JP2001503264A - Nucleic acid sequence encoding β-C-4-oxygenase required for astaxanthin biosynthesis obtained from Haematococcus pluvialis - Google Patents

Abstract

  (57) [Summary] The present invention relates generally to a biotechnological method for producing (3S, 3'S) astaxanthin. Specifically, the present invention relates to a peptide having β-C-4-oxygenase activity; a DNA segment encoding the peptide; an RNA segment encoding the peptide; a vector and a recombinant DNA molecule containing the DNA segment; A host cell or organism containing the recombinant DNA molecule or DNA segment; and a biotechnological method for producing (3S, 3'S) astaxanthin or a food additive containing (3S, 3'S) astaxanthin by using the host.

Description

DETAILED DESCRIPTION OF THE INVENTION Nucleic acid sequence encoding β-C-4-oxygenase required for astaxanthin biosynthesis from Haematococcus pluvialis Field and background of the invention The present invention relates generally to a biotechnological method for producing (3S, 3'S) astaxanthin. Specifically, the present invention relates to a peptide having β-C-4-oxygenase activity; a DNA segment encoding the peptide; an RNA segment encoding the peptide; a vector and a recombinant DNA molecule containing the DNA segment; A host cell or organism containing a recombinant DNA molecule or DNA segment; and a biotechnological method for producing (3S, 3'S) astaxanthin or a food additive containing (3S, 3'S) astaxanthin by using the host. Carotenoids, such as astaxanthin, are natural pigments that underlie many of the yellow, orange, and red colors found in living organisms. Carotenoids are widely distributed in nature and have two major biological functions in various biological systems: carotenoids act as light-harvesting pigments in photosynthesis and protect against damage from photo-oxidation. Such and further biological functions of carotenoids, their industrially important role, and their biosynthesis are described below. As part of a concentrating antenna, carotenoids can absorb photons and transfer their energy to chlorophyll, thus helping to capture light in the 450-570 nm range [Cogdell RJ and Frank HA (187 ) How carotenoids function in photosynthetic bacteria Biochim Biophys Acta 895: 63-79; Cogdell R (1988) Function of pigments in chloroplasts Goodwin TW, Ed., Plant Pigments, 183-255, Academic Press, London Frank HA, Violette CA, Trautman JK, Slueve AP, Owens TG and Albrecht AC (1991) Carotenoids in photosynthesis: Structure and photochemistry Pure Appl Chem 63: 109-114; Frank HA, Farhoosh R, D ecoster B and Christensen RL ( 1992) Molecular features controlling the efficiency of energy transfer from carotenoids to chlorophyll in photosynthesis Murata N, Ed., Research in Photosynthesis, Vol. I, pp. 125-128, Kluwer, Dordrecht; and Cogdell RJ and Gardiner AT (1992). 3) Function of carotenoids in photosynthesis. See Meth Enzymol 214: 185-193]. Carotenoids are an essential component of the protein-dye complex of the light-harvesting antenna in photosynthetic organisms, but are also important components of the photosynthetic reaction center. Most of the total carotenoids are localized within the light-harvesting complex II [Bassi R, Pinaw B, Dainese P and Marquartt J (1993) Photosystem II carotenoid binding protein Eur J Biochem 212: 297- 302]. The identity of the carotenoprotein with photosynthetic activity and its precise position in the light-harvesting system is unknown. Carotenoids in the chlorophyll-protein complex of the photochemically active thermophilic cyanobacterium Synechococcus sp. Were examined by linear dichroism spectroscopy of oriented samples [Breton J and Katos (1987) in photosystem II. Dye Orientation: Low Temperature Linear Dichroism Study of Core Particles Isolated from Synechococcus sp. And Its Chlorophyll-Protein Subunits See Biochim Biophys Acta 892: 99-107]. These complexes mainly contained a β-carotene pool that absorbs about 505 nm and 470 nm, where the β-carotene pool is oriented near the membrane plane. In the photochemically inactive chlorophyll-protein complex, β-carotene absorbs about 495 and 465 nm, and the molecule is oriented perpendicular to the plane of the membrane. Evidence has revealed that carotenoids bind to photosystem (PS) II in cyanobacteria [Suzuki R and Fujita Y (1977) Photobleaching of carotenoids induced by the action of photosynthetic reaction center II: DCMU-sensitive Plant Cell Physiol 18: 625-631; and isolation and characterization of Newman PJ and Sherman LA (1978) photosystem I and II membrane particles from the cyanobacterium Synechococcus cedrorum. Biochim Biophys Acta 503: 343-361] . Two molecules of β-carotene are present in the reaction center core of PSII [Ohno T, Satoh K and Katoh S (1986) Chemical composition of the oxygen releasing complex purified from the thermophilic cyanobacterium Synechocococus sp. Biochim Biophys Acta 852 : 1-8; Gounaris K, Chapman DJ and Barber J (1989) Isolation and characterization of the D1 / D2 / cytochrome b-559 complex from Synechocystis PCC 6803 Biochim Biophys Acta 973: 296-301; and Ne well RW, van Amerongen H, Barber J and van Grondelle R (1993) Spectroscopic characterization of photosystem II reaction centers using polarized light: Evidence for β-carotene excitons in PSII reaction centers. See Biochim Biophys Acta 1057: 232-238. thing]. However, their exact functions remain unknown. [Review: Satoh K (1992) Structure and Function of Photosystem II Reaction Center Murata N, Ed., Research in Photosynthesis, Volume II, pp. 3-12, Kluwer, Dordrechht ]. Such β-carotene molecules with two attached molecules have demonstrated protection from photodamage in the isolated PSII reaction center of chlorophyll P680 [De Las Rivas J, Tel fer A and Barber J (1993) 2 The attached β-carotene molecule protects P680 from photodamage in the isolated PSII reaction center, see Biochim Biophys Acta 1142: 155-164]. This is thought to be related to the protection of PSII against degradation of the D1 subunit [Sandmann G (1993) Genes and enzymes involved in phytoene desaturation to lycopene (Summary), 10th International Symposium on See Ca rotenoids, Trondheim CLI-2]. The light-harvesting pigment of the highly purified oxygen-releasing PSII complex of the thermophilic cyanobacterium Synechococcus sp. Consists of 50 chlorophyll a and 7 β-carotene molecules and does not contain xanthophyll molecules [Ohno T, Satoh K. And Katoh S (1986) Chemical composition of the oxygen releasing b-complex purified from the thermophilic cyanobacterium Synechococcus sp. Biochim Biophys Acta 852: 1-8]. β-carotene has been shown to play a role in the assembly of green algal active PSII [Humbeck K, Romer S and Senger H (1989) Evidence for an essential role of carotenoids in the assembly of active PSII. Planta 179: 242 -250]. The PSI complex isolated from Phormidium luridum containing 40 chlorophylls per P700 contained on average 1.3 molecules of β-carotene [Thornber JP, Alberte RS, Hunter FA, Shiozawa JA and Kan KS (1976) Organizing Chlorophyll in Photosynthetic Units of Plants See Brookhaven SympBiology 28: 132-148]. When preparing PSI particles from Synechococcus sp. Strain PCC 6301 containing 130 ± 5 antenna chlorophylls per P700, 16 carotenoids were detected [Lundell DJ, Glazer AN, Melis A and Malkin R]. (1985) Cyanobacterial photosystem I complex features J Biol Chem 260: 646-654]. Significant amounts of β-carotene and xanthophylls, cryptoxanthin and isocryptoxanthin were detected in the dye-protein complex of the PSI of the thermophilic cyanobacterium Synechococcus elongatus (Coufal J, Hladik J and Sofrova D (1989) cyanobacteria Carotenoid Content of the Dye-Protein Complex of Synechococcus elongatus Photosystem I See Photosynthetica 23: 603-616]. The structure of the subunit protein-complex of the cyanobacterium Synechococcus sp. Consisting of four polypeptides (62, 60, 14 and 10 kDa) contained approximately 10 β-carotene molecules per P700 [Takahashi Y. Multiple forms of the P700-chlorophyll a-protein complex of Hirota K and Katoh S (1985) Synechococcus sp .: iron, quinone and carotenoid content. See Photosynth Res 6: 183-192]. This carotenoid is exclusively bound to large polypeptides with functional antenna chlorophyll a. The fluorescence excitation spectra of these complexes suggested that β-carotene acts as an efficient antenna for PSI. As mentioned above, a further essential function of carotenoids is to protect the photosynthetic apparatus from photo-oxidation reactions caused by chlorophyll in the excited triplet state. Carotenoid molecules having π-electron conjugation of nine or more carbon-carbon double bonds can absorb triplet energy from chlorophyll, thus preventing the formation of harmful singlet oxygen radicals. it can. In Synechococcus sp., Triplet-state carotenoids are monitored at the closed PSII center, and its kinetics of about 25 nanoseconds production is due to energy transfer from chlorophyll triplets in the antenna [Schlodder E and Brettel K ( 1988) Initial charge separation of closed photosystem II with 11 nanosecond lifetime: see flash absorption spectroscopy on the oxygen-evolving photosystem II complex of Synechococcus. Biochim Biophys Acta 933: 22-34]. This process, which has a lower yield compared to the yield of radical pair formation, is believed to play a role in protecting chlorophyll from damage due to overexcitation. The protective role of carotenoids in vivo has been elucidated by the use of bleaching herbicides such as norflurazon, which inhibits carotenoid biosynthesis in all organisms that undergo oxygenic photosynthesis [review: Sandmann G and Boger P (1989)]. Inhibition of carotenoid biosynthesis by herbicides Boger P and Sandmann G, eds., Target Sites of Herbicide Action, pages 25-44, CRC Press, Boca Raton, Florida]. Norflurazon treatment in the light reduces both carotenoid and chlorophyll levels, while chlorophyll levels are unaffected in the dark. Inhibition of photosynthetic efficiency in Oscillatoria agardhii cells treated with a pyridinone herbicide (fluridone) is due to the relative abundance of mixoxanthophyll, zeaxanthin and β-carotene, which results in the photo-oxidation of chlorophyll molecules. [See Canto de Loure I, Dubacq JP and Thomas JC (1987) Nitrogen deficiency effects on cyanobacterial pigments and lipids Plant Physiol 83: 838-843]. Zeaxanthin has been shown to be required to dissipate the excess excitation energy of antenna chlorophyll in a non-radiative manner [Demmig-Adams B (1990) Carotenoids and photoprotection in plants: the role of xanthophyll zeaxanthin Biochim Biophys Acta 1020: 1-24; and Demmig-Adams B and Adams WW III (1990) High energy states that quench the fluorescence of carotenoids zeaxanthin and chlorophyll] Photosynth Res 25: 187-197]. In algae and plants, photoinduced deepoxidation of violaxanthin, which produces zeaxanthin, is involved in the photoprotection process [Review: Demmig-Adams B and Adams WW III (1992). Protection and other responses Ann Rev Plant Physiol Plant Mol Biol 43: 599-626]. The photoinduced deepoxidation of violaxanthin and its reverse reaction in the dark are known as the "xanthophyll cycle" [Demmig-Adams B and Adams WWIII (1992) Photoprotection of plants against high light stress And other responses Ann Rev Plant Physiol Plant Mol Biol 43: 599-626]. Cyanobacterial lichens, with cyanobacteria as the main symbiotic algae, are susceptible to high-intensity light, possibly because they do not contain any zeaxanthin and, possibly, are unable to dissipate non-radiative energy; Lichens (algal lichens), which are symbiotic algae, contain zeaxanthin and are more resistant to high light stress (Demmig-Adams B, Adams WW III, Green TGA, Czygan FC and Lange OL (1990) algae Differences in sensitivity to light stress of two lichens forming a symbiotic group (lichen partner with xanthophyll cycle and lichen partner without xanthophyll cycle) Oecologia 84: 451-456; Demmig-Adams B And Adams WWII I (1993) Sunto-adapted leaf xanthophyll cycle, protein turnover and high light resistance Plant Physiol 103: 1413-1420; and Demmig-Adams B (1 990) Carotenoids and photoprotection in plants: the role of xanthophyllzeaxanthin. See Biochim Biophys Acta 1020: 1-24]. In contrast to algae and plants, cyanobacteria do not have a xanthophyll cycle. However, cyanobacteria do contain sufficient amounts of zeaxanthin and other xanthophylls to support photoprotection of chlorophyll. Several other functions are found in carotenoids. The results showing the accumulation of β-carotene in UV-resistant mutants of the cyanobacterium Gloeocapsa alpicola suggest that carotenoids protect against damaging species generated by near ultraviolet (UV) radiation [Buckley CE and Houghton JA (1976) Study on the effect of near-ultraviolet radiation on pigmentation of the cyanobacterium Gloeocapsa alpicola. See Arch Microbiol 107: 93-97]. This was more clearly demonstrated in Escherichia coli cells producing carotenoids (Tuveson RW and Sandmann G (1993) Cloned carotenoids expressed in Escherichia coli against phototoxic molecules activated by near ultraviolet light) See Genetic Protection Meth Enzymol 214: 323-330]. Carotenoids are efficient antioxidants because they can scavenge oxygen radical species, thus protecting cells from oxidative damage. Such functions of carotenoids are important in almost all organisms [Krinsky NI (1989) Antioxidant function of carotenoids Free Radical Biol Med 7: 617-635; and Palozza P and Krinsky NI (1992) in vivo and in vitro Antioxidant Action of Carotenoids-Review Meth Enzymol 213: 403-420]. Other cellular functions can be affected by carotenoids, albeit indirectly. Although carotenoids in cyanobacteria are not major phototactic photoreceptors, the effects of carotenoids on the phototactic response observed in Anab aena variabilis are due to singlet oxygen radicals, which can act as signal intermediates in this system. [Nultsch W and Schuchart H (1985) Phototactic reaction chain model Arch Microbiol 142: 180-184 of the cyanobacterium Anabaenavariabilis]. In flowers and fruits, carotenoids facilitate pollinator attraction and seed dispersion. This latter aspect is strongly linked to agriculture. The type and extent of pigmentation in fruits and flowers is one of the most important attributes of many crops. This is primarily because the color of these products often determines their appeal to consumers, thereby increasing their market value. Because carotenoids are non-toxic, they have important commercial uses as colorants in the food industry [see Bauernfeind JC (1981) Carotenoids as colorants and vitamin A precursors, Academic Press, London]. The red color of the tomato fruit is provided by lycopene which accumulates in chromoplasts during the fruit ripening period. Tomato extracts containing a high percentage of lycopene (greater than 80% of dry weight) are commercially produced worldwide for industrial use as a food colorant. Furthermore, meat, feathers or fish and bird eggs exhibit the color of a given food carotenoid. Thus, carotenoids are frequently used in poultry food additives and aquaculture. Certain cyanobacterial species (eg, Spirulina sp. [See Recent Advances in the Treatment of Microalgae Sommer TR, Potts WT and Morrissy NM (1990) Cultured Hydrobiologia 204/205: 435-443]) And cultured in aquaculture to produce human food supplements. Thus, the content of carotenoids (mainly β-carotene) in these cyanobacteria is closely related to biotechnology in a commercial sense. Most carotenoids contain 8 C _Five C constructed from isoprenoid units ₄₀ And contains a series of conjugated double bonds. Carotene is either a linear or cyclic molecule containing no oxygen atoms and containing one or two terminal rings. Xanthophylls are oxygenated derivatives of carotenes. Various glucosylated carotenoids and carotenoid esters have been identified. C ₄₀ The skeleton further elongates to C ₄₅ Or C ₅₀ It can produce carotenoids or can be shortened to produce apocarotenoids. Some non-photosynthetic bacteria also have C ₃₀ Synthesize carotenoids. General background on carotenoids can be found in Goodwin TW (1980) The Biochemistry of the Carotenoids, Volume 1 (2nd Edition), Chapman and Hall, New York; and Goodwin TW and Britton G (1988) Carotenoid distribution and analysis. Goodwin TW, Plant Pigments, pp. 62-132, Academic Press, New York. To date, over 640 different natural carotenoids have been characterized. Thus, carotenoids are responsible for most of the various shades of yellow, orange and red found in microorganisms, fungi, algae, plants and animals. Carotenoids are synthesized by all photosynthetic organisms and some non-photosynthetic bacteria and fungi, but are also widely distributed throughout the animal kingdom through food intake. Carotenoids are synthesized de novo from isoprenoid precursors only in photosynthetic organisms and some microorganisms, and they typically accumulate in protein complexes in the photosynthetic membrane, in the cell membrane and in the cell wall. As detailed in FIG. 1, in the β-carotene biosynthetic pathway, four enzymes convert the central isoprenoid pathway geranylgeranyl pyrophosphate to β-carotene. Carotenoids are produced from the general isoprenoid biosynthetic pathway. Although this pathway has been known for decades, very recently, mainly using genetics and molecular biology, several molecular mechanisms involved in carotenoid biosynthesis have been elucidated. This is due to the fact that most enzymes involved in the conversion of phytoene to carotene and xanthophylls are unstable and the activity of membrane-bound proteins is lost upon solubilization [Beyer P, Weiss G and Kleinig H (1985) Solubilization of the membrane-bound carotene synthase from daffodil chromoplasts and its reconstitution Eur J Biochem 153: 341-346; and Bramley PM (1985) In vitro biosynthesis of carotenoids Adv Lipid Res21 : 243-279]. However, solubilization of a carotene synthase from Synechocystis sp. PCC 6714 strain that retains partial activity was reported [Bramley PM and Sandmann G (1987) Solubilization of carotene synthase of Aphanocapsa Phytochem 26: 1935-1939 checking]. There is no true in vitro system of carotenoid biosynthesis that allows a direct assay of enzymatic activity. A cell-free carotenoid producing system has been developed [see Clarke IE, Sandmann G, Bramley PM and Boger P (1982) carotene biosynthesis with isolated photosynthetic membranes FEBS Lett 140: 203-206] and adapted to cyanobacteria [Cartotenoid biosynthesis by Aphanocapsa homogenate linked to Sandmann G and Bramley PM (1985) Phycomyces blakesleeanus phytoene production system Planta 164: 259-263; and in vitro and in vivo xanthophylls by Bramley PM and Sandmann G (1985) cyanobacterium Aphanocapsa Biosynthesis at Phytochem 24: 2919-2922]. Reconstitution of phytoene desaturase of Synechococcus sp. Strain PCC 7942 in liposomes was performed after purification of the polypeptide expressed in Escherichia coli [Fraser PD, Linden H and Sandmann G (1993) of recombinant Synechococcus phytoendesaturase. Purification from Escherichiacoli overexpressing strain and its reactivation See Biochem J 291: 687-692]. Referring to FIG. 1, carotenoids are synthesized from isoprenoid precursors. The central pathway of isoprenoid biosynthesis can be seen to be initiated by the conversion of acetyl-CoA to mevalonic acid. C _Five Molecular D ^Three Isopentenyl pyrophosphate (IPP) is formed from mevalonic acid, which is the basic component of all long-chain isoprenoids. After IPP isomerizes to dimethylallyl pyrophosphate (DMAPP), three molecules of IPP are further bonded to form C ₂₀ The molecule keranylgeranyl pyrophosphate (GGPP) is produced. Such 1'-4 condensation reactions are catalyzed by prenyltransferases [Kleinig H (1989) Role of plastids in isoprenoid biosynthesis See Ann Rev Plant Physiol Plant Mol Biol 40: 39-59]. It has been shown in plants that all reactions from DMAPP to GGPP are carried out by this same enzyme (GGPP synthase) [Dogbo O and Camara B (1987) Isopentenyl pyrophosphate isomerase and Capsicum of geranylgeranyl pyrophosphate synthase. Purification by affinity chromatography from chromoplasts Biochim Biophys Acta 920: 140-148; and Laferriere A and Beyer P (1991) Purification of geranylkeranyl diphosphate synthase from Sinapis alba Ethioplast Biochim Biophys Acta 216: 156-163 See]. The first step specific to carotenoid biosynthesis is the production of prephytoene pyrophosphate (PPPP) by head-to-head condensation of two molecules of GGPP. GGPP is able to remove 15-cis-phytoene (colorless C ₄₀ (Hydrocarbon molecules). This two-step reaction is catalyzed by the soluble enzyme phytoene synthase, an enzyme encoded by a single gene (crtB), in both cyanobacteria and plants [Chamovitz D, Misawa N, Sandmann G and Hirschberg J ( 1992) Molecular cloning of a gene encoding cyanobacterial phytoene synthase (carotenoid biosynthetic enzyme) and expression in Escherichia coli FEBS Lett 296: 305-310; Ray JA, Bird CR, Maunders M, Grierson D and Schuch W (1987) Sequence of pTOM5 (cDNA associated with maturation obtained from tomato) Nucl Acids Res 15: 10587-10588; phytoene synthase complex of Camara B (1993) plant-three enzymes of components, immunology and biosynthesis Meth Enzymol 214: 352-365]. All subsequent steps in the pathway occur within the membrane. Phytoene is converted to lycopene via phytofluene, zeta-carotene and neurosporene by four desaturation (dehydrogenation) reactions. Each time it is desaturated, the number of conjugated double bonds increases by two, resulting in an increase in the number of conjugated double bonds from three in phytoene to eleven in lycopene. Little is known about the molecular mechanism of enzymatic dehydrogenation of phytoene compared to others [Jones BL and Porter JW (1986) Carotene biosynthesis in higher plants CRC Crit Rev Plant Sci 3: 295-324; Beyer P, Mayer M and Kleinig H (1989) Molecular oxygen and geometric isomerism of intermediates are essential for carotene desaturation and cyclization in daffodil chromoplasts Eur J Biochem 184: 141-150 checking]. In cyanobacteria, algae and plants, the first two desaturation reactions (15-cis-phytoene to ζ-carotene) are catalyzed by one membrane-bound enzyme (phytoene desaturase) [ Jones BL and Porter JW (1986) Carotene biosynthesis in higher plants CRC Crit Rev Plant Sci 3: 295-324; and Beyer P, Mayer M and Kleinig H (1989) See Eur J Biochem 184: 141-150, which is essential for carotene desaturation and cyclization in daffodil chromoplasts]. Since the ζ-carotene product is mainly in the all-trans configuration, cis-trans isomerization is considered at this desaturation step. The primary structure of the cyanobacterial phytoene desaturase polypeptide is conserved relative to the primary structure of the algal and plant phytoene desaturase polypeptides (greater than 65% identical residues are found) [Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) One polypeptide that catalyzes the conversion of phytoene to ζ-carotene is regulated at the transcriptional stage during tomato ripening Proc Natl Acad Sci USA 89: 4962. -4966; Pecker I, Chamovitz D, Mann V, Sandmann G, Boger P and Hirschberg J (1993) Molecular characterization of carotenoid biosynthesis in plants: Tomato phytoene desaturase gene Murata N, Ed., Research in Phytosynthesis, Volume III , Pages 11-18, Kluwer, Dordrecht]. In addition, the same inhibitors block the phytoene desaturases of the two systems [Sandmann G and Boger P (1989) Inhibition of carotenoid biosynthesis by herbicides, edited by Boger P and Sandmann G, Target Sites of Herbicide Action, 25- 44 p., CRC Press, Boca Raton, Florida]. Thus, enzymes that catalyze the desaturation of phytoene and phytofluene in cyanobacteria and plants probably have similar biochemical and molecular properties and differ from those of phytoene desaturases of other microorganisms. One such difference is that Rhodobacter capsulatus, Erwinia sp. Or fungal phytoene desaturase converts phytoene to neurosporene, lycopene or 3,4-dehydrolycopene, respectively. Desaturation of phytoene in daffodil chromoplasts [Beyer P, Mayer M and Kleinig H (1989) Geometrical isomeric states of molecular oxygen and intermediates indicate carotene desaturation and cyclization in daffodil chromoplasts See Eur J Biochem 184: 141-150, which is essential for the reaction], for the cell-free system of Synechococcus sp. Strain PCC 7942 [Sandmann G and Kowalczyk S (1989) in vitro carotene production and in Anacystis. Characteristics of the Phytoene Desaturase Reaction See Biochem Biophys Res Com 163: 916-921], although oxygen is not directly involved in this reaction, but is a molecular entity that is considered as the ultimate electron acceptor. Relies on oxygen. In cyanobacteria, the mechanism of dehydrogenase-electron transferase was favored over that of dehydrogenase-monooxygenase (Sandmann G and Kowalczyk S (198 9) Characterization of carotene production in vitro and phytoene desaturase reaction in Anacystis. Biochem Biophys Res Com 163: 916-921]. A conserved FAD binding motif is present in all phytoene desaturases whose primary structure has been analyzed [Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) Conversion of phytoene to カ ロ -carotene. One polypeptide that catalyzes is regulated at the transcriptional stage during tomato fruit ripening Proc Natl Ac Sci USA 89: 4962-4966; Pecker I, Chamovitz D, Mann V, Sandmann G, Boger P and Hirschberg J (1993) Molecular characterization of carotenoid biosynthesis in plants: see Tomato phytoene desaturase gene Murata N, Research in Phytosynthesis, Vol. III, pp. 11-18, Kluwer, Dordrecht]. The pepper phytoene desaturase enzyme has been shown to contain protein-bound FAD [Huguney P, Romer S, Kuntz M and Camara B (1992) catalyzes the synthesis of phytofluene and ζ-carotene in Capsicum chromoplasts Characterization and Molecular Cloning of Flavo Proteins See Eur J Biochem 209: 399-407]. Since phytoene desaturase is located in the membrane, soluble redox components are further expected. This virtual component is NAD (P) ⁺ ([Mayer MP, Nievelstein V and Beyer P (1992) Purification and characterization of NADPH-dependent oxidoreductase from chromoplasts of Narcissus pseudonarcissus-Plant Physiol Biochem 30: 389-398, a redox mediator thought to be involved in carotene desaturation. ), Or another electron and hydrogen carrier (such as quinone). Synechocystis sp. The cell position of phytoene desaturase in PCC 6714 and Anabaenavariabilis ATCC 29413 was determined to be primarily within (85%) photosynthetic thylakoid membranes using specific antibodies [Serrano A, Gimenez P, Scinnidt A and San. dmann G (1990) Immunocytochemical localization and function determination of photoautotrophic prokaryotic phytoene desaturylase J Gen Microbiol 136: 2465-2469]. In cyanobacteria, algae and plants, ζ-carotene is converted to lycopene via neurosporene. Little is known about its enzymatic mechanism, but it is predicted to be performed by one enzyme [Linden H, Vioque A and Sandmann G (1993)], a carotenoid biosynthesis gene encoding the caroten desaturase Anaba. ena PCC 7120, isolated by heterologous complementation See FEMS Microbiol Lett 106: 99-104]. Anabaena sp. The deduced amino acid sequence of PCC 7120 strain ζ-carotene desaturase contains a motif similar to the dinucleotide binding motif found in phytoene desaturase. Lycopene is converted to β-carotene by two cyclization reactions. Synechococcus sp.PCC strain 7942 (Cunningham FX Jr, Chamovitz D, Misawa N, Gantt E and Hirschberg J (1993) Cloning of cyanobacterial lycopene cyclase (enzyme catalyzing β-carotene biosynthesis) gene and its expression in Escherichia coli See functionally expressed F EBS Lett 328: 130-138] and plants [Camara B and Dogbo O (1986) Demonstration of lycopene cyclase and solubilized Plant Physiol 80: 172-184 from Capsicum chromoplast membranes. ], There is evidence that these two cyclization reactions are catalyzed by one enzyme (lycopene cyclase). This membrane-bound enzyme is inhibited by the triethylamine compounds CPTA and MPTA [Sandmann G and Boger P (1989) Inhibition of carotenoid biosynthesis by herbicides Boger P and Sandmann G eds., Target Sites of Herbicide Action, 25-44 , CRC Press, Boca Raton, Florida]. Cyanobacteria perform only a β-cyclization reaction and therefore do not contain ε-carotene, δ-carotene and α-carotene and their oxygenated derivatives. The β-ring forms a `` carbonium '' when the terminal C-1,2 double bond of a linear lycopene molecule is folded at the C-5,6 double bond and hydrogen is subsequently lost from C-6. Formed by producing an "ion" intermediate. Acyclic carotene in which the bond between 7, 8 is not a double bond has been reported. Thus, complete desaturation, such as lycopene, or at least half the molecule, such as neurosporene, is essential for the reaction. Lycopene cyclization involves a dehydrogenation reaction that does not require oxygen. The cofactors required for this reaction are unknown. The dinucleotide binding domain may be Synechococcus sp. Which suggests NAD (P) or FAD as a coenzyme for lycopene cyclase. It was found in the lycopene cyclase polypeptide of PCC 7942 strain. The addition of various oxygen-containing side groups, such as hydroxy, methoxy, oxo, epoxy, aldehyde or carboxylic acid moieties, results in the formation of various xanthophyll species. Little is known about the formation of xanthophylls. The hydroxylation of β-carotene requires molecular oxygen in the mixed-function oxidase reaction. A cluster of genes encoding enzymes of all pathways is identified by the purple photosynthetic bacterium Rhodobacter capsulatus [Armstrong GA, Alberti M, Leach F and Hearst JE (1989) protein product nucleotide sequence, composition, And properties Mol. Gen Gent 216: 254-268] and non-photosynthetic bacteria Erwinia herbicola [Sandmann G, Woods WS and Tuveson RS (1990) Identification of carotenoids in Erwinia herbicola and Escherichia coli transformants FEMS Micobiol Lett 71: 77- 82; Hundle BS, Beyer P, Kleining H, Englert H and Hearst JE (1991) Carotenoids of the Escherichia coli HB101 strain having the Erwinia herbicola and Erwinia herbicola carotenoid gene cluster Photochem Photobiol 54: 89-93; and Schnurr G, Shmidt A and Sandmann G (1991) Mapping of a carotenogenic gene cluster from Erwinia herbicola and functional identification of six genes. See FEMS Microbiol Lett 78: 157-162] and Erwinia uredovora [Misawa N, Nakagawa M , Kobayashi K, Yamano S, IzawaI, Nakamura K and Harashima K (1990) Erwinia uredovora mosquito by functional analysis of gene products in Escherichia coli Elucidation J Bacteriol of Tenoido biosynthetic pathway 172: 6704-6712 see] have been cloned from. Two genes, GGPP synthase al-3 [Nelson MA, Morelli G, Caratoli A, Romano N and Macino G (1989) Nerospora crassa carotenoid biosynthesis gene (albino -3) Molecular cloning Mol Cell Biol 9: 1271-1276; and Carattoli A, Romano N, Ballario P, Morelli G and Macino G (1991) Neurospora crassa carotenoid biosynthesis gene (alblno3) J Biol Chem 266: 5854-5859 See Endocaturase al-1 [Schmidhauser TJ, Lauter FR, Russo VEA and Yanofsky C (1990) cloning, sequencing and light regulation of light-regulated Molcel l (Neurospora cra ssa carotenoid biosynthesis gene). Biol 10: 5064-5070] has been cloned from Neurospora crassa. However, attempts to clone these genes from cyanobacteria or plants using these genes as heterologous molecular probes were unsuccessful. This is because sufficient sequence similarity was not found. The first "plant-type" gene for carotenoid synthase has been cloned from cyanobacteria using a molecular genetic approach. In the first step for cloning the gene for phytoene desaturase, a number of mutants resistant to norflurazon, a phytoene desaturase-specific inhibitor, were isolated in Synechococcus sp. Strain PCC 7942 [Linden H, Sandmann G, Chamovitz D, Hirschberg J and Boger P (1990) Biochemical characteristics of Synechococcus mutants selected for the bleaching herbicide norflurazon. See Pestic Biochem Physiol 36: 46-51]. The gene conferring norflurazon resistance was then cloned by transforming the wild strain to herbicide resistance [Chamovitz D, Pecker I and Hirschberg J (1991) Molecular basis for herbicide norflurazon resistance Plant Mol Biol 16: 967-9 74; Chamovitz D, Pecker I, Sandmann G, Boger P and Hirschberg J (1990) Cloning the norflurazon resistance gene in cyanobacteria Z Naturforsch 45c: 482-486]. Several supporting strains have shown that the cloned gene (formerly called pds and now crtP) encodes phytoene desaturase. The most crucial was the functional expression of phytoene desaturase activity in transformed Escherichia coli cells (Linden H, Misawa N, Cha movitz D, Pecker I, Hirschberg J and Sandmann G (1991) different phytoene desaturase genes. Of functional complementation and accumulated carotene in Escherichia coli of Z Naturforsch 46c: 1045-1051; and Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) Conversion of phytoene to ζ-carotene. One polypeptide that catalyzes is regulated at the transcriptional stage during tomato ripening, see Proc Natl Acad Sci USA 89: 4962-4966]. The crtP gene was also cloned from Synechocystis sp. strain PCC 6803 using a similar method [Martinez-Ferez IM and Vioque A (1992) Nucleotide sequence of the phytoene desaturase gene from Synechocystis sp. PCC 6803 and resistance to the herbicide norflurazon Features of the novel mutant Plant Mol Biol 18: 981-983]. Subsequently, algae [Pecker I, Chamovitz D, Mann V, Sandmann G, Boger P and Hirschberg J (1993) Molecular characteristics of carotenoid biosynthesis in plants: Tomato phytoene desaturase gene Murata N (ed.) Research in Photosynthesis, III, pp. 11-18, Kluwer, Dordrecht] and higher plants [Bartley GE, Viitanen PV, Pecker I, Chamovitz D, Hirschberg J and Scolinik PA (1991) Photosynthetic bacteria of soybean cDNA encoding phytoene desaturase. Cloning and expression in E. coli, Enzyme of the carotenoid biosynthetic pathway Proc Natl Acad Sci USA 88: 6532-6536; and Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) Phytoene to ζ-carotene One polypeptide that catalyzes the conversion is a heterologous gene from Proc Natl Acad Sci USA 89: 4962-4966, which is regulated at the transcriptional stage during tomato ripening] The cyanobacterial crtP gene was used as a molecular probe for cloning. Syne chococcus sp. PCC 7942 strain and Synechococcus sp. The phytoene desaturase of PCC 6803 strain consists of 474 and 467 amino acid residues, respectively, and its sequence is highly conserved (74% identity and 84% similarity). The calculated molecular weight is 51 kDa, but is slightly hydrophobic (hydropathic index -0.2), which does not include a hydrophobic region long enough to penetrate lipid bilayers. The primary structure of the cyanobacterial phytoene desaturase has a high degree of conservation (61% identity and 81% similarity) to the enzyme from the green alga Dunalliela barda wil; [Pecker I, Chamovitz D, Mann V, Sandmann G, Boger P and Hirschberg J (1993) Molecular characterization of carotenoid biosynthesis in plants: see the tomato phytoene desaturase gene Murata N (ed.) Research in Photosynthesis, Vol. III, 11-18 Sada, Kluwer, Dordrecht]). There is one polypeptide that catalyzes the conversion of tomato [Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) phytoene to ζ-carotene, at the transcriptional stage during tomato ripening. Regulated Proc Natl Acad Sci USA 89: 4 962-4966], pepper [Hugueney P, Romer S, Kuntz M and Camara B (1992) Cap sicum Chromoplast synthesis of phytofluene and ζ-carotene. Catalyze Lab protein characteristics and molecular cloning See Eur J Biochem 209: 399-407] and soybean [encoding phytoene desaturase, Bartley GE, Viitanen PV, Pecker I, Chamovit z D, Hirschberg J and Scolinik PA (1991) Molecular cloning and expression of soybean cDNA in photosynthetic bacteria, see Carotenoid Biosynthetic Pathway Enzyme Proc Natl Acad Sci USA 88: 6532-6536] (62-65% identical and about 79% similar; Chamovitz D (1993) Run Molecular analysis of the early stages of carotenoid biosynthesis in algae: phytoene synthase and phytoene desaturase Ph.D. Thesis, see The Hebrew University of Jerusalem]) and have a high degree of conservation. Eukaryotic phytoene desaturases are larger (64 kDa); however, they are processed during transport to plastids and mature to a size comparable to that of cyanobacterial enzymes. There is a high degree of structural similarity in the carotenoids of Rhodobacter caps ulatus, Erwinia sp. and Neurospora crassa, including phytoene desaturase in the crtI gene product [Review: Armstrong GA, Hundle BS and Hearst JE (1993) Photosynthesis and non-photosynthesis] Meth Enzymol 214: 297-311] Evolutionary conservation and structural similarity of carotenoid biosynthetic gene products from organisms. As noted above, a high degree of conservation of the primary structure of phytoene desaturase also exists among oxygen-producing photosynthetic organisms. However, except for the amino-terminal FAD binding sequence, there is little sequence similarity between the "plant-type" crtP gene product and the "bacterial-type" phytoene desaturase (crtI gene product; 19-23% identity and 42 ~ 47% similarity). crtP and crtl are not derived from the same ancestral gene and are postulated to have arisen independently via convergent evolution (Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) One polypeptide that catalyzes the conversion to ζ-carotene is regulated at the transcriptional stage during tomato ripening, see Proc Natl Acad Sci USA 89: 4962-4966]. This hypothesis is supported by the different dehydrogenation sequences catalyzed by the two types of enzymes and their different sensitivities to inhibitors. Although not as evident as in the case of phytoene desaturases, similar differences are observed in the structure of phytoene synthase between one cyanobacterium and a plant and another microorganism. The crtB gene (formerly psy) encoding phytoene synthase was identified in a chemom adjacent to crtP and in the same operon of Synechococcus sp. Strain PCC 7942 [Bartley GE, Viitanen PV, Pecker I, Chamovitz D, Molecular cloning and expression in photosynthetic bacteria of soybean cDNA encoding Hirschberg J and Scolinik PA (1991) phytoene desaturase, see Proc Natl Acad Sci USA 88: 6532-6536, an enzyme of the carotenoid biosynthetic pathway]. This gene encodes a 307 amino acid 36 kDa polypeptide with a hydrophobicity index of -0.4. The deduced amino acid sequence of the cyanobacterial phytoene synthase is highly conserved with tomato phytoene synthase (57% identical and 70% similar; the sequence of Ray JA, Bird CR, Maunders M, Grierson D and Schuch W (1987) pTOP Tomato maturation-associated cDNA Nucl Acids Res 15: 10587-10588]) is not as highly conserved with the crtB sequences of other bacteria (29-32% identical to 10 gaps in alignment and 48 ~ 50% similar). Both types of enzymes contain two conserved sequence motifs that are also found in prenyltransferases of various organisms [Bartley GE, Viitanen PV, Pecker I, Chamovitz D, Hirschberg J and Scolinik PA (1991) phytoene desaturase Cloning and expression of soybean cDNA encoding photosynthesis in photosynthetic bacteria, carotenoid biosynthetic pathway enzyme Proc Natl Acad Sci USA 88: 6532-6536; Carattoli A, Romano N, Ballario P, Morelli G and Macino G (1991) Neurospora crassa carotenoid biosynthesis gene (albino 3) J Biol Chem 266: 5854-5859; Armstrong GA, Hundle BS and Hearst JE (1993) Evolutionary conservation and structural similarity of carotenoid biosynthesis gene products from photosynthetic and non-photosynthetic organisms Meth Enzymol 214: 297-311; and Chamovitz D (1993) Molecular analysis of the early stages of carotenoid biosynthesis in cyanobacteria: phytoene synthase And phytoene desaturase Ph.D.Thesis, see The Hebrew University of Jerusalem]. These regions of the polypeptide are thought to be involved in the binding and / or elimination of pyrophosphate during the condensation of two GGPP molecules. Erwinia sp. By screening an expression library of cyanobacterial genomic DNA in cells of Echerichia coli having the crtB and crtE genes and the cyanobacterium crtP, the crtQ gene encoding ζ-carotene desaturase from Anabaena sp. zds) [Linden H, Vioque A and Sandmann G (1993) Isolation of a carotenoid biosynthesis gene encoding 遺 伝 子 -carotene desaturase from Anabaena PCC 7120 by heterologous complementation FEMS Microbiol Lett 106: 99-104 See]. Since these Echerichia coli cells produce ζ-carotene, colonies with a red-brown pigment that produces lycopene against the yellowish background of cells that produce ζ-carotene can be identified. The putative ζ-carotene desaturase from Anabaena strain PCC 7120 is a 56 kDa polypeptide consisting of 499 amino acid residues. Surprisingly, its primary structure is not conserved with phytoene desaturases of the "plant type" (crtP gene product), but has considerable sequence similarity to bacterial type enzymes (crtI gene product). [See Sandmann G (1993) Genes and enzymes involved in the desaturation of phytoene to lycopene (summary), 10th International Symposium on Carotenoids, Trondheim CL1-2]. The cyanobacterial crtQ gene and the crtI gene of other microorganisms may have evolved from a common ancestor in evolution. Using essentially the same cloning strategy as for crtP, the lycopene cyclase gene (formerly lcy) was cloned from Synechococcus sp. Strain PCC 7942. The gene was isolated by transformation of wild-type herbicide resistance by using the lycopene cyclase inhibitor 2- (4-methylphenoxy) -triethylamine hydrochloride (MPTA) [Cunningham FX Jr, Chamovitz D, Misawa N, Gantt E and Hirschberg J (1993) Cloning of cyanobacterial lycopene cyclase (enzyme catalyzing β-carotene biosynthesis) gene and its functional expression in Escherichia coli FEBS Lett 328: 130-138]. Lycopene cyclase is the product of one gene product, which catalyzes the double cyclization of lycopene to β-carotene. Synechococcus sp. Strain PCC 7942 is a 46 kDa polypeptide of 411 amino acid residues. It has no sequence similarity to the crtY gene product (Lycopene cyclase) from Erwinia uredovora or Erwinia herbicola. The genes for β-carotene hydroxylase (crtZ) and zeaxanthin glycosylase (crtX) were expressed in Erwinia herbicola [Hundle B, Alberti M, Nievelstein V, Beyer P, Kleinig H, Armstrong GA, Burke DH and Hearst JE (1994) Escherichia coli. Functional issues of expressed Erwinia herbicola Eho10 carotenoid gene Mol Gen Genet 254: 406-416; Hundle BS, Obrien DA, Alberti M, Beyer P and Hearst JE (1992) Functional expression of zeaxanthin glucosyltransferase from Erwinia herbicola and See the proposed phosphate binding site Proc Natl Acad Sci USA 89: 9321-9325] and Erwinia uredovora [Misawa N, Nakagawa M, Kobayashi K, Yamano S, Izawa I, Nakamura K and Harashima K (1990 ) Elucidation of Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products in Escherichia coli. See J Bacteriol 172: 6704-6712] I have. As the oxidized form of β-carotene, the ketocarotenoid astaxanthin (3,3′-dihydroxy-β, β-carotene-4,4′-dione) in aquatic crustaceans was first described. Later, astaxanthin was found very frequently in many marine animals and algae. However, very few animals can synthesize astaxanthin de novo from other carotenoids, and most of them are obtained from food. In the plant kingdom, astaxanthin mainly occurs in several species of cyanobacteria, algae and lichens. However, it is rarely found in petals of higher plants [see Goodwin TW (1980) The Biochemistry of the Carotenoids, Volume 1 (2nd ed.), Chapman and Hall, London and New York]. The function of astaxanthin as a potent animal antioxidant has been demonstrated [see Mik i W (1991) Biological function and activity of animal carotenoids Pure Appl Chem 63: 141]. Astaxanthin is a potent inhibitor of lipid peroxidation and plays an active role in protecting biological membranes from oxidative damage [In vivo and in vitro antioxidant effects of Palozza P and Krinsky NI (1992) carotenoids- And review of Method s Enzymol 213: 403-420; and Kurashige M, Okimasu E, Inove M and Utsumi K (1990) Inhibition of oxidative damage of biological membranes by astaxanthin Physiol Chem Phys Med NMR 22:27]. The chemopreventive effects of astaxanthin have also been investigated, and it has been shown that astaxanthin significantly reduces the incidence of induced bladder cancer in mice [Tanaka T, Morishita Y, Suzui M, Kojima T, Okumura A And Mori H (1994) Chemoprevention of mouse bladder carcinogenesis by the natural carotenoid astaxanthin Carcinogenesis 15:15]. Astaxanthin has also been demonstrated to exert immunomodulatory effects by enhancing antibody production [Jyonouchi H, Zhang L and Tomita Y (1993) Study of immunomodulatory effects of carotenoids II. Nutr Cancer 19: 269 that enhances the production of antibodies to T-dependent antigens in vitro without promoting carcinogenesis; and Jyonouchi H, Hill JR, Yoshifumi T and Good RA (1991) Studies on the immunomodulatory effects of carotenoids I mice Effect of β-carotene and astaxanthin on lymphocyte function and cell surface marker expression in vitro culture system. See Nutr Cancer 19:93]. Although the full biomedical properties of astaxanthin have not yet been elucidated, early results suggest that astaxanthin may play a key role in cancer and tumor prevention and elicit a positive response from the immune system . Astaxanthin is the major carotenoid pigment in salmon and shrimp and imparts attractive coloring to eggs, meat and skin [Torrisen OJ, Hardy RW, Shearer KD (1989) Coloring of salmon-carotenoid deposits and metabolism in salmon Crit Rev Aquatic Sci 1: 209]. Global salmon yields in 1991 were approximately 720,000 MT, of which 25-30% were produced in various aquaculture facilities [Meyers SP (1994) Global aquaculture, feed formulation and the development of the role of carotenoids Pure Appl Chem 66: 1069]. Yield is expected to rise to 460,000 MT by 2000 [See Biorn dahl T (1990) Economics of salmon farming, Blackwell Scientific, Oxford, page 1]. The red coloration of the salmon meat contributes to the appeal to consumers and thus affects the price of the final product. Animals are unable to synthesize carotenoids and acquire pigment via the food chain from primary producers (marine algae and phytoplankton). Animals grown in intensive cultures usually do not have optimal color. Therefore, carotenoid-containing nutrients are artificially added in aquaculture, which adds considerable cost to the producer. Astaxanthin is the most expensive carotenoid compound used commercially (market price as of 2,500-3,500 $ / kg as of 1995). Astaxanthin is mainly used as a nutritional supplement and colors a wide range of aquatic animals. In the Far East, it is also used in poultry feed to typically color chickens. Astaxanthin is a desirable and effective colorant in the food industry and is also valuable in cosmetics. Recently, astaxanthin was reported to be an effective antioxidant in humans, and therefore a desirable edible additive. The availability of natural (3S, 3'S) astaxanthin is limited. It has been commercially extracted from several crustacean species [Torrisen OJ, Hardy RW, Sheerer KD (1989) Coloring of salmon-carotenoid deposits and metabolism in salmon Crit Rev Aquatic Sci 1: 209. See]. The (3R, 3'R) stereoisomer of astaxanthin is produced by Phaffia [Yeast Species, Andrews AG, Phaff HJ and Starr MP (1976) Carotenoids of Phaffia, fermenting yeast Phytochemistry with red pigment, Vol. 5, See pages 1003-1007]. Currently, a synthetic astaxanthin comprising a 1: 2: 1 mixture of (3S, 3'S)-, (3S, 3'R)-and (3R, 3'R) -isomers is being manufactured by Hoffman-La Roche. And sold at a high price (approximately $ 2,500 / Kg) under the name "CAROPHYLLPink" [Mayer H (1994) Opinion on Carotenoid Synthesis Pure & Appl Chem 6, Vol. 931-938]. Recently, a novel gene for keto compound biosynthesis, named crtW, was isolated from marine bacteria Agrobacterium auranticacum and Alcaligenes PC-1 that produce ketocarotenoids such as astaxanthin. When the crtW gene was induced into Eschrichia coli engineered to accumulate β-carotenoids by the Erwinia carotenoid-generating gene, Eschrichia coli transformants synthesized canthaxanthin (a precursor in the astaxanthin synthesis pathway) [Misawa N, Kajiwar a S, Kondo K, Yokoyama A, Satomi Y, Saito T, Miki W and Ohtani T (1995) Canthaxanthan biosynthesis by conversion of methylene to a keto group in β-carotene by one gene Biochemical and biophysical research communications 210, 867-876]. Therefore, it is desirable to find a relatively inexpensive source of (3S, 3'S) astaxanthin used as a feed supplement in aquaculture and as an expensive chemical for various other industrial applications. Although astaxanthin is synthesized in various bacteria, fungi and algae, an important constraint on the use of ecosystems for its production is the yield of these systems compared to chemical synthesis. And the extraction method is expensive in these systems. One way to solve these problems is to increase the production efficiency of astaxanthin production in ecosystems using recombinant DNA technology. This allows the production of astaxanthin in genetically engineered hosts, which, in higher plants, grow easily and are easy to extract. Furthermore, the production of astaxanthin in genetically engineered hosts makes it possible, for example, to use astaxanthin produced in aquaculture applications where there is no need for extraction by selecting the appropriate host. Accordingly, a nucleic acid segment encoding β-C-4-oxygenase (enzyme that converts β-carotene to canthaxanthin), and a recombinant vector molecule containing the nucleic acid of the present invention, and these vector molecules or (3S, 3 ′S) The need for a host cell or transgenic organism transformed or transfected with a DNA segment for the biotechnological production of astaxanthin is widely recognized and it is of great advantage to have one. Other features and advantages of the invention will be apparent from the following description and from the claims. Summary of the Invention A general object of the present invention is to provide a biotechnological method for the production of (3S, 3'S) astaxanthin. It is a particular object of the present invention to provide β-C-4-oxygenase activity and a DNA segment encoding this peptide to enable biotechnological production of astaxanthin and xanthophyll. It is a further object of the present invention to provide an RNA segment encoding a polypeptide comprising an amino acid sequence corresponding to said peptide. It is a further object of the present invention to provide a vector and a recombinant DNA molecule comprising said DNA segment. It is a further object of the present invention to provide a host cell containing said recombinant DNA molecule. Another object of the present invention is to provide a host transgenic organism containing said recombinant DNA molecule or said DNA segment in a cell. Another object of the present invention is to provide a host transgenic organism that expresses β-C-4-oxygenase activity in chloroplast and / or chromoplast-containing tissues. Another object of the present invention provides a food additive for animal or human consumption, including said host cells or transgenic organisms. Another object of the present invention provides a method for producing astaxanthin using said host cell or transgenic organism. A further object of the present invention is a method for producing canthaxanthin, echinenone, cryptoxanthin, isocryptoxanthin, hydroxyechinenone, zeaxanthin, adonirubin and / or adnixanthin using said host cells or transgenic organisms. I will provide a. Further objects and advantages of the present invention will become clear from the description hereinafter. In one embodiment, the present invention relates to a DNA segment encoding a polypeptide comprising an amino acid sequence corresponding to the Haematococcus pluvialis crtO gene. In a further embodiment, the present invention relates to an RNA segment encoding a polypeptide comprising an amino acid sequence corresponding to the Haematococcus Pluvialis crtO gene. In another embodiment, the present invention relates to a polypeptide comprising an amino acid sequence corresponding to the Haematococcus pluvialis crtO gene. In a further embodiment, the present invention relates to a recombinant DNA molecule encoding a polypeptide corresponding to the Haematococcus pluvialis crtO gene. In another embodiment, the present invention relates to a host cell containing said recombinant DNA molecule or DNA segment. In a further embodiment, the invention relates to a host transgenic organism containing said recombinant DNA molecule or said DNA segment in a cell. In another embodiment, the present invention relates to a method for producing astaxanthin using said host cell or said transgenic organism. In another embodiment, the present invention relates to a method for producing other xane and fill. In another embodiment, the invention relates to a method for obtaining high expression of a transgene in a plant, especially a chromoplast-containing cell. In one further embodiment, the present invention relates to a method for transporting a carotenoid biosynthetic enzyme encoded by a transgene to chromoplasts. BRIEF DESCRIPTION OF THE FIGURES In the present specification, the present invention will be described only by way of example with reference to the accompanying drawings. Here: FIG. 1 is a general biomedical pathway of β-carotene biosynthesis, describing all molecules in the pathway (where IPP is isopentenyl pyrophosphate, DMAPP is dimethylallyl pyrophosphate, GPP Is geranyl pyrophosphate, FPP is farnesyl pyrophosphate, GGPP is geranylgeranyl pyrophosphate, PPPP is prephytoene pyrophosphate); FIG. 2 shows the nucleotide sequence of the crtO cDNA of the present invention using GCG software (CRTOA.SEQ). And a cDNA clone cloned by KaJiwara et al. (CTROJ.SEQ) [Kajiwara S, Kakizono T, Saito T, Kondo K, Ohtani T, Nishio N, Nagai S and Misawa N (1995) Haem Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from atococcus pluvialis and astaxanthin synthesis in Escherichia coli See Plant Molec Biol 29: 343-352] (where (: ) Indicates the same, (-) indicates a gap, and nucleotide numbers are according to SEQ ID NO: 4 for CRTOA.AMI and Kajiwara et al. For CRT OJ.AMI); FIG. 3 shows the present invention using GCG software. FIG. 3 is an identity map between the amino acid sequence encoded by the crtO cDNA (CRTOA.AMI) and the amino acid sequence encoded by the cDNA cloned by Kajiwara et al. (CRTOJ.AMI) [Kajiwara S, Kakizono T, Saito T, Kondo K, Ohtani T, Nishio N, Nagai S and Misawa N (1995) Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis and astaxanthin synthesis in Escherichia coli Plant Molec Biol 29: 343-352 (Where (:) indicates the same, (-) indicates a gap, and the amino acid number is as shown in SEQ ID NO: 4 for CRTOA.AMI and Kajiwara et al. For CRTOJ.AMI. FIG. 4 is a schematic diagram of a pACYC184-derived plasmid designated pBCAR, which contains the genes crtE, crtB, crtI and crtY of Erwinia herbicola, which are genes for β-carotene in Escherichia coli cells. FIG. 5 is a schematic diagram of a plasmid derived from pACYC184, designated pZEAX, which contains the crtE, crtB, crtI, crtY and genes of Erwini aherbicola and these genes are zeaxanthin in Escherichia coli cells. FIG. 6 shows that pBluescriptSK named pHPK ^- FIG. 1 is a schematic diagram of a plasmid derived from Haematococcus pluvialis, containing a full-length cDNA insert encoding a β-carotene C-4-oxygenase enzyme designated crtO from SEQ ID NO: 1. The cDNA was identified by the complementary color of Escherichia coli cells; FIG. 7 is a schematic representation of the pACYC184-derived plasmid designated pCANTHA. The plasmid contained the cDNA encoding β-C-4-oxygenase from Haematococcus pluvia lis which was isolated from the plasmid pHPK of FIG. 6 and inserted into the PstI site of the coding sequence of the crtZ gene of the plasmid pZEAX of FIG. This recombinant plasmid had the crtE, crtB, crtI, crtY genes of Erwinia herbicola and the crtO gene of Haematococcus plu vialis, all of which were derived by inserting a 1.2 kb PstI-PstI DNA fragment Required for the production of canthaxanthin in Escherichia coli cells; FIG. 8 is a schematic diagram of the pACYC184-derived plasmid designated pASTA. The plasmid contained the cDNA of β-C-4-oxygenase from Haematococcus pluvialis inserted into the PstI site 600 bp downstream of the crt E gene of plasmid pZEAX, isolated from plasmid pHPK in FIG. This recombinant plasmid had the crtE, crtB, crtI, crtY, crtZ genes of Erwinia herbicola and the crtO gene of Haematococcus pluvialis, which were derived by inserting a 1.2 kb PstI-PstI DNA fragment of All are required for the production of astaxanthin in Escherichia coli cells; FIG. 9 is a schematic representation of the plasmid derived from pBR328, designated PAN3.5-KETO. The plasmid was isolated from the plasmid pHPK of FIG. 6 and designated as pPAN35D5 [Hirschberg J, Ohad N, Pecker I and Rahat A (1987) of a herbicide resistant mutant of the cyanobacterium Synechococcus R2.Z. Isolation and characteristics described in Naturforsch 42c: 102-112]. 1.2 kb PstI- containing the cDNA of β-C-4-oxygenase from Haematococcus cus pluvialis inserted into the PstI site present in the β-lactamase gene of It was induced by inserting a PstI DNA fragment. This has the psbAI gene from the cyanobacterium Synechococcus PCC7942 in the plasmid vector pBR328 (Hirschberg J, Ohad N, Pecker I and Rahat A (1987) Isolation of a herbicide-resistant mutant of the cyanobacterium Synechococcus R2.Z. And the feature Naturforsch 42c: 102-112]; this recombinant plasmid has the crtO gene of Haematococcus pluvialis, which is required for the production of astaxanthin in Synechococcus PCC7942 cells; FIG. 10 is named pBIB Is a schematic diagram of a T-DNA region of a Ti binary plasmid (Escherichia coli NAgro bacterium) described in Becker D (1990) Binary vector Nucleic Acids Research 18: 230 which enables exchange of a plant selectable marker with a reporter gene. ]. This vector is a derivative of the Ti plasmid pBI101 [Jeff esrson AR, Kavanagh TA and Bevan WM (1987) GUS fusion: β-glucuronidase as a sensitive and versatile fusion gene marker in higher plants The EMCO J.6: 3901-3907 Described in]. (Where B _R And B _L Are the right and left boundaries of the T-DNA region, respectively, pAg7 is the polyadenylation site of gene 7 of the Agrobacterium Ti plasmid, and pAnos is the 250 bp DNA fragment containing the polyadenylation site of the nopaline synthase gene of Agrobacterium. NPTII is a 1,800 bp long DNA fragment encoding kanamycin resistance, and pnos is a 300 bp long DNA fragment containing the promoter sequence of the nopaline synthase gene of Agrobacterium (where pAnos is the DNA fragment of Agrobacterium). FIG. 11 is a schematic diagram of a T-DNA region of a Ti binary plasmid (Escherichia coli, Agrobacterium) named pPTBIB, which contains a polyadenylation site of the palin synthase gene). This is the genomic DNA sequence of the tomato species Lycope rsicon esculentum designated PT (nucleotides 1-1448 of the Pds gene, Mann V, Pecker I and Hirschberg J (1994) Phytoendesaturase (Pds) from tomato (Lycopersicon esculentum). Cloning and characterization of the gene for Pant Molecular Biology 24: 429-434). It contains in the HindIII-SmaI site of the binary plasmid vector pBIB of FIG. 10 the promoter region of the Pds gene and the coding region of the amino-terminal region of the polypeptide PDS which acts as a penetrating peptide for transport to chloroplasts and chromoplasts. (Where B _R And B _L , PAg7, pAnos, NPT II, pnos and pAnos are defined as above); FIG. 12 is a schematic representation of the T-DNA region of a Ti binary plasmid (E. coli, Agrobacterium) designated pPTCRTOBIB. . This was prepared by cloning a 1,110 nucleotide long Eco47III-NcoI fragment of the cDNA of crtO from H. pluvialis into the SmaI site of plasmid pPTBIB in FIG. 11, so that the coding nucleotide sequence at the amino terminus of PDS was exist in the same reading frame of crtO (where B _R And B _L , PAg7, pAnos, NPT II, pnos and pAnos are defined as above, PT is the promoter, and the tomato and CRTO penetrating peptide coding sequence is H. FIG. 13 is the nucleotide sequence of crtO from pluvialis (nucleotides 211 to 1321 of SEQ ID NO: 1); FIG. 13 shows Southern DNA blot analysis of HindIII-digested genomic DNA extracted from wild-type (WT) and crtO tomato transgenic plants. Is shown. Transgenic plant origins are named 2, 3, 4, 6, 9 and 10 and, according to the present invention, are essentially the same as described in Sambrook et al., Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. The crtO cDNA was used as a radioactive probe as described in 1989 (where the size of the marker (M) DNA fragment is indicated on the left in kilobase pairs (kb) and the expected position of the internal T-DNA HindIII fragment ( Arrows) deduced from the sequence of pPTPDSBIB shown in FIG. 12 containing the crtO cDNA sequence); FIG. 14 shows the biosynthetic pathway of astaxanthin; FIG. 15 shows flowers from wild-type tobacco plants and the present invention. 1 shows flowers from transgenic tobacco plants. Description of the preferred embodiment The present invention generally relates to a method for producing (3S, 3'S) astaxanthin. Specifically, the present invention relates to a peptide having β-C-4-oxygenase activity; a DNA segment encoding the peptide; an RNA segment encoding the peptide; a vector and a recombinant DNA molecule containing the DNA segment; A host cell or organism containing the recombinant DNA molecule or DNA segment; and a biotechnological method for producing (3S, 3'S) astaxanthin or a food additive containing (3S, 3'S) astaxanthin by using the host. The unicellular freshwater green alga Haematococcus pluvialis can produce large amounts of () when exposed to unfavorable growth conditions or after different environmental stresses such as phosphate or nitrogen starvation, high concentrations of salts in the growth medium, or high intensity of light. Accumulates 3S, 3'S) astaxanthin [Yong YYR and Lee YK (1991) Phycologia 30 257-261; Droop MR (1954) Arch Microbiol 20: 391-397; and Andrews AG, Borch G, Liaaen-Jensen S and Snatzke G (1974) Acta Chem Scand B28: 730-736]. During this process, the algal vegetative cells form cysts and change their color from green to red. The present invention provides cloning of a cDNA (designated crtO) from Haematococcus pluvialis encoding β-C-4-oxygenase (enzyme that converts β-carotene to canthaxanthin), and expresses β-carotene hydroxylase Disclosed is expression in a heterologous system (eg, the Erwinia herbicola crtZ gene product), which results in the production of (3S, 3'S) astaxanthin. The crtO cDNA and its encoded peptide with β-C-4-oxygenase activity are novel nucleic acid and amino acid sequences, respectively. The cloning method of crtO cDNA used a strain of Escherichia coli, which was subjected to genetic manipulation to produce β-carotene, and a cDNA library of Haematococcu s pluvialis was transfected into this strain and expressed. Visual screening of Escherichia coli cells with reddish brown pigment has identified canthaxanthin-producing transformants. Thus, the cloned cDNA has been expressed in two heterologous systems (Escherichia coli and Synechococcus PCC7942 cells), both capable of producing β-carotene, and further engineered (Erwinia herbicola crtZ gene product) or endogenous. It has been shown that both of these systems can produce (3S, 3'S) astaxanthin, which may include sex β-carotene hydroxylase activity. The crtO cDNA or its protein product is a nucleic acid or amino acid that is significant relative to the nucleic acid or amino acid sequence of crtW, which produces ketocarotenoids such as astaxanthin, and protein products isolated from the marine bacteria Agrobacterium auranticacum and Alcaligenes PC-1. Does not show sequence similarity [Misawa N, Kajiwara S, Kondo K, Yokoyama A, Satomi Y, Saito T, Miki W and Ohtani T (1995) Canta by conversion of methylene to a keto group in β-carotene by one gene. See Xanthan Biosynthesis Biochemical and biophysical research communications, volume 209, pp. 867-876]. However, the crtO cDNA and its protein product are substantially identical to the nucleic acid and amino acid sequence of a recently cloned cDNA encoding a 320 amino acid protein product with β-carotene oxygenase activity isolated from Haematococcus pluvialis. Showing nucleic acid and amino acid sequence identity to (Kajiwara S, Kakizono T, Saito T, Kondo K, Ohtani T, Nishio N, Nagai S and Misawa N (1995) Isolation of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis and Functional Identification and Astaxanthin Synthesis in Escherichia coli See Plant Molec Biol 29: 343-352]. Nevertheless, as shown in FIG. 2, the crtO cDNA (CRTOA.SEQ in FIG. 2) and the cDNA described by Kajiwara et al. (CRTOJ.SEQ in FIG. 2) [see references above]. The degree of sequence identity between the two was 75.5%, and as shown in FIG. 3, the crtO cDNA protein product (CRTOA.AMI in FIG. 3) and the protein described by Ka jiwara et al. (CRTOJ.AMI in FIG. 3). The degree of sequence identity between is 78%. This was determined using GCG software. Therefore, as described in detail below, the crtO cDNA in either a system that is easy to grow and can be used directly as an additive in fish feed or a system that allows for a simple and inexpensive astaxanthin extraction procedure is possible. Can be used to biotechnically produce (3S, 3'S) astaxanthin. In one embodiment, the present invention provides a polypeptide comprising the Haematococcus pluvialis crtO gene and amino acid sequences corresponding to allelic and species variants and functionally naturally occurring and / or artificially induced variants thereof. Related to the DNA segment to be encoded As used herein and in the claims that follow, the expression “allelic and species variants and functionally naturally occurring and / or artificially induced variants thereof” refers to DNA (or to any of the following). RNA) or means known in the art for obtaining it. However, the terms “variant” and “variant” indicate the presence of sequence differences (ie, mutations). In the invention herein and in the following claims, the sequence variation is 77-80%, preferably 80-85%, more preferably 85-90%, most preferably 90-100%. Are the same nucleotides. In a preferred embodiment, the DNA segment comprises the sequence set forth in SEQ ID NO: 1. In another embodiment, the DNA segment encodes the amino acid set forth in SEQ ID NO: 4. The present invention also relates to SEQ ID NO: 1 or SEQ ID NO: 2 under highly stringent conditions [eg, as described in Sambrook et al., Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1989]. Comprising a sequence that hybridizes to a nucleic acid probe comprising at least 15, preferably at least 50, more preferably at least 100, even more preferably at least 200, even more preferably at least 500 contiguous nucleotides as described. And a pure DNA segment characterized by: Alternatively, a DNA segment of the present invention may be characterized in that it can hybridize under low stringent conditions to a nucleic acid probe comprising the coding sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (nucleotides 166 to 1152). . Examples of such low stringency conditions are described in Sambrook et al., And use lower hybridization temperatures, such as, for example, 20 ° C., lower than those used for the above high stringency conditions. The DNA segments of the present invention may also be characterized as being capable of hybridizing under high stringency conditions to a nucleic acid probe comprising the coding sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (nucleotides 166 to 1152). The present invention also provides synthetically generated oligonucleotides (eg, oligodeoxyribonucleotides or oligoribonucleotides and analogs thereof) that can hybridize to at least 10 nucleotide sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Including. In another embodiment, the present invention provides a polypeptide comprising the Haematococcus pluvialis crtO gene and amino acid sequences corresponding to allelic and species variants and functionally naturally occurring and / or artificially derived variants thereof. For the RNA segment encoding In a preferred embodiment, the RNA segment comprises the sequence set forth in SEQ ID NO: 2. In another preferred embodiment, the RNA segment encodes the amino acid sequence set forth in SEQ ID NO: 4. The present invention also provides under high stringency conditions at least 15, preferably at least 50, more preferably at least 100, even more preferably at least 200, even more, SEQ ID NO: 1 or SEQ ID NO: 2 It comprises a pure RNA segment, characterized in that it comprises a sequence that hybridizes to a nucleic acid probe preferably comprising at least 500 contiguous nucleotides. Alternatively, the RNA segment of the present invention is characterized in that it can hybridize under low stringent conditions to a nucleic acid probe containing the coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (nucleotides 166 to 1152). obtain. Further, the RNA segment of the present invention is also characterized in that it can hybridize under high stringent conditions to a nucleic acid probe containing the coding sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (nucleotides 166 to 1152). obtain. In another embodiment, the present invention provides polypeptides comprising the Haematococcus pluvialis crtO gene and amino acid sequences corresponding to allelic, species variants and functionally naturally occurring and / or artificially derived variants thereof. About. In a preferred embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4. The present invention includes any peptide that is homologous to the polypeptide (ie, 80-85%, preferably 85-90%, more preferably 90-100% of the same amino acids). As used herein and in the claims that follow, the term "homologous" refers to sequence identity between two peptides. When a monomeric subunit of the same amino acid is occupied at both positions in two compared sequences, they are homologous at that position. Homology between two sequences is a function of the number of homologous positions shared by the two sequences. For example, if eight out of ten positions in two sequences are occupied by the same amino acid, the two sequences are 80% homologous. Other polypeptides included in the invention include allelic variants, homologs of other species, naturally occurring variants, derived variants and under high or low stringency conditions (see above) It is a peptide encoded by DNA that hybridizes to the coding region (nucleotides 166 to 1152) shown in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the present invention relates to a recombinant DNA molecule comprising a vector (eg, a plasmid or viral vector) and a DNA segment encoding a polypeptide described above. In a preferred embodiment, the DNA segment is in a vector operably linked to a promoter. In a further embodiment, the present invention relates to a host cell containing said recombinant DNA molecule or DNA segment. Suitable host cells include both prokaryotic cells (such as bacteria, including Escherichia coli) and lower eukaryotic cells (eg, yeast) and higher eukaryotic cells (eg, algae, plant or animal cells). Introduction of the recombinant molecule into cells can be performed using methods known in the art such as, but not limited to, transfection, transformation, microinjection, and gene bombardment. Cells made to contain the recombinant DNA molecule can proliferate to form colonies, or can be made to differentiate to form differentiated organisms. Recombinant DNA molecules may be transiently contained within cells (e.g., by processes known in the art, such as transient transfection); It is preferably stably contained in the cell (by a process known in the art such as described above). In a preferred embodiment, the cells are producing β-carotene endogenously, or have been engineered to produce β-carotene by genetic engineering means, wherein the cells are endogenous or engineered β-carotene. Contains carotene hydroxylase activity. Such cells may be used as food additives for animal (eg, salmon) and human consumption. Further, such cells may be used to extract astaxanthin and / or other xanthophylls, as described below. In a further embodiment, the invention relates to a host transgenic organism (eg, a higher plant or animal) containing the recombinant DNA molecule or the DNA segment in a cell. Introduction of a recombinant molecule or DNA segment into a host transgenic animal can be performed using methods known in the art. In a preferred embodiment, the host organism is producing β-carotene endogenously, or has been engineered to produce β-carotene by genetic engineering means, preferably the host organism is endogenous or genetically engineered. Contains engineered β-carotene hydroxylase activity. Such organisms may be used as food additives for animal (eg, salmon) and human consumption. Further, such organisms may be used to extract astaxanthin and / or other xanthophylls, as described below. In another embodiment, the present invention relates to a method for producing astaxanthin using said host cell or transgenic organism. In another embodiment, the present invention relates to the use of the above-described host cell or transgenic organism in the use of canthaxanthin, echinenone, cryptoxanthin, isocryptoxanthin, hydroxyechinenone, zeaxanthin, adonirubin, 3-hydroxyechinenone, 3 The present invention relates to a method for producing xanthophylls such as' -hydroxyechinenone and / or adonixanthin. For these purposes, provided are cells or transgenic organisms as described above. Host cells or organisms are made to grow under conditions favorable to produce astaxanthin and the additional xanthophylls described above, which are then extracted by methods known in the art. In another embodiment, the invention encodes a polypeptide comprising an amino acid sequence corresponding to the Haematococcus pluvialis crtO gene, allelic and species variants and functionally naturally occurring or artificially derived variants thereof. Transgenic plants that express a transgene. Preferably, expression is highest in chromoplast-containing tissues. In another embodiment, the invention relates to a first DNA segment encoding a polypeptide for directing a protein to a plant chloroplast or chromoplast (eg, derived from the tomato Pds gene) and Haematococcus pluvialis. A set comprising a second DNA segment in the reading frame encoding a polypeptide comprising an amino acid sequence corresponding to the crtO gene, allelic and species variants and functionally naturally occurring and artificially derived variants thereof Pertaining to a recombinant DNA vector. In another embodiment, the invention relates to a first DNA segment comprising a promoter that is highly expressible in plant chloroplasts or chromoplast-containing tissues (eg, derived from the Pds gene of tomato) and Haematococcus plu vialis crtO. The present invention relates to a recombinant DNA vector comprising a second DNA segment encoding a polypeptide comprising the amino acid sequence corresponding to the gene, allelic and species variants and functionally naturally occurring and artificially derived variants thereof. The present invention will be described with reference to the following examples together with the above description. Examples Details of the following protocols and experiments are set forth in the following examples. Algae and growth conditions. Haematococcus pluvialis (Culture Collection of Algae and Protozoa, Windermere, UK) was a gift from Liverpool John Moores University by Dr. Andrew Young. Algae suspension cultures are grown in liquid medium as described by Nichols and Bold [see Nichols HW, Bold HC (1964) Trichsarcina polymorpha gen et sp nov J Phycol 1: 34-39]. Was. For the introduction of astaxanthin biosynthetic cells, cells were harvested, washed with water and resuspended in nitrogen-depleted medium. Cultures were exposed to continuous light (75 μE / m ^Two / s photon flux) in a 250 ml Erlenmeyer flask on a rotary stirrer at 25 ° C and 80 rpm. Construction of cDNA library. The construction of a Haematococcus pluvialis cDNA library was described in more detail in Lotan and Hirschberg (1995) FEBS letters 364: 125-128. Briefly, total RNA was extracted from algal cells grown under nitrogen depletion conditions (cell color was reddish brown) for 5 days. Cells were harvested from 50 ml cultures and their RNA content was extracted using Ti reagent (Molecular Research Center, INC). Poly-An RNA was isolated by two cycles of fractionation on oligo dT-cellulose (Boehinger). Final yield was 1.5% of total RNA. Uni-ZAP ^TM A cDNA library was constructed on the XR vector using a ZAP-cDNA synthesis kit (both from Stratagene). For amplification of the cDNA library, Escherichia coli cells of the XL1-Blue MRF ′ strain (Stratagene) were used. Plasmids and Escherichia coli strains. Plasmid pPL376, containing the gene required for carotenoid biosynthesis in the bacterium Erwinia hrbicola, was obtained from Tubesson (for details on plasmid pPL376, see Tubesson RW, Larson RA and Kagan J (1988) near-UV light and phototoxic molecules. Role of cloned carotenoid genes expressed in Escherichia coli in protection against inactivation, see J Bacteriol 170: 4675-4680]. Cells of the Escherichia coli JM109 strain harboring plasmid pPL376 accumulate a bright yellow carotenoid, zeaxanthin glycoside. In the first step, a 1.1 kb SalI-SalI fragment was deleted from this plasmid to inactivate the crtX gene encoding zeaxanthin glucosyltransferase. In the second step, the 0.8 kb fragment was deleted by BamHI partial digestion of the plasmid DNA followed by self-ligation to inactivate crtZ encoding β-carotene hydroxylase. A 7.4 kb fragment was prepared by BglII partial digestion and cloned into the BamHI site of the vector pACYC184. As shown in FIG. 4, the resulting recombinant plasmid had the crtE, crtB, crtI and crtY genes and was named pBCAR [Lotan and Hirschberg (1995) FEBS letters 364: 125-128]. Plasmid pBCAR was transfected into Escherichia coli SOLR strain cells (Stratagene). Colonies that appeared on Luria Broth (LB) medium containing chloramphenicol [described in Sambrook et al., Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1989] have this plasmid, A dark yellow-orange color due to the accumulation of carotene. As can be seen in FIG. 5, a further plasmid was constructed, named pZEAX, which allows for the synthesis and accumulation of zeaxanthin in Escherichia coli [for this plasmid, Lotan and Hirschberg (1995) FEBS letters 3 64: 125- 128 in detail]. The SOLR strain from Escherichia coli cells was used as a host for the pZEAX plasmid. Escherichia coli cells were grown on LB medium (as described above) at 37 ° C. in the dark on a 225 rpm rotary shaker. Ampicillin (50 μg / ml) and / or chloramphenicol (30 μg / ml) (both from Sigma) were added to the medium for selection of appropriate transformed cells. As seen in FIG. 6, plasmid pHPK containing the full-length cDNA of the β-carotene C-4-oxygenase enzyme was identified by complementarity as described in Lotan and Hirschberg (1995) FEBS letters 364: 125-128. (See description below). A 1.2 kb PstI-PstI DNA fragment containing the cDNA of β-C-4-oxygenase from Haemat ococcus pluvialis was isolated from plasmid pHPK and inserted into the PstI site in the coding sequence of the crtZ gene of plasmid pZEAX. This recombinant plasmid was named pCAN THA. This plasmid is shown in FIG. The same 1.2 kb PstI-PstI DNA fragment was also inserted into the PstI site 600 bp downstream of the crtE gene of plasmid pZEAX. The obtained recombinant plasmid was named pASTA. This plasmid is shown in FIG. The same 1.2 kb PstI-PstI DNA fragment was also inserted into the PstI site present in the β-lactamase gene of plasmid pPAN35D5 (Hirschberg J, Ohad N, Pecker I and Rahat A (1987) Synechococcus R2.Z Isolation and characterization of herbicide-resistant mutants of Naturforsch 42c: 102-112]. This has the psbAI gene from the cyanobacterium Synechococcus PCC7942 in the plasmid vector pBR328 [Hirschberg J, Ohad N, Pecker I and Rahat A (1987) A single herbicide resistant mutant of the cyanobacterium Synechococcus R2.Z. Separation and characteristics Naturforsch 42c: 102-112]. This plasmid was designated as PAN3.5-KETO. This plasmid is shown in FIG. This plasmid was used in transformation of Synechococcus PCC7942 cells according to the procedure described by Golden [Golden SS (1988) Classical and Gene Transfer-Based Methods for Cyanobacterial Mutations Methods Enzymol 167: 714-727]. Excision of phage library and screening of β-carotene oxygenase gene. Mass excision of the cDNA library prepared as described above was performed using ExAssis helper phage (Stratagene) in cells of the SOLR strain of Escherichia coli harboring plasmid pBCAR. The excised library in phagemid form was placed on LB plates containing 1 mM isopropylthio-β-D-galactosidase (IPTG), 50 μg / ml ampicillin and 30 μg / ml chloramphenicol at about 100-150 per plate. Placed at a density that gives rise to colonies. Plates were incubated overnight at 37 ° C. and for another 2 days at room temperature. The plates were then kept at 4 ° C until colonies were screened for color change. Plasmid for high expression of crtO in chromoplasts. As seen in FIGS. 10-11, the genome of the tomato species Lycopersicon esculentum containing the coding sequence of the amino-terminal region of the polypeptide PDS, which acts as a promoter for the Pds gene and a penetrating peptide for transport to chloroplasts and chromoplasts. DNA sequence (nucleotides 1-1448 of the Pds gene [Mann V, Pecker I and Hirschberg J (1994) Cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum) described in Plant Molecular Biology 24: 429-434. Has been cloned into the HindIII-SmaI site of the binary vector pBIB [described in the binary vector Nucleic Acids Research 18: 230, which allows the exchange of a Becker D (1990) plant selectable marker for a reporter gene]. This is shown in FIG. The recombinant plasmid was named pPTBIB. This is shown in FIG. As seen in FIG. A 1,110 nucleotide long Eco47III-NcoI fragment (nucleotides 211-1321 of SEQ ID NO: 1) containing the cDNA of crtO from pluvialis was subcloned into the SmaI site of plasmid pPTBIB (FIG. 11). As a result, the coding nucleotide sequence at the amino terminus of Pds is in the same reading frame as crtO. The recombinant plasmid was named pPTCRTOBIB. Transgenic higher plant formation. pPTCRTOBIB DNA was extracted from E. coli and transformed into cells of Agrobacterium tumefaciens strain EHA105 [Dower JW, Miller FJ and Raggsale WC (1988) E. coli by high voltage electroporation. High efficiency transformation of Nuc. Acids Res. 18: 6127-6145]. Agrobacterium cells were grown at 28 ° C. in LB medium supplemented with 50 μg / ml streptomycin and 50 μg / ml kanamycin as selection agents. Agrobacterium cells harboring pPTCRTOBIB can be harvested from suspension cultures during the stationary phase of growth, Horsch RB, Fry JE, Hoffmann NL, Eicholtz D, Rogers SG and Fraley RT, a simple and general method for transferring genes to plants Science (1985) 227: 1229-1231; and Jeffesrson AR, Kavanagh TA and Bevan WM (1987) GUS fusion: β-glucuronidase as a sensitive and versatile fusion gene marker in higher plants The EMCO J. 6: 3901-3907 Was used for transformation as described in Leaf explants of the Nicotiana tobaccum NN strain were infected with transformed Agrobacterium cells and kanamycin-resistant transgenic plants were recreated according to the protocols described in Horsch et al. (1985) and Jefferson et al. (1987), supra. Referring to FIG. 13, the presence of the DNA sequence of the crtO gene construct in fully grown regenerated plants was determined by DNA Southern blot analysis. For this purpose, DNA is extracted from the leaves [extraction of restrictable DNA from plants of the genus Kanazawa and Tsusumi (1992) Nelumbo Plant Plant Biology Reports 10: 316-318], and the endonuclease HindIII And the fragments were size separated by gel electrophoresis and hybridized with a radiolabeled crtO sequence (SEQ ID NO: 1). Each transgenic plant tested contained at least one copy of the crt Oc DNA sequence, a 1.75 kb band (arrow) resulting from an internal HindIII-HindIII fragment of pPTCRTOBIB T-DNA, an additional band resulting from partial digestion, Additional bands of varying size (depending on the insertion position of the plant genome) and a 1.0 kb band originating from the tobacco plant itself (and thus also appearing in the negative control WT lane) were generated. Sequence analysis. DNA sequence analysis was performed by the dideoxy method [DNA sequencing with Sanger F, Nicklen S and Coulsen AR (1977) chain growth end inhibitors. See Proc Natl Acad Sci USA 74: 5463-5467]. Carotenoid analysis. Aliquots of Escherichia coli cells grown in LB liquid medium were centrifuged at 13,000 g for 10 minutes, washed once with water and centrifuged again. After removing the water, the cells were resuspended in 70 μl of acetone and incubated at 65 ° C. for 15 minutes. The sample was centrifuged again at 13,000 g for 10 minutes and the supernatant containing the carotenoids was placed in a clean tube. The carotenoid extract was converted to nitrogen (N _Two ) Air-dried under steam and stored at -2 ° C until needed for analysis. Carotenoids from plant tissues were extracted by mixing 0.5-1.0 gr of tissue with 100 μl of acetone, followed by incubation at 65 ° C. for 15 minutes, and then processing the samples as described above. Acidified reverse phase C18 column, Spherisorb ODS-2 (silica 5 μm The product was subjected to high performance liquid chromatography (HPLC). The mobile phase was injected at a rate of 1.5 ml / min with a three-phase Merck-Hitachi L-6200A high pressure pump. The mobile phase consisted of a formulated solvent system containing hexane / dichloromethane / isopropyl alcohol / triethylamine (88.5: 10: 1.5: 0.1, v / v). A peak was detected at 470 nm using a Waters 996 photodiode array detector. Individual carotenoids were identified by their retention times and their typical absorption spectra in comparison to chemically pure β-carotene, zeaxanthin, echinenone, canthaxanthin, adnirubin and astaxanthin standard samples (the latter). Were donated by Liverpool John Moores University by Dr. Andrew Young). Thin layer chromatography (TLC) was performed on silica gel 60 F254 plates (Merck) using ethyl acetate / benzene (7: 3, v / v) as eluent. Visible absorption spectra were recorded on a Shimazu UV-160A spectrophotometer. All spectra were recorded in acetone. The fine structure of the spectrum was expressed as% III / II [Britton, G (1995) UV / visible spectroscopy: Carotenoids; Volume IB, Spectroscopy Briton G, Liaaen-Jensen S and Pfander HBirkhauser Verlag, Basel, 13-62. page]. Isolation and identification of carotenoids extracted from E. coli cells was performed on silica TLC with high absorption (R _f (Lowest value). FIG. 14 shows the structure of the carotenoid and the biosynthetic pathway of astaxanthin. Hereinafter, the details refer to carotenoids numbered 1 to 9 in FIG. β-carotene (1). R _f 0.92 (inseparable from certification (1)). R _t .VISλ _max nm: (428), 452, 457,% III / II = 0. Echinenone (2). R _f 0.90 (inseparable from certification (2)). R _t .VISλ _max nm: 455,% III / II = 0. Canthaxanthin (3). R _f 0.87 (inseparable from certification (3)). R _t .VISλ _max nm: 470,% III / II = 0. β-cryptoxanthin (4). R _f 0.83. R _t .VISλ _max nm: (428), 451, 479,% III / II = 0. Adonirubin (5). R _f 0.82 (inseparable from certification (5)). R _t .VISλ _max nm: 476,% III / II = 0. Astaxanthin (6). R _f 0.79 (inseparable from certification (6)). R _t .VISλ _max nm: 477,% III / II = 0. Adonixanthin (7). R _f 0.72. R _t .VISλ _max nm: 464,% III / II = 0. Zeaxanthin (8). R _f 0.65 (inseparable from certification (8)). R _t .VISλ _max nm: (428), 451, 483,% 111/11 = 27. Hydroxyechinenone (9). R _f 0.80. R _t .VISλ _max nm: 464,% III / II = 0. Chirality arrangement. The chirality configuration of astaxanthin was determined by HPLC of the derived diastereoisomeric camphanate of astaxanthin [Renstrom B, Borch G, Skulberg M and Liaaen-Jensen S (1981) from (3S, 3S 'from Haematococcus plu vialis). ) Optical purity of astaxanthin Phytochem 20: 256l-2565]. The analysis demonstrated that Escherichia coli cells synthesized pure (3S, 3S ') astaxanthin. Example 1 Cloning of β-C-4-Oxygenase Gene A cDNA library was constructed in a λZAP II vector from poly-An RNA of Haematococcus pluvialis cells which had been induced to synthesize astaxanthin by nitrogen depletion as described above. . The entire library was excised into β-carotene accumulating cells of the Escherichia coli SOLR strain carrying the plasmid pBCAR (shown in FIG. 4). Screening for the β-carotene oxygenase gene was based on color visualization of colonies 3 mm in diameter in size. Astaxanthin and other oxidized forms of β-carotene (ie, xanthophyll) have different shades of color and can therefore be detected from the yellow β-carotene background. The screen contained approximately 100,000 colonies, which grew on LB media plates containing ampicillin and chloramphenicol, selecting for both the λZAP II vector and the pBCAR plasmid in a plasmid-grown form. Some colonies showed different shades, but one showed a pronounced red-brown pigment. This colony was presumed to contain the xanthophyll biosynthesis gene and was selected in the following examples for further analysis as described below. Example 2 Analysis of β-C-4-oxygenase activity in Escherichia coli Red-brown colonies presumed to contain xanthophyll biosynthesis genes (see Example 1 above) were streaked and analyzed further. First, a plasmid DNA was prepared from a red-brown colony of a recombinant ZAPII plasmid having a cDNA that causes xanthophyll synthesis in Escherichia coli, and the plasmid DNA was transfected into Escherichia coli of XLI-Blue strain, and ampicillin was transfected. Isolated by selection on containing media. This plasmid was named pHPK (pHPK is a λZAP II vector containing an insert isolated from a red-brown colony) and used to express β-carotene-producing Escherichia coli cells (plasmid pBCAR shown in FIG. 4). , The formation of red-brown colonies was observed. Carotenoids were extracted from the transformants and host cells (as a control) with acetone, and analyzed by HPLC. HPLC analysis of carotenoids of β-carotene-synthesizing host bacteria (Escherichia coli SOLR strain having the plasmid pBCAR shown in FIG. 4) revealed only a trace amount of β-carotene in the transformed cells as compared with the red-brown colonies. Although not found, a new major peak for canthaxanthin and another small peak for echinenone appeared [Lotan and Hirschberg (1995) FEBS letters 364: 125-128]. These results indicate that the cDNA designated crtO in the plasmid encodes an enzyme with β-C-4-oxygenase activity that converts β-carotene to canthaxanthin via echinenone (see FIG. 14). That). Thus, it is concluded that one enzyme catalyzes this two-step ketotransformation by acting symmetrically on the 4 and 4 'carbons of the β and β' rings, respectively. Example 3 Production of Astaxanthin in Escherichia coli Cells Whether β-carotene hydroxylase (eg, the product of the crtZ gene of Erwinia herbicola) can convert canthaxanthin thus produced to astaxanthin, and / or -To determine whether zeaxanthin converted from β-carotene by carotene hydroxylase can be converted to astaxanthin by β-C-4-oxygenase, the crto cDNA of Haematococcu s pluvialis was isolated from Erwinia herbicola. It was expressed in Escherich ia coli cells together with the crtZ gene. For this purpose, Escherichia coli cells of the SOLR strain were transformed with either plasmid pASTA containing both crtZ and crtO, as shown in FIG. 8, or pHPK containing crtO, as shown in FIG. Transfected with either pZEAX containing crtZ as indicated. The carotenoids of the resulting transfected cells were extracted and analyzed by HPLC as described above. The results (shown in Table 1) show the composition of carotenoids extracted from cells containing plasmid pASTA. Similar carotenoid composition was observed in Escherichia coli cells having pHPK and pZEAX. The results shown in Table 1 show that the carotenoids treating either the β-terminal group or the 4-keto-terminal group at the C-3 and C-3 'carbons are hydroxylation reactions catalyzed by the crtZ gene product. Demonstrates that it acts as a substrate for Hydroxylation of β-carotene and canthaxanthin produces zeaxanthin and astaxanthin, respectively. These hydroxylations result in the formation of astaxanthin, an intermediate ketocarotenoid, 3-hydroxyechinenone, adonixanthin and adnirubin. These results further indicate that astaxanthin can be produced in heterologous cells by expressing the crtO gene along with the gene encoding β-carotene hydroxylase. Example 4 Sequence analysis of the β-carotene C-4-oxykenase gene The full length of the cDNA insert in plasmid pHPK (1771 base pairs), determined in the presence of a poly A tail, was subjected to nucleotide sequence analysis. The sequence set forth in Sequence Listing 1 and its translation into the amino acid sequence set forth in Sequence Listing 3 were deposited with the EMBL database on May 1, 1995, resulting in EMBL accession numbers X86782 and X86783, respectively. An open reading frame (ORF) of 825 nucleotides (nucleotides 166 to 1152 of SEQ ID NO: 3) was identified within this sequence. The ORF encodes an enzyme β-carotene C-4-oxygenase having 329 amino acids as set forth in SEQ ID NO: 4, which has been demonstrated by its functional expression in Escherichia coli cells (see above example). See Example 3). The gene for this enzyme was named crtO. Example 5 Transformation of Cyanobacteria with crtO The plasmid DNA of pPAN3.5-KATO shown in FIG. 9 was transfected into cells of the cyanobacterium Synechococcus PCC7942 according to the method described by Golden [Golden SS (1988) classical Mutation of Cyanobacteria Methods Enzymol 167: 714-727] by a method based on genetic and gene transfer. Cyanobacterial cells were placed on Petri dishes containing BG11 containing chloramphenicol. Chloramphenicol-resistant Synechococcus PCC7942 colonies that appeared 10 days later were analyzed for carotenoid content. As described in detail in Table 2 below, HPLC analysis of these cells indicated that the major carotenoid components of the cells were β-carotene, echinenone, canthaxanthin, adnirubin and astaxanthin. A similar analysis of the wild-type strain and Synechococcus PCC7942 transfected (and thus unable to produce active protein) with a plasmid in which the orientation of the crtO gene was reversed (not shown), showed that echinenone, canthaxanthin, adnirubin and astaxanthin were No production was shown. These results demonstrate that crtO of Haematococcus pluvialis can be expressed in cyanobacteria, and that its expression provided the β-C-4-oxygenase enzyme activity required for the conversion of β-carotene to canthaxanthin. I do. This result indicates that the endogenous β-carotene hydroxylase of Syn echococcus PCC7942 can convert the produced canthaxanthin to astaxanthin. The carotenoid biosynthetic pathway is similar in all green photosynthetic organisms [FIGS. 1 and 10 and Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992), which catalyzes the conversion of phytoene to ζ-carotene. Two polypeptides are regulated at the transcriptional stage during tomato fruit ripening, see Proc Natl Acad Sci USA 89: 4962-4966], so any polypeptide that also expresses endogenous β-carotene hydroxylase It is speculated that by expressing crtO in tissues, astaxanthin can be produced in algae and higher plants. In addition, crtO expression in any tissue that expresses either endogenous or engineered β-carotene hydroxylase by any organism that contains either the endogenous or engineered β-carotene biosynthetic pathway By doing so, it is speculated that astaxanthin can be produced. Example 6 Determination of the Chirality Configuration of Astaxanthin Produced in a Heterogeneous System The chirality configuration of astaxanthin produced by the Escherichia coli cells described in Example 3 above and by the cyanobacterium Synchococcus PCC7942 cells described in Example 5 above Was determined by HPLC of the derived diastereoisomeric camphanate of astaxanthin [Renstrom B, Borch G, Skulberg M and Liaa en-Jensen S (1981) Optical characterization of (3S, 3S ') astaxanthin from Haematococcus pluvialis. Purity Phytochem 20: 2561-2565]. Analysis demonstrated that the Escherichia coli and Synchococcus PCC7942 cells described above synthesized pure (3S, 3S ′) astaxanthin. Example 7 Transformation of higher plants with crtO Producing native astaxanthin in higher plants has two expected benefits. First, as a pure chemical, astaxanthin is widely used as a feed additive for fish. It may be a suitable food colorant for human consumption and may have applications in the cosmetics industry as well. Second, the inclusion of astaxanthin in vivo in flowers and fruits provides an attractive pink / red color that increases their appearance and / or nutritional value. In flowers and fruits, carotenoids are usually synthesized and accumulate at high concentrations in chromoplasts (typical pigmented plastids), thus providing their organs with a typical dark color. Inducing the synthesis of astaxanthin in chromoplasts allows the accumulation of high concentrations of this ketocarotenoid. Overexpression of the carotenoid biosynthesis gene results in increased carotenoid levels in chloroplasts, or other changes in carotenoid composition in chloroplasts damage thylakoid membranes, impair photosynthesis, and are therefore harmful to plants. is there. In contrast, increasing carotenoid levels or altering carotenoid composition in chromoplasts does not affect plant viability or the weekly amount of fruit and flowers. Thus, as described above, the crtO gene isolated from the alga Haematococcus pluvialis is used in higher plants using gene transfer techniques in such a way that the expression of the gene is positively regulated, especially in chromoplast-containing cells. Transplanted. For this purpose, a T-DNA containing binary plasmid vector as shown in FIG. 12 was used in E. coli to encode the promoter and the coding DNA sequence of the penetrating peptide encoded by Pds from the tomato species Lycopersicon esculen tum (Hluvialis (Ligated to the coding sequence of crtO from E. coli). If this DNA construct is introduced into tobacco (Nicotiana tabacum NN) plants via Agrobacterium-mediated transformation to form transgenic plants, as described in the methods above, the plants will in particular have floral tissue (chromosome). (Plast-containing cells) to gain the ability to acidify ketocarotenoids. It should be noted that the Pds gene promoter may direct transcription and thus may be expressed especially in chloroplast and / or chromoplast-containing tissues of plants. It should further be noted that the penetrating peptide encoded by a portion of the Pds coding sequence may direct the binding protein (ie, in frame) to plant chromoplasts and / or chloroplasts. As shown in FIG. 15, in chromoplast-containing cells, such as dense gland tissue of tobacco flowers, this DNA construct is present at higher concentrations of astaxanthin and other ketocarotenoids that change color from normal yellow to red. Induces the accumulation of The concentration and composition of carotenoids in chloroplast-containing tissues such as leaves and chromoplast-containing tissues such as flowers were determined in transgenic plants and compared to normal non-transformed plants. The carotenoid composition in the leaves (chloroplast-containing tissues) and flower dense gland tissues (chromoplast-containing tissues) of wild-type and transgenic tobacco plants was determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) as described above. ). Table 3 below summarizes the total carotenoid concentrations in leaves (chloroplast-containing tissue) and dense glandular tissues (chromoplast-containing tissue) of wild-type and transgenic tobacco plants. The percentages of carotenoid composition in leaves of wild-type and transgenic tobacco plants are summarized in Table 4 below. The percentages of carotenoid composition in the dense glands of wild-type and transgenic tobacco plants are summarized in Table 5 below. In particular, note the increased content of hydroxyechinenone, 3'hydroxyechinenone, adonirbin, adonixanthin and astaxanthin in the chromoplast-containing tissue of transgenic tobacco plants. Thus, the present invention provides a peptide having β-C-4-oxygenase activity; a DNA segment encoding the peptide; an RNA segment encoding the peptide; a vector and a recombinant DNA molecule containing the DNA segment; By providing a host containing a recombinant DNA molecule or DNA segment; and a biotechnological method for producing (3S, 3'S) astaxanthin or a food additive containing (3S, 3'S) astaxanthin by using the host, It overcomes the disadvantages of the currently known features by enabling relatively inexpensive biotechnological production of 3S, 3'S) astaxanthin. While the invention has been described with a limited number of embodiments, it will be apparent that many modifications, changes and other applications of the invention may be made.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 9/02 9/02 C12P 7/38 C12P 7/38 C12Q 1/68 A C12Q 1 / 68 C12N 5/00 A // (C12N 15/09 ZNA C12R 1:89) (C12P 7/38 C12R 1:19) (C12P 7/38 C12R 1:89) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Rotan Termer Israel, Kinneret 15105 (72) Inventor Harker Mark 92461 Jerusalem, Israel, Nal Kiss 9

Claims (1)

[Claims]   1. A nucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4 A DNA segment containing a reotide sequence.   2. The nucleotide sequence is an allelic variant, a species variant, a naturally occurring variant Selected from the group consisting of artificially induced variants and combinations thereof. 2. The DNA segment according to claim 1, which is a variant.   3. 2. The method of claim 1, wherein the nucleotide sequence comprises the sequence of SEQ ID NO: 1. DNA segment.   4. The nucleotide sequence   2. The D of claim 1, wherein the sequence comprises nucleotides 166-1152 of SEQ ID NO: 1. NA segment.   5. Nucleic acids that hybridize to nucleic acid probes under high stringency conditions A DNA segment comprising an otide sequence, wherein the probe comprises a small number of SEQ ID NO: 1. A DNA segment comprising at least 15 contiguous nucleotides.   6. Nucleic acids that hybridize to nucleic acid probes under low stringency conditions A DNA segment containing an otide sequence, wherein the probe is the nucleus of SEQ ID NO: 1. A DNA segment comprising leotides 166-1152.   7. Nucleic acids that hybridize to nucleic acid probes under high stringency conditions A DNA segment comprising an otide sequence, wherein the probe comprises a small number of SEQ ID NO: 2. A DNA segment comprising at least 15 contiguous nucleotides.   8. Nucleic acids that hybridize to nucleic acid probes under low stringency conditions A DNA segment containing an otide sequence, wherein the probe is a nucleoside of SEQ ID NO: 2. A DNA segment comprising leotides 166-1152.   9. A nucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4 RNA segment containing a reotide sequence.   10. Wherein said nucleotide sequence is an allelic variant, a species variant, a naturally occurring modification. Selected from the group consisting of body, artificially induced variants and combinations thereof. The RNA segment according to claim 9, which is a variant to be obtained.   11. 10. The method of claim 9, wherein the nucleotide sequence comprises the sequence of SEQ ID NO: 2. RNA segment listed.   12. The nucleotide sequence corresponds to nucleotides 166 to 1152 of SEQ ID NO: 2. The RNA segment of claim 9 comprising.   13. Nucleic acids that hybridize to nucleic acid probes under high stringency conditions An RNA segment comprising a reotide sequence, The probe comprises at least 15 contiguous nucleotides of SEQ ID NO: 1. RNA segment.   14． Nucleic acids that hybridize to nucleic acid probes under low stringency conditions An RNA segment comprising a reotide sequence, wherein the probe is An RNA segment comprising the nucleotides 166-1152.   15. Nucleic acids that hybridize to nucleic acid probes under high stringency conditions An RNA segment comprising a reotide sequence, wherein said probe comprises a small number of SEQ ID NO: 2. An RNA segment comprising at least 15 contiguous nucleotides.   16. Nucleic acids that hybridize to nucleic acid probes under low stringency conditions An RNA segment comprising a reotide sequence, wherein the probe is An RNA segment comprising the nucleotides 166-1152.   17． Haematococcus pluvialiscrtO gene, pair And species variants and their functionally naturally occurring and artificially induced A polypeptide comprising an amino acid sequence corresponding to the derived variant.   18. The method according to claim 17, wherein the amino acid sequence is the sequence of SEQ ID NO: 4. Polypeptide.   19. A polypeptide comprising an amino acid sequence homologous to the sequence of SEQ ID NO: 4.   20. A polypeptide containing the amino acids encoded by the DNA segments The DNA segment is under the conditions of low stringency and Hybridizes to nucleotides 166-1152, and the polypeptide is β-C- A polypeptide having 4-oxygenase activity.   21. A recombinant vector DNA molecule comprising the DNA segment according to claim 2.   22. A host comprising the recombinant vector DNA molecule of claim 21, wherein the host comprises the recombinant vector DNA molecule of claim 21. The host, wherein the host is selected from the group consisting of a cell and an organism.   23. A host comprising the DNA segment according to claim 2, wherein the host comprises: A host selected from the group consisting of cells and organisms.   24. Astaxanthin, canthaxanthin, echinenone, cryptoxanthi , Isocryptoxanthin, hydroxyechinenone, zeaxanthin, Selected from the group consisting of rubin or adonixanthin and combinations thereof A method for producing xanthophylls, comprising:   (A) providing a host according to claim 22;   (B) Propagation for producing the xanthofail in the host Providing conditions; and   (C) extracting the xanthophyll from the host;   Including, methods.   25. Astaxanthin, canthaxanthin, echinenone, isocryptoxa , Cryptoxanthin, hydroxyechinenone, zeaxanthin, Selected from the group consisting of rubin or adonixanthin and combinations thereof A method for producing xanthophylls, comprising:   (A) providing the host of claim 23;   (B) providing the host with growth conditions for producing the xanthofail; Process; and   (C) extracting the xanthophyll from the host;   Including, methods.   26. 23. The host of claim 22, wherein said host is used as a food additive.   27. 24. The host of claim 23, wherein said host is used as a food additive.   28. Haematococcus pluvialiscrtO gene, pair And species variants, or their functionally naturally occurring or artificially induced variants Introduction to a polypeptide encoding an amino acid sequence corresponding to the derived variant Transgenic plants that express genes.   29. 29. The method of claim 28, wherein said expression is greatest in chromoplast-containing tissues. The transgenic plant as described.   30. To direct proteins to plant chloroplasts or chromoplasts DNA segment encoding a polypeptide of Haematoc occus pluvialiscrtO gene, allelic and species variants, or Correspond to their functionally naturally occurring and artificially derived variants DNA segment in a second frame encoding a polypeptide comprising an amino acid sequence Recombinant DNA vector containing the   31. The first DNA segment is derived from a tomato Pds gene; A recombinant DNA vector according to claim 30.   32. Highly expressed in plant chloroplast or chromoplast-containing tissues First DNA segment containing a possible promoter and Haematoco ccus pluvialiscrtO gene, allelic and species variants, or Corresponding to their functionally naturally occurring and artificially derived variants. Recombination comprising a second DNA segment encoding a polypeptide comprising a amino acid sequence DNA vector.   33. The first DNA segment is derived from a tomato Pds gene; A recombinant DNA vector according to claim 30.