carotenoids and the like can also be confirmed visually.

  For example, a gene encoding an enzyme that synthesizes astaxanthin from β-carotene is introduced into the plastid genome, and expressed plastid genome modified lettuce, cauliflower, cabbage, rapeseed, potato, tomato, carrot, rice, soybean, Tobacco or the like is preferable as a plant in the present invention. That is, the plastid genome-modified plant is preferably lettuce, cauliflower, cabbage, rapeseed, potato, tomato, carrot, rice, soybean, or tobacco. Among them, the plastid genome-modified plant in the present invention is particularly preferably a chloroplast genome-modified plant, and most preferably, a carotenoid 3,3′-hydroxyl-introducing enzyme, a carotenoid 4,4′-keto group-introducing enzyme and an isoenzyme. This is lettuce expressed by introducing a gene encoding each pentenyl diphosphate isomerase into the chloroplast genome. Examples of the lettuce include head lettuce such as Berkeley, Cisco, Kaiser, Legacy; and leaf lettuce such as green leaf, frill lettuce, and green wave, and head lettuce is preferable, and among them, Berkeley is more preferable. For example, the chloroplast genome modified lettuce described in Non-Patent Document 14 or 15 is also suitably used as the plastid genome modified plant in the present invention.

  A gene encoding the carotenoid 4,4'-keto group-introducing enzyme is introduced into the plastid genome, and the expressed plastid genome-modified plant usually produces an astaxanthin fatty acid ester in the transformed plastid. Astaxanthin fatty acid ester has antioxidant, anti-aging effects (suppression of skin photoaging, prevention of macular degeneration, etc.), anti-fatigue effects (reduction of eye strain and muscle fatigue, etc.), anti-life habits Has pathological effects (prevention of diabetic nephropathy, oxidation of LDL (bad cholesterol), improvement of blood fluidity, suppression of blood pressure increase), anti-inflammatory action, anti-cancer action, etc. (eg Toshikazu Yoshikawa, Naito) Supervised by Yuji, functions and applications of astaxanthin, CM Publishing, 2012). Furthermore, the astaxanthin fatty acid ester has higher intestinal absorbability at the time of oral administration than the free form (astaxanthin), and is useful as a functional food material and the like.

  By the extraction method of the present invention, for example, a carotenoid containing an astaxanthin fatty acid ester is obtained from a plastid genome-modified plant obtained by introducing a gene encoding the carotenoid 4,4′-keto group-introducing enzyme or the like into the plastid genome of a plant. It can be extracted efficiently. For this reason, such plastid genome-modified plants can be suitably used for producing astaxanthin fatty acid esters.

4). TECHNICAL FIELD The present invention relates to a plastid genome modified plant obtained by introducing a gene encoding a carotenoid 4,4′-keto group-introducing enzyme into a plastid genome of a plant. Of the present invention for the production of astaxanthin fatty acid esters. Preferable embodiments of the plastid genome-modified plant and its production method are as described above, and together with a gene encoding a carotenoid 4,4′-keto group transducing enzyme, a carotenoid 3,3′-hydroxyl transducing enzyme, and isopentenyl A plastid genome-modified plant obtained by introducing a gene encoding each enzyme of diphosphate isomerase into the plastid genome of a plant is preferable.
Each gene and its preferred embodiment are as described above. The plastid genome-modified plant is preferably chloroplast genome-modified lettuce.

  The produced plastid genome-modified plant or tissue thereof is usually cultivated or cultured to produce an astaxanthin fatty acid ester in the plant or tissue thereof. The method for cultivating or culturing the plastid genome-modified plant or its tissue may be appropriately set according to the type of plant and the like, and is not particularly limited.

  The astaxanthin fatty acid ester may be a diester, a monoester, or a mixture of a diester and a monoester. The diastaxanthin fatty acid ester is preferably an astaxanthin myristic acid ester or palmitic acid ester.

  For example, if the plastid genome-modified plant is a chloroplast genome-modified lettuce, its leaves and the like, usually as an astaxanthin fatty acid ester as carotenoid, astaxanthin educt, frityra xanthine, 4-ketoanthaxanthin, Such as lactucaxanthin. Therefore, these carotenoid mixtures can be obtained by the extraction method. In the case of chloroplast genome modified lettuce, astaxanthin fatty acid ester is usually contained in an amount of about 50 to 90% by weight, preferably about 50 to 80% by weight, in all carotenoids extracted by the extraction method. Astaxanthin fatty acid ester is usually a mixture of monoester and diester, and is myristic acid ester or palmitic acid ester. Such carotenoid mixtures can be produced by plastid genome modified plants.

  A mixture of carotenoids produced by a plastid genome-modified plant is suitably used for, for example, feeds for health and functional foods, cultured fish, poultry and the like. Further, each carotenoid can also be isolated from a mixture of carotenoids by a known separation or purification method such as HPLC.

  The plastid genome-modified plant or its tissue is, for example, a raw material containing astaxanthin fatty acid ester or the like, or subjected to treatment such as heating, drying, shredding, powdering, etc. It is also suitably used as feed for fish and poultry.

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

<Test Example 1>
(Preparation of plasmid for astaxanthin production)
CrtW and CrtZ derived from the marine bacterium Brevundimonas sp. Strain SD212 are a combination of enzymes that efficiently convert β-carotene to Asta (Patent Document 2).

Since the original crtW and crtZ genes derived from Brevundimonas sp. SD212 have a high GC content of 70%, we chemically synthesized crtW and crtZ that were changed to plant-type codon usage (the encoded amino acid sequence was identical to the original one). ). The synthesized sequences of crtW and crtZ are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. The base sequences of chemically synthesized crtW and crtZ (SEQ ID NO: 1 and SEQ ID NO: 3) can be obtained from DDBJ accession number AB377271 and accession number AB377272, respectively.

The pLD200-ZW used for preparing the plasmid plasmid pRL-crtZW for producing astaxanthin will be described.
Hasunuma et al. Introduced the genes crtW and crtZ ( SEQ ID NO: 1 and SEQ ID NO: 3 respectively ) directly into the chloroplast genome of non-food crop tobacco ( Nicotiana tabacum ) under the control of the rrn promoter. Modified (Chloroplast-Genome Modified; hereinafter referred to as CGM) tobacco was obtained (Non-Patent Document 13). CGM tobacco transformed with the plasmid pLD200-ZW (non-patent document 13) inserted downstream of the rrn promoter in the order of crtZ and crtW and attached with a terminator of rps16 downstream showed high Asta productivity. The production amount of Asta in this CGM tobacco was the maximum, reaching 5.44 mg per 1 g of dry weight of leaves, but all produced Asta was free (Non-patent Document 13). Even though 99% of carotenoids were converted to ketocarotenoids such as Asta (74% of all carotenoids) in the leaves of this CGM tobacco, CGM tobacco was able to synthesize and grow normally. The plasmid pLD200-ZW is a vector for transforming tobacco chloroplast into Not I-P rrn - aadA -T psbA -P rrn - crtZ - crtW - Eco RI-T rps16 - Sal I fragment (Non-patent Document 13). It is a plasmid inserted into the Not I- Sal I site between rbcL and accD gene (derived from tobacco chloroplast genome) in pLD200 . P and T represent a promoter and a terminator, respectively. aadA is a streptomycin / spectinomycin adenyltransferase gene, which confers resistance to spectinomycin (or streptomycin) to the host (marker gene). The base sequence of tobacco chloroplast transformation vector pLD200 is disclosed in DDBJ Accession No. CS165378. Incidentally, the foregoing Not I-P rrn - aadA -T psbA -P rrn - crtZ - crtW - Eco RI-T rps16 - Sal I fragment, PCR grown Bam HI- crtZ the crtZ and crtW chemically synthesized described above as a template - The Xba I- crtW - Eco RI fragment is transformed into the plasmid pLD7-rrnP-MCS (base) containing the Not I-P rrn - aadA -T psbA -P rrn - Bgl II- Xba I- Eco RI (MCS) -T rps16 - Sal I fragment. Bgl II- Eco those made by inserting the RI site (prepared plasmid name pLD7-rrnP-crtZ-crtW sequences disclosed in DDBJ accession No. AB375764); non-Patent Document 13). In addition, the sequence upstream of the start codon ATG of crtZ and crtW gene in the PCR-grown Bam HI- crtZ - Xba I- crtW - Eco RI fragment is the same after each restriction site, BamHI / XbaI site-AAAGAGGAGAAATTACATATG (SEQ ID NO: 7). This sequence contains ribosome-binding sites that are chloroplast and efficient in translation.

The present inventors used pRL200 (FIG. 4) produced by Kanemoto et al. As a vector for transformation of lettuce chloroplasts (H. Kanamoto, A. Yamashita, H. Asao, S. Okumura, H. et al.). Takase, M. Hattori, A. Yokota, K. Tomizawa, Transgenic Research 15: 205-217, 2006). pRL200 has a Not I- Nhe I- Sal I cloning site between the rbcL and accD genes derived from the lettuce chloroplast genome (FIG. 4). Cut out the Not I-P rrn - aadA -T psbA -P rrn - crtZ - crtW - Eco RI-T rps16 - Sal I fragment in the plasmid pLD200-ZW and insert it into the Not I- Sal I site of this vector pRL200. pRL-crtZW was prepared.

Since the original idi gene from Paracoccus sp. N81106 has a high GC content of 70%, the idi was changed to a plant-type codon and was chemically synthesized (the encoded amino acid sequence was the same as the original). The chemically synthesized idi sequence is shown in SEQ ID NO: 5. The base sequence (SEQ ID NO: 5) of this chemically synthesized idi can be obtained from DDBJ Accession No. AB453829. Next, Mfe I- idi - Eco RI fragment was synthesized by PCR using this chemically synthesized gene as a template. The sequence upstream of the start codon ATG of the idi gene in this DNA fragment is Mfe I site-AAAGAGGAGAAATTACATATG (SEQ ID NO: 8). This sequence contains ribosome-binding sites that are chloroplast and efficient in translation. This Mfe I- idi - Eco RI fragment was inserted into the Eco RI site of plasmid pLD7-rrnP-crtZ-crtW (Non-patent Document 13) to obtain plasmid pLD7-rrnP-crtZ-crtW-idi. Next, the Not I-P rrn - aadA -T psbA -P rrn - crtZ - crtW - idi - Eco RI-T rps16 - Sal I fragment in this plasmid was excised and inserted into the Not I- Sal I site of the lettuce vector pRL200. Plasmid pRL-crtZWidi was prepared (FIG. 5).

<Test Example 2>
(Transformation of lettuce chloroplasts)
Lettuce ( Lactuca sativa L. var. Berkeley) leaves (chloroplasts) were transformed with the plasmids pRL-crtZW and pRL-crtZWidi prepared in Test Example 1. Specifically, it was performed as follows.

The seeds of wild-type lettuce Berkeley ( Lactuca sativa L. var. Berkeley) were purchased from Takii seedlings. This lettuce seed is immersed in 70% ethanol for 2 minutes to be sterilized, washed thoroughly with sterilized water, 0.8% plant agar (manufactured by Wako Pure Chemical Industries), 0.1% MS vitamin solution (manufactured by Sigma Aldrich) And 3% sucrose (manufactured by Wako Pure Chemical Industries, Ltd.) and placed on MS medium (Murashige and Skoog, Physiologia Plantarum 15: 473-497, 1962) (hereinafter referred to as lettuce medium) adjusted to pH 5.8. Then, aseptic culture was performed at 20 ° C. in an artificial weather chamber (Sanyo Growth Cabinet). Cut the leaf tissue after 3-4 weeks of culture into lettuce medium containing 0.5 mg / L benzylaminopurine (Sigma Aldrich) and 0.1 mg / L naphthalene acetic acid (Sigma Aldrich). After standing overnight in a dark place, using the two astaxanthin-producing plasmids (pRL-crtZW and pRL-crtZWidi) prepared in Test Example 1, gene transfer was performed by the particle bombardment method (particle gun method). went. Kanamoto et al. (H. Kanamoto, A. Yamashita, H. Asao, S. Okumura, H. Takase, M. Hattori, A. Yokota, K. Tomizawa, Transgenic Research 15: 205-217, 2006 ). The leaf tissue after gene transfer was allowed to stand in the dark for 2 days and sectioned, 0.5 mg / L benzylaminopurine, 0.1 mg / L naphthalene acetic acid, 500 mg / L polyvinylpyrrolidone, and 50 mg / L spec It was placed on a lettuce medium containing tinomycin dihydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) and selected for 3 to 8 weeks to obtain a chloroplast-transformed lettuce in vitro plant.

<Test Example 3>
(Acquisition of the chloroplast genome modification lettuce T ₁₎
Among the chloroplast-transformed lettuce in vitro plants obtained in Test Example 2, reddish leaves were selected, 0.5 mg / L benzylaminopurine, 0.1 mg / L naphthalene acetic acid, and 50 mg / L. Were placed on a lettuce medium containing spectinomycin dihydrochloride, and in vitro plants were redifferentiated via adventitious buds and grown on a lettuce medium. Among the in vitro plants, the process of selecting reddish leaves and redifferentiating the in vitro plants via adventitious buds by the same operation as described above was repeated 2-3 times. The thus obtained chloroplast-transformed lettuce in vitro plant with more reddish leaves was a line transformed with the plasmid pRL-crtZWidi. One of these in vitro plants was transferred to soil, acclimatized for several weeks in an artificial meteorograph, and then transferred to a specific net room. The lettuce transformant, ie, the Chloroplast-Genome Modified lettuce (CGM lettuce) grew smoothly, made pink flowers bloom and added many seeds. This is referred to as T ₁ seed. This T ₁ seed showed a germination rate of 99%, which was higher than the germination rate of non-transformants (93%) (Non-patent Document 15). The following experiment was conducted using CGM lettuce leaves grown from the T ₁ seed.

<Comparative Example 1>
(Extraction and purification of carotenoids from chloroplast genome modified lettuce)
Paul D. Fraser et al. (PD Fraser, M. Elisabete, S. Pinto, DE Holloway, PM Bramley, Plant Journal, 24: 551-558, 2000) extracts carotenoids from plant tissues. This method is often used. For example, it was used also in the non-patent documents 12 to 15. This method has been optimized by examining several extraction conditions such as the type of solvent. The plant tissue is ground in a mortar and then added in the order of methanol and chloroform to extract the pigment.

The present inventors tried to extract the whole carotenoid by the method of Paul Fraser et al. Using leaves (0.2 g) of a chloroplast genome modified lettuce (CGM lettuce) T ₁ plant. However, in this method, can not be extracted efficiently dye from the leaves of CGM lettuce T ₁ plants, the brown color remained in fractions of leaves should be already extracted.

<Example 1>
(Extraction and purification of carotenoids from chloroplast genome modified lettuce)
For the extraction of carotenoids, methanol or chloroform-methanol or acetone is often used. Therefore, after crushing the leaves (0.2 g) of the CGM lettuce T ₁ plant with a mortar, extraction with acetone was attempted, but the acetone extract was only slightly brown. Therefore, the inventors adopted a method in which the chopped leaves were passed through with acetone and then soaked in chloroform-methanol and crushed in a mortar there. By combining this extract with the slightly colored acetone pretreatment liquid, adding hexane and water, and separating with a separatory funnel, the carotenoid pigment can be completely collected in the upper organic solvent layer. It was. Specifically, it was performed as follows.

Several leaves (5.11 g) of CGM lettuce T ₁ plants were cut out and chopped, then 20 mL of acetone was added and dehydrated at room temperature for 5 hours. The acetone solution was slightly colored (brown). After lettuce leaves were taken out from acetone and transferred to a mortar, 50 mL of chloroform-methanol (1: 1) was added stepwise and ground well, followed by extraction at room temperature for 6 hours. After the acetone solution and chloroform-methanol (1: 1) solution were combined, the solution was transferred to a separatory funnel, and 200 mL of hexane and water (hexane: water 1: 1 (volume ratio)) were added thereto for liquid separation. The colored organic solvent layer (upper layer) was separated.

  As a control, leaves of non-transformed lettuce (6.86 g) were cut out, carotenoid pigments were similarly extracted, and a colored organic solvent layer (upper layer) was separated.

Each organic solvent layer was dried over anhydrous sodium sulfate, and then the absorption method (K. Schiedt, S. Liaaen-Jensen, Isolation and analysys, in Carotenoids (G. Britton, S. Liaaen-Jensen, H. Pfander (Eds. ) Birkhauser, Basel; 1995; Vol. 1A, pp 81-108) The total carotenoid content was measured using a molecular extinction coefficient at 470 nm for the determination of ketocarotenoids, including astaxanthin (fatty acid esters and free forms). E (absorbance at 1 cm, 1% (w / v))) was used as 2100. For quantification of carotenoids other than ketocarotenoids, molecular extinction coefficient ( E (1 cm, 1% (w / v) at 447 nm) was used. )) Was used as 2).
The total carotenoid content of CGM lettuce leaves and control lettuce leaves was 230 μg and 86 μg per 1 g (wet weight), respectively. CGM lettuce was 2.67 times more than control lettuce.

The above extracted solution was concentrated to dryness with an evaporator, and then each carotenoid component was separated and purified by HPLC.
HPLC conditions:
HPLC column Cosmosil 5SL-II 4.6 × 250 mm
Moving bed 0-20 minutes Acetone / hexane (2: 8), 20 minutes later acetone / hexane (4: 6), flow rate 1.0 ml / min, detection 450 nm
The composition of each carotenoid was determined from the peak area of HPLC.

(Identification and quantification of carotenoids produced by modified chloroplast genome lettuce)
The results of HPLC analysis are shown in FIGS. Each carotenoid was fractionated and identified by UV-VIS, FAB MS, ¹ H-NMR, and CD (FIGS. 2 and 3; each spectral data is described later).

Each peak in FIG. 2 was identified as follows.
1: (3 S , 3 ' S ) -Astaxanthin diesters, 2: Canthaxanthin, 3:: (3 S , 3' S ) -Astaxanthin monoesters, 4: (3 S ) -Adonirubin, 5: (3 ' S )- Asteroidenone, 6: (3 S , 3 ' S ) -Astaxanthin, 7: Fritschiellaxanthin, 9: Lactucaxanthin, 10: 4-Ketoantheraxanthin, 11: cis -4-Ketoantheraxanthin. 12: Chlorophyll degradation product.

Each peak in FIG. 3 was identified as follows.
1: β-Carotene, 2: β-Cryptoxanthin, 3: Lactucaxanthin, 4: Lutein, 5: Zeaxanthin, 6: Lutein-5,6-epoxide, 7: Antheraxanthin, 8: Chlorophyll degradation product, 9: 9'- cis -Neoxanthin.

  Table 1 shows the carotenoid composition of CGM lettuce leaves and control lettuce leaves that were revealed as a result. The biosynthetic pathway of various carotenoids in CGM lettuce and the functions of various carotenoid synthases are shown in FIG.

^* Diesters with myristic acid: palmitic acid and diesters with myristic acid: diesters with palmitic acid (19:47:34)
^** Astaxanthin myristic acid ester: palmitic acid ester (45:55)

Through these studies, the present inventors have produced 2.67 times as much total carotenoids as normal lettuce in CGM lettuce, of which 67.4% are Asta fatty acid esters (49.2%: diester, 18.2%: monoester) The fatty acid part of the Asta fatty acid ester is myristate or palmitic acid, and as an ketocarotenoid other than the Asta fatty acid ester (10%), Fritiella It was found that xanthine (6%), canthaxanthin (5.3%), 4-ketoanthelaxanthin (4.3%), etc., and lactucaxanthin (3.8%), a carotenoid specific to lettuce, were also included (Table) 1). Today, it has been reported that Asta is synthesized in various agricultural crops by advanced biotechnology. In any case, it is an example of production of Asta educt (Non-patent Documents 5 and 10-15). As in the case of CGM lettuce this time, it was an unexpected finding that Asta fatty acid ester was synthesized in large quantities and contained more than half of the total carotenoids. This is the first finding that has been clarified by examining the efficient extraction and purification of carotenoids from CGM lettuce. Since Asta produced by H. pluvialis, which is actually sold as a health food material, is also a fatty acid ester, this is a health food material for carotenoids extracted from CGM lettuce (and CGM lettuce itself in the future). It was convenient in considering the use of. In addition, CGM lettuce also produces rare carotenoids such as frithielaxanthin, 4-ketoanthelaxanthin and lactucaxanthin, but there are no other organisms that produce carotenoid mixtures of these combinations, so these rare carotenoids are The total carotenoids in CGM lettuce (called multi-carotenoids) cannot be realized by other production methods. In addition, the total production of Asta in CGM lettuce is 180 μg / g wet weight in terms of free form. Not only among the recombinant food crops produced so far, but also Asta-containing foods such as salmon and salmon (for example, The content of Asta in salmon and salmon roe was the highest level compared to 20-38 μg / g wet weight in terms of free form. In addition, 4-ketoantheraxanthin is a novel carotenoid identified for the first time in CGM tobacco leaves (Non-patent Document 13), and Frithielaxanthin is a rare carotenoid contained in a trace amount in Japanese crab and acategani (Non-patent Document 1). Reference 1). The CGM lettuce produced this time can be easily grown in plant plants for recombinant plants, so it is not only expected as a stable source of other rare ketocarotenoids in addition to Asta, but also the physiological role of ketocarotenoids in higher plants It is expected to become a model plant for exploring.

(Spectral data of carotenoids produced by modified chloroplast genome lettuce)
The spectral data of the carotenoid identified from the chloroplast genome modification lettuce are shown.
(3 S , 3 ' S ) -Astaxanthin diester: UV-VIS: 470 nm (Et ₂ O) .FAB-MS: m / z 1072 (Astaxanthin dimyristate), m / z 1044 (Astaxanthin myristyl palmitate), m / z 1016 (Astaxanthin dipalmitate. Ratio of Astaxanthin dimyristate and Astaxanthin myristyl palmitate to Astaxanthin dipalmitate by ionic strength of MS (19:47:34). ¹ H NMR δ (CDCl ₃ ): 0.88 (t, J = 7.5 Hz, CH ₃ in fatty acid moiety), 1.23 (6H, s, CH ₃ -17, 17 '), 1.26 (s, CH ₂ in fatty acid moiety), 1.34 (6H, s CH ₃ -16, 16'), 1.70 (t, J = 7.5 Hz, CH ₂ in fatty acid moiety), 1.91 (6H, s, CH ₃ -18, 18 '), 1.99 (6H, s, CH ₃ -19, 19'), 2.00 (6H, s, CH ₃ -20, 20 '), 2.01 (2H, overlapped, H-2, 2' eq), 2.07 (2H, dd J = 12, 6 Hz, H-2, 2 'ax), 2.08 (t, J = 7.5 Hz, CH ₂ in fatty acid moiety), 2.43 (m, CH ₂ in fatty acid moiety), 5.54 (2H, dd, J = 13.5, 5.5 Hz, H-3, 3 '), 6.20 (2H, d, J = 16 Hz, H-7, 7 '), 6.30 (2H, d, J = 11.5 Hz, H-10, 10'), 6.32 (2H, m, H-14, 14 '), 6.40 (2H, d, J = 16 Hz, H-8, 8 '), 6.45 (2H, d, J = 15.5 Hz, H-12, 12'), 6.65 (2H, dd, J = 15.5, 11.5 Hz, H-11, 11 '), 6.67 (2H, m, H-15, 15'). CD λ nm (Δε): 240 (-20.0), 252 (0), 270 (+20.0), 284 (0) , 314 (-34.0), 355 (0), 372 (+3.0).

Canthaxanthin: UV-VIS: 470 nm (Et ₂ O). FAB-MS: m / z 564 [M] ⁺ .
(3 S , 3 ' S ) -Astaxanthin monoester: UV-VIS: 470 nm (Et ₂ O). FAB-MS: m / z 834 (Astaxanthin myristate), m / z 806 (Astaxanthin palmitate. Ratio of Astaxanthin myristate to Astaxanthin dipalmitate (45:55). ¹ H-NMR δ (CDCl ₃ ): 0.88 (t, J = 7.5 Hz, CH ₃ in fatty acid moiety), 1.21 (3H, s, CH ₃ -17 '), 1.23 (3H, s, CH ₃ -17), 1.26 (s, CH ₂ in fatty acid moiety), 1.32 (3H, s, CH ₃ -16'), 1.34 (3H, s, CH ₃ -16 ), 1.70 (t, J = 7.5 Hz, CH ₂ in fatty acid moiety), 1.82 (1H, dd, J = 13, 13 Hz, H-2'ax), 1.91 (3H, s, CH ₃ -18) , 1.95 (3H, s, CH ₃ -18 '), 1.99 (6H, s, CH ₃ -19, 19'), 2.00 (6H, s, CH ₃ -20, 20 '), 2.01 (1H, overlapped, H-2 eq), 2.07 (1H, dd, J = 12, 6 Hz, H-2 ax), 2.08 (t, J = 7.5 Hz, CH ₂ in fatty acid moiety), 2.16 (1H, dd, J = 12, 5.5 Hz, H-2 'eq), 2.43 (m, CH ₂ in fatty acid moiety), 3.69 (1H, d, J = 1.5 Hz, OH-3'), 4.33 (1H, ddd, J = 13.5 , 5.5, 1.5 Hz, H-3 '), 5.54 (1H, dd, J = 13.5, 5.5 Hz, H-3), 6.20 (1H, d, J = 16 Hz, H-7), 6.21 (2H, d, J = 16Hz, H-7 '), 6.30 (2H, d, J = 11.5 H z, H-10, 10 '), 6.31 (2H, m, H-14, 14'), 6.40 (2H, d, J = 16 Hz, H-8), 6.43 (2H, d, J = 16 Hz , H-8 '), 6.45 (2H, d, J = 15.5 Hz, H-12, 12'), 6.66 (2H, dd, J = 15.5, 11.5 Hz, H-11, 11 '), 6.67 (2H , m, H-15, 15 '). CD λ nm (Δε): 240 (-20.0), 252 (0), 270 (+20.0), 284 (0), 314 (-34.0), 355 (0) , 372 (+3.0).

(3 S ) -Adonirubin: UV-VIS: 470 nm (Et ₂ O). FAB-MS: m / z 580 [M] ⁺ . CD λ nm (Δε): 220 (+12.0), 230 (0), 234 (-12.0), 252 (0), 275 (+10.0), 283 (0), 310 (-17.0), 340 (0), 370 (+4.4).
(3 ' S ) -Asteroidenone: UV-VIS: 455 nm (Et ₂ O). FAB-MS: m / z 566 [M] ⁺ .
(3 S , 3 ' S ) -Astaxanthin: UV-VIS: 470 nm (Et ₂ O). FAB-MS: m / z 596 [M] ⁺ . CD λ nm (Δε): 240 (-20.0), 252 (0), 270 (+20.0), 284 (0), 314 (-34.0), 355 (0), 372 (+3.0).

Fritschiellaxanthin: UV-VIS: 452 nm (Et ₂ O). FAB-MS: m / z 582 [M] ⁺ .
4-Ketoantheraxanthin: UV-VIS: 452 nm (Et ₂ O). FAB-MS: m / z 598 [M] ⁺ .
cis -4-Ketoantheraxanthin: UV-VIS: 448 nm (Et ₂ O). FAB-MS: m / z 598 [M] ⁺ .
Lactucaxanthin: UV-VIS: 416, 439, 468 nm (Et ₂ O). FAB-MS: m / z 568 [M] ⁺ .
Lutein: UV-VIS: 422, 445, 474 nm (Et ₂ O). FAB-MS: m / z 568 [M] ⁺ .

Claims (25)

  (I) a step of dehydrating a carotenoid-producing plant or tissue thereof by contacting with a dehydrating solvent, (II) a step of extracting the dehydrated plant or tissue thereof with an extraction solvent to obtain a carotenoid-containing extract, and (III) Water and an organic solvent are added to the solvent for dehydration brought into contact with the plant or its tissue in step (I) and the carotenoid-containing extract obtained in step (II), and the mixture is separated to contain carotenoids. A method for extracting carotenoid from a plant or tissue thereof, comprising a step of recovering an organic solvent layer.   The extraction method according to claim 1, wherein the carotenoid contains a ketocarotenoid.   The extraction method according to claim 1 or 2, wherein the carotenoid contains an astaxanthin fatty acid ester.   The extraction method according to claim 3, wherein the astaxanthin fatty acid ester is a mixture of a diester and a monoester.   The extraction method according to any one of claims 1 to 4, wherein the dehydrating solvent is at least one organic solvent selected from the group consisting of acetone, ethanol, methanol, and 2-propanol. .   The extraction method according to any one of claims 1 to 5, wherein the extraction solvent is a polar organic solvent.   The organic solvent used in step (III) is at least one organic solvent selected from the group consisting of hexane, diethyl ether, petroleum ether, ethyl methyl ether, and dimethyl ether. The extraction method described in 1.   The extraction method according to any one of claims 1 to 7, wherein in step (I), the minced plant or the tissue thereof is contacted with a dehydrating solvent.   The carotenoid-producing plant or tissue thereof is a plastid genome-modified plant or tissue obtained by introducing a gene encoding a carotenoid 4,4′-keto group-introducing enzyme into a plant plastid genome. The extraction method according to any one of claims 1 to 8.   A gene encoding a carotenoid 4,4′-keto group-introducing enzyme is obtained from (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or (ii) a nucleotide sequence complementary to a DNA comprising the nucleotide sequence of SEQ ID NO: 1. And a DNA encoding a polypeptide having an activity of introducing a keto group into the 4th and / or 4 'positions of the β ring of a carotenoid having a β ring. The extraction method according to claim 9, wherein the extraction method is characterized.   Modification of plastid genome obtained by introducing a carotenoid-producing plant or tissue thereof into a plastid genome by introducing genes encoding carotenoid 3,3′-hydroxyl transferase and isopentenyl diphosphate isomerase The extraction method according to claim 9 or 10, which is a plant or a tissue thereof.   The gene encoding the carotenoid 3,3′-hydroxyl transferase is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 3 or (ii) a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 3. And a DNA encoding a polypeptide that hybridizes under stringent conditions and has an activity of introducing a hydroxyl group into the 3rd and / or 3'position carbon of a carotenoid having a β ring. Item 12. The extraction method according to Item 11.   A gene encoding isopentenyl diphosphate isomerase is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 5 or (ii) a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 5; The extraction method according to claim 11 or 12, which is a DNA encoding a polypeptide that hybridizes under a gentle condition and has an isomerase activity for converting isopentenyl diphosphate to dimethylallyl diphosphate. .   A plastid genome-modified plant is obtained by introducing DNA containing the nucleotide sequence of SEQ ID NO: 1, DNA containing the nucleotide sequence of SEQ ID NO: 3, and DNA containing the nucleotide sequence of SEQ ID NO: 5 into the plastid genome of the plant. It is a thing, The extraction method as described in any one of Claims 9-13 characterized by the above-mentioned.   The extraction method according to any one of claims 9 to 14, wherein the plant is lettuce, cauliflower, cabbage, rapeseed, potato, tomato, carrot, rice, soybean, or tobacco.   Use of a plastid genome-modified plant obtained by introducing a gene encoding a carotenoid 4,4'-keto group-introducing enzyme into a plant plastid genome to produce an astaxanthin fatty acid ester.   A gene encoding a carotenoid 4,4′-keto group-introducing enzyme comprises (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, or (ii) a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 DNA that encodes a polypeptide that hybridizes with DNA under stringent conditions and has an activity of introducing a keto group into the 4-position and / or 4'-position of the β-ring of a carotenoid having a β-ring The use according to claim 16.   The plastid genome-modified plant is a plastid genome-modified plant obtained by further introducing into the plastid genome a gene encoding each of the carotenoid 3,3'-hydroxyl transferase and isopentenyl diphosphate isomerase enzymes. Use according to claim 16 or 17, characterized in that   The gene encoding the carotenoid 3,3′-hydroxyl transferase is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 3 or (ii) a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 3. And a DNA encoding a polypeptide that hybridizes under stringent conditions and has an activity of introducing a hydroxyl group into the 3rd and / or 3'position carbon of a carotenoid having a β ring. Item 19. Use according to Item 18.   A gene encoding isopentenyl diphosphate isomerase is (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 5 or (ii) a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence of SEQ ID NO: 5; The use according to claim 18 or 19, which is a DNA encoding a polypeptide that hybridizes under a gentle condition and has an isomerase activity for converting isopentenyl diphosphate to dimethylallyl diphosphate.   What is obtained by introducing a plastid genome-modified plant into a plant plastid genome by introducing DNA comprising the nucleotide sequence of SEQ ID NO: 1, DNA comprising the nucleotide sequence of SEQ ID NO: 3, and DNA comprising the nucleotide sequence of SEQ ID NO: 5 Use according to any one of claims 16 to 20, characterized in that   The use according to any one of claims 16 to 21, wherein the astaxanthin fatty acid ester is a mixture of a diester and a monoester.   The use according to any one of claims 16 to 22, wherein the astaxanthin fatty acid ester is astaxanthin myristic acid ester or palmitic acid ester.   24. Use according to any of claims 16 to 23, characterized in that the plant is lettuce, cauliflower, cabbage, rapeseed, potato, tomato, carrot, rice, soybean or tobacco.   The use according to any one of claims 16 to 24, wherein the plastid genome-modified plant is a chloroplast genome-modified lettuce obtained by introducing the gene into a chloroplast genome.