AU732842B2 - Nucleic acid sequence encoding beta-C-4-oxygenase from haematococcus pluvialis for the biosynthesis of astaxanthin - Google Patents

Description

WO 98/18910 PCT/US97/17819 NUCLEIC ACID SEQUENCE ENCODING BETA-C-4-OXYGENASE FROM HAEMATOCOCCUS PLUVIALIS FOR THE BIOSYNTHESIS OF ASTAXANTHIN FIELD AND BACKGROUND OF THE INVENTON The present invention relates, in general, to a biotechnological method for production of (3S,3'S) astaxanthin. In particular, the present invention relates to a to peptide having a P-C-4-oxygenase activity; a DNA segment coding for this peptide; an RNA segments coding for this peptide; a recombinant DNA molecule comprising a vector and the DNA segment; a host cell or organism containing the above described recombinant DNA molecule or DNA segment; and to a method of biotechnologically producing (3S,3'S) astaxanthin or a food additive containing (3S,3'S) astaxanthin, using the host.

Carotenoids, such as astaxanthin, are natural pigments that are responsible for many of the yellow, orange and red colors seen in living organisms.

Carotenoids are widely distributed in nature and have, in various living systems, two main biological functions: they serve as light-harvesting pigments in photosynthesis, and they protect against photooxidative damage. These and additional biological functions of carotenoids, their important industrial role, and their biosynthesis are discussed hereinbelow.

As part of the light-harvesting antenna, carotenoids can absorb photons and transfer the energy to chlorophyll, thus assisting in the harvesting of light in the range of 450 570 nm [see, Cogdell RJ and Frank HA (1987) How carotenoids function in photosynthestic bacteria. Biochim Biophys Acta 895: 63-79; Cogdell R (1988) The function of pigments in chloroplasts. In: Goodwin TW (ed) Plant Pigments, pp 183-255. Academic Press, London; Frank HA, Violette CA, Trautman JK, Shreve AP, Owens TG and Albrecht AC (1991) Carotenoids in photosynthesis: structure and photochemistry. Pure Appl Chem 63: 109-114; Frank HA, Farhoosh R, Decoster B and Christensen RL (1992) Molecular features that control the efficiency of carotenoid-to-chlorophyll energy transfer in photosynthesis. In: Murata N (ed) Research in Photosynthesis, Vol I, pp 125-128.

Kluwer, Dordrecht; and, Cogdell RJ and Gardiner AT (1993) Functions of carotenoids in photosynthesis. Meth Enzymol 214: 185-193]. Although carotenoids are integral constituents of the protein-pigment complexes of the lightharvesting antennae in photosynthetic organisms, they are also important components of the photosynthetic reaction centers.

WO 98/18910 PCT/US97/17819 2 Most of the total carotenoids is located in the light harvesting complex II [Bassi R, Pineaw B, Dainese P and Marquartt J (1993) Carotenoid binding proteins of photosystem II. Eur J Biochem 212: 297-302]. The identities of the photosynthetically active carotenoproteins and their precise location in lightharvesting systems are not known. Carotenoids in photochemically active chlorophyll-protein complexes of the thermophilic cyanobacterium Synechococcus sp. were investigated by linear dichroism spectroscopy of oriented samples [see, Breton J and Kato S (1987) Orientation of the pigments in photosystem II: lowtemperature linear-dichroism study of a core particle and of its chlorophyll-protein to subunits isolated from Synechococcus sp. Biochim Biophys Acta 892: 99-107].

These complexes contained mainly a p-carotene pool absorbing around 505 and 470 nm, which is oriented close to the membrane plane. In photochemically inactive chlorophyll-protein complexes, the p-carotene absorbs around 495 and 465 nm, and the molecules are oriented perpendicular to the membrane plane.

Evidence that carotenoids are associated with cyanobacterial photosystem (PS) II has been described [see, Suzuki R and Fujita Y (1977) Carotenoid photobleaching induced by the action of photosynthetic reaction center II: DCMU sensitivity. Plant Cell Physiol 18: 625-631; and, Newman PJ and Sherman LA (1978) Isolation and characterization of photosystem I and II membrane particles from the blue-green alga Synechococcus cedrorum. Biochim Biophys Acta 503: 343-361]. There are two p-carotene molecules in the reaction center core of PS II [see, Ohno T, Satoh K and Katoh S (1986) Chemical composition of purified oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp. Biochim Biophys Acta 852: 1-8; Gounaris K, Chapman DJ and Barber J (1989) Isolation and characterization of a D1/D2/cytochrome b-559 complex from Synechocystis PCC6803. Biochim Biophys Acta 973: 296-301; and, Newell RW, van Amerongen H, Barber J and van Grondelle R (1993) Spectroscopic characterization of the reaction center of photosystem II using polarized light: Evidence for p-carotene excitors in PS II reaction centers. Biochim Biophys Acta 1057: 232-238] whose exact function(s) is still obscure [reviewed by Satoh K (1992) Structure and function of PS II reaction center. In: Murata N (ed) Research in Photosynthesis, Vol. II, pp. 3-12. Kluwer, Dordrecht]. It was demonstrated that these two coupled P-carotene molecules protect chlorophyll P680 from photodamage in isolated PS II reaction centers [see, De Las Rivas J, Telfer A and Barber J (1993) 2-coupled p-carotene molecules protect P680 from photodamage in isolated PS II reaction centers. Biochim. Biophys. Acta 1142: 155-164], and this may be related to the protection against degradation of the Dl subunit of PS II [see, Sandmann G (1993) Genes and enzymes involved in the desaturation WO 98/18910 PCT/US97/17819 3 reactions from phytoene to lycopene. (abstract), 10th International Symposium on Carotenoids, Trondheim CL1-2]. The light-harvesting pigments of a highly purified, oxygen-evolving PS II complex of the thermophilic cyanobacterium Synechococcus sp. consists of 50 chlorophyll a and 7 p-carotene, but no xanthophyll, molecules [see, Ohno T, Satoh K and Katoh S (1986) Chemical composition of purified oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp. Biochim Biophys Acta 852: p-carotene was shown to play a role in the assembly of an active PS II in green algae [see, Humbeck K, Romer S and Senger H (1989) Evidence for the essential role of to carotenoids in the assembly of an active PS II. Planta 179: 242-250].

Isolated complexes of PS I from Phormidium luridum, which contained chlorophylls per P700, contained an average of 1.3 molecules of p-carotene [see, Thornber JP, Alberte RS, Hunter FA, Shiozawa JA and Kan KS (1976) The organization of chlorophyll in the plant photosynthetic unit. Brookhaven Symp Biology 28: 132-148]. In a preparation of PS I particles from Synechococcus sp.

strain PCC 6301, which contained 130 5 molecules of antenna chlorophylls per P700. 16 molecules of carotenoids were detected [see, Lundell DJ, Glazer AN, Melis A and Malkin R (1985) Characterization of a cyanobacterial photosystem I complex. J Biol Chem 260: 646-654]. A substantial content of p-carotene and the xanthophylls cryptoxanthin and isocryptoxanthin were detected in PS I pigmentprotein complexes of the thermophilic cyanobacterium Synechococcus elongatus [see, Coufal J, Hladik J and Sofrova D (1989) The carotenoid content of photosystem 1 pigment-protein complexes of the cyanobacterium Synechococcus elongatus. Photosynthetica 23: 603-616]. A subunit protein-complex structure of PS I from the thermophilic cyanobacterium Synechococcus sp., which consisted of four polypeptides (of 62, 60, 14 and 10 kDa), contained approximately 10 3carotene molecules per P700 [see, Takahashi Y, Hirota K and Katoh S (1985) Multiple forms of P700-chlorophyll a-protein complexes from Synechococcus sp.: the iron, quinone and carotenoid contents. Photosynth Res 6: 183-192]. This carotenoid is exclusively bound to the large polypeptides which carry the functional and antenna chlorophyll a. The fluorescence excitation spectrum of these complexes suggested that P-carotene serves as an efficient antenna for PS I.

As mentioned, an additional essential function of carotenoids is to protect against photooxidation processes in the photosynthetic apparatus that are caused by the excited triplet state of chlorophyll. Carotenoid molecules with 7r-electron conjugation of nine or more carbon-carbon double bonds can absorb triplet-state energy from chlorophyll and thus prevent the formation of harmful singlet-state oxygen radicals. In Synechococcus sp. the triplet state of carotenoids was WO 98/18910 PCT/US97/17819 4 monitored in closed PS II centers and its rise kinetics of approximately nanoseconds is attributed to energy transfer from chlorophyll triplets in the antenna [see, Schlodder E and Brettel K (1988) Primary charge separation in closed photosystem II with a lifetime of 11 nanoseconds. Flash-absorption spectroscopy with oxygen-evolving photosystem II complexes from Synechococcus. Biochim Biophys Acta 933: 22-34]. It is conceivable that this process, that has a lower yield compared to the yield of radical-pair formation, plays a role in protecting chlorophyll from damage due to over-excitation.

The protective role of carotenoids in vivo has been elucidated through the 0o use of bleaching herbicides such as norflurazon that inhibit carotenoid biosynthesis in all organisms performing oxygenic photosynthesis [reviewed by Sandmann G and Boger P (1989) Inhibition of carotenoid biosynthesis by herbicides. In: Boger P and Sandmann G (Eds.) Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton, Florida]. Treatment with norflurazon in the light results in a decrease of both carotenoid and chlorophyll levels, while in the dark, chlorophyll levels are unaffected. Inhibition of photosynthetic efficiency in cells of Oscillatoria agardhii that were treated with the pyridinone herbicide, fluridone, was attributed to a decrease in the relative abundance of myxoxanthophyll, zeaxanthin and 3carotene, which in turn caused photooxidation of chlorophyll molecules [see, Canto de Loura I, Dubacq JP and Thomas JC (1987) The effects of nitrogen deficiency on pigments and lipids of cianobacteria. Plant Physiol 83: 838-843].

It has been demonstrated in plants that zeaxanthin is required to dissipate, in a nonradiative manner, the excess excitation energy of the antenna chlorophyll [see, Demmig-Adams B (1990) Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 1-24; and, Demmig- Adams B and Adams WW III (1990) The carotenoid zeaxanthin and high-energystate quenching of chlorophyll fluorescence. Photosynth Res 25: 187-197]. In algae and plants a light-induced deepoxidation of violaxanthin to yield zeaxanthin, is related to photoprotection processes [reviewed by Demmig-Adams B and Adams WW III (1992) Photoprotection and other responses of plants to high light stress. Ann Rev Plant Physiol Plant Mol Biol 43: 599-626]. The light-induced deepoxidation of violaxanthin and the reverse reaction that takes place in the dark, are known as the "xanthophyll cycle" [see, Demmig-Adams B and Adams WW III (1992) Photoprotection and other responses of plants to high light stress. Ann Rev Plant Physiol Plant Mol Biol 43: 599-626]. Cyanobacterial lichens, that do not contain any zeaxanthin and that probably are incapable of radiationless energy dissipation, are sensitive to high light intensity; algal lichens that contain zeaxanthin are more resistant to high-light stress [see, Demmig-Adams B, Adams WO 98/18910 PCT/US97/17819 WW III, Green TGA, Czygan FC and Lange OL (1990) Differences in the susceptibility to light stress in two lichens forming a phycosymbiodeme, one partner possessing and one lacking the xanthophyll cycle. Oecologia 84: 451-456: Demmig-Adams B and Adams WW III (1993) The xanthophyll cycle, protein turnover, and the high light tolerance of sun-acclimated leaves. Plant Physiol 103: 1413-1420; and, Demmig-Adams B (1990) Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim Biophys Acta 1020: 1-24].

In contrast to algae and plants, cyanobacteria do not have a xanthophyll cycle.

However, they do contain ample quantities of zeaxanthin and other xanthophylls that can support photoprotection of chlorophyll.

Several other functions have been ascribed to carotenoids. The possibility that carotenoids protect against damaging species generated by near ultra-violet (UV) irradiation is suggested by results describing the accumulation of 3-carotene in a UV-resistant mutant of the cyanobacterium Gloeocapsa alpicola [see, Buckley CE and Houghton JA (1976) A study of the effects of near UV radiation on the pigmentation of the blue-green alga Gloeocapsa alpicola. Arch Microbiol 107: 93- 97]. This has been demonstrated more elegantly in Escherichia coli cells that produce carotenoids [see, Tuveson RW and Sandmann G (1993) Protection by cloned carotenoid genes expressed in Escherichia coli against phototoxic molecules activated by near-ultraviolet light. Meth Enzymol 214: 323-330]. Due to their ability to quench oxygen radical species, carotenoids are efficient antioxidants and thereby protect cells from oxidative damage. This function of carotenoids is important in virtually all organisms [see, Krinsky NI (1989) Antioxidant functions of carotenoids. Free Radical Biol Med 7: 617-635; and, Palozza P and Krinsky NI (1992) Antioxidant effects of carotenoids in vivo and in vitro an overview. Meth Enzymol 213: 403-420]. Other cellular functions could be affected by carotenoids, even if indirectly. Although carotenoids in cyanobacteria are not the major photoreceptors for phototaxis, an influence of carotenoids on phototactic reactions, that have been observed in Anabaena variabilis, was attributed to the removal of singlet oxygen radicals that may act as signal intermediates in this system [see, Nultsch W and Schuchart H (1985) A model of the phototactic reaction chain of cyanobacterium Anabaena variabilis.

Arch Microbiol 142: 180-184].

In flowers and fruits carotenoids facilitate the attraction of pollinators and dispersal of seeds. This latter aspect is strongly associated with agriculture. The type and degree of pigmentation in fruits and flowers are among the most important traits of many crops. This is mainly since the colors of these products WO 98/18910 PCT/US97/17819 6 often determine their appeal to the consumers and thus can increase their market worth.

Carotenoids have important commercial uses as coloring agents in the food industry since they are non-toxic [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 during fruit ripening in chromoplasts. Tomato extracts, which contain high content (over 80% dry weight) of lycopene, are commercially produced worldwide for industrial use as food colorant. Furthermore, the flesh, feathers or eggs of fish and birds assume the color of the dietary carotenoid provided, and thus carotenoids are frequently used in dietary additives for poultry and in aquaculture. Certain cyanobacterial species, for example Spirulina sp. [see, Sommer TR, Potts WT and Morrissy NM (1990) Recent progress in processed microalgae in aquaculture. Hydrobiologia 204/205: 435-443], are cultivated in aquaculture for the production of animal and human food supplements. Consequently, the content of carotenoids, primarily of 3carotene, in these cyanobacteria has- a major commercial implication in biotechnology.

Most carotenoids are composed of a C40 hydrocarbon backbone, constructed from eight C5 isoprenoid units and contain a series of conjugated double bonds. Carotenes do not contain oxygen atoms and are either linear or cyclized molecules containing one or two end rings. Xanthophylls are oxygenated derivatives of carotenes. Various glycosilated carotenoids and carotenoid esters have been identified. The C40 backbone can be further extended to give C45 or carotenoids, or shortened yielding apocarotenoids. Some nonphotosynthetic bacteria also synthesize C30 carotenoids. General background on carotenoids can be found in Goodwin TW (1980) The Biochemistry of the Carotenoids, Vol. 1, 2nd Ed. Chapman and Hall, New York; and in Goodwin TW and Britton G (1988) Distribution and analysis of carotenoids. In: Goodwin TW (ed) Plant Pigments, pp 62-132. Academic Press, New York.

More than 640 different naturally-occurring carotenoids have been so far characterized, hence, 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 as well as several nonphotosynthetic bacteria and fungi, however they are also widely distributed through feeding throughout the animal kingdom.

Carotenoids are synthesized de novo from isoprenoid precursors only in photosynthetic organisms and some microorganisms, they typically accumulate in WO 98/18910 PCT/US97/17819 7 protein complexes in the photosynthetic membrane, in the cell membrane and in the cell wall.

As detailed in Figure 1, in the biosynthesis pathway of p-carotene, four enzymes convert geranylgeranyl pyrophosphate of the central isoprenoid pathway to p-carotene. Carotenoids are produced from the general isoprenoid biosynthetic pathway. While this pathway has been known for several decades, only recently, and mainly through the use of genetics and molecular biology, have some of the molecular mechanisms involved in carotenoids biogenesis, been elucidated. This is due to the fact that most of the enzymes which take part in the conversion of phytoene to carotenes and xanthophylls are labile, membrane-associated proteins that lose activity upon solubilization [see, Beyer P, Weiss G and Kleinig H (1985) Solubilization and reconstitution of the membrane-bound carotenogenic enzymes from daffodile chromoplasts. Eur J Biochem 153: 341-346; and, Bramley PM (1985) The in vitro biosynthesis of carotenoids. Adv Lipid Res 21: 243-279].

However, solubilization of carotenogenic enzymes from Synechocystis sp. strain PCC 6714 that retain partial activity has been reported [see, Bramley PM and Sandmann G (1987) Solubilization of carotenogenic enzyme of Aphanocapsa.

Phytochem 26: 1935-1939]. There is no genuine in vitro system for carotenoid biosynthesis which enables a direct essay of enzymatic activities. A cell-free carotenogenic 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 for cyanobacteria [see, Sandmann G and Bramley PM (1985) Carotenoid biosynthesis by Aphanocapsa homogenates coupled to a phytoene-generating system from Phycomyces blakesleeanus. Planta 164: 259-263; and, Bramley PM and Sandmann G (1985) In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa. Phytochem 24: 2919-2922]. Reconstitution of phytoene desaturase from Synechococcus sp. strain PCC 7942 in liposomes was achieved following purification of the polypeptide, that had been expressed in Escherichia coli [see, Fraser PD, Linden H and Sandmann G (1993) Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli. Biochem J 291: 687-692].

Referring now to Figure 1, carotenoids are synthesized from isoprenoid precursors. The central pathway of isoprenoid biosynthesis may be viewed as beginning with the conversion of acetyl-CoA to mevalonic acid. D 3 -isopentenyl pyrophosphate (IPP), a C5 molecule, is formed from mevalonate and is the building block for all long-chain isoprenoids. Following isomerization of IPP to dimethylallyl pyrophosphate (DMAPP), three additional molecules of IPP are WO 98/18910 PCT/US97/17819 8 combined to yield the C20 molecule, geranylgeranyl pyrophosphate (GGPP).

These 1'-4 condensation reactions are catalyzed by prenyl transferases [see, Kleinig H (1989) The role of plastids in isoprenoid biosynthesis. Ann Rev Plant Physiol Plant Mol Biol 40: 39-59]. There is evidence in plants that the same enzyme, GGPP synthase, carries out all the reactions from DMAPP to GGPP [see, Dogbo O and Camara B (1987) Purification of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthase from Capsicum chromoplasts by affinity chromatography. Biochim Biophys Acta 920: 140-148; and, Laferriere A and Beyer P (1991) Purification of geranylgeranyl diphosphate to synthase from Sinapis alba etioplasts. Biochim Biophys Acta 216: 156-163].

The first step that is specific for carotenoid biosynthesis is the head-to-head condensation of two molecules of GGPP to produce prephytoene pyrophosphate (PPPP). Following removal of the pyrophosphate, GGPP is converted to phytoene, a colorless C40 hydrocarbon molecule. 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 [see, Chamovitz D, Misawa N, Sandmann G and Hirschberg J (1992) Molecular cloning and expression in Escherichia coli of a cyanobacterial gene coding for phytoene synthase, a carotenoid biosynthesis enzyme. FEBS Lett 296: 305-310; Ray JA, Bird CR, Maunders M, Grierson D and Schuch W (1987) Sequence of pTOM5, a ripening related cDNA from tomato. Nucl Acids Res 15: 10587-10588; Camara B (1993) Plant phytoene synthase complex component 3 enzymes, immunology, and biogenesis. Meth Enzymol 214: 352-365]. All the subsequent steps in the pathway occur in membranes. Four desaturation (dehydrogenation) reactions convert phytoene to lycopene via phytofluene, -carotene, and neurosporene. Each desaturation increases the number of conjugated double bonds by two such that the number of conjugated double bonds increases from three in phytoene to eleven in lycopene.

Relatively little is known about the molecular mechanism of the enzymatic dehydrogenation of phytoene [see, Jones BL and Porter JW (1986) Biosynthesis of carotenes in higher plants. CRC Crit Rev Plant Sci 3: 295-324; and, Beyer P, Mayer M and Kleinig H (1989) Molecular oxygen and the state of geometric iosomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts-Eur J Biochem 184: 141-150]. It has been established that in cyanobacteria, algae and plants the first two desaturations, from 15-cis-phytoene to c-carotene, are catalyzed by a single membrane-bound enzyme, phytoene desaturase [see, Jones BL and Porter JW (1986) Biosynthesis of carotenes in higher plants. CRC Crit Rev Plant Sci 3: 295- WO 98/18910 PCTIUS97/17819 9 324; and, Beyer P, Mayer M and Kleinig H (1989) Molecular oxygen and the state of geometric iosomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts. Eur J Biochem 184: 141-150].

Since the (-carotene product is mostly in the all-trans configuration, a cis-trans isomerization is presumed at this desaturation step. The primary structure of the phytoene desaturase polypeptide in cyanobacteria is conserved (over 65% identical residues) with that of algae and plants [see, Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing the conversion of phytoene to (-carotene is transcriptionally regulated during tomato lo fruit 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: the phytoene desaturase gene in tomato. In: Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18.

Kluwer, Dordrectht]. Moreover, the same inhibitors block phytoene desaturase in the two systems [see, Sandmann G and Boger P (1989) Inhibition of carotenoid biosynthesis by herbicides. In: Boger P and Sandmann G (eds) Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton, Florida]. Consequently, it is very likely that the enzymes catalyzing the desaturation of phytoene and phytofluene in cyanobacteria and plants have similar biochemical and molecular properties, that are distinct from those of phytoene desaturases in other microorganisms. One such a difference is that phytoene desaturases from Rhodobacter capsulatus, Erwinia sp. or fungi convert phytoene to neurosporene, lycopene, or 3,4-dehydrolycopene, respectively.

Desaturation of phytoene in daffodil chromoplasts [see, Beyer P, Mayer M and Kleinig H (1989) Molecular oxygen and the state of geometric iosomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts. Eur J Biochem 184: 141-150], as well as in a cell free system of Synechococcus sp. strain PCC 7942 [see, Sandmann G and Kowalczyk S (1989) In vitro carotenogenesis and characterization of the phytoene desaturase reaction in Anacystis. Biochem Biophys Res Cor 163: 916-921], is dependent on molecular oxygen as a possible final electron acceptor, although oxygen is not directly involved in this reaction. A mechanism of dehydrogenase-electron transferase was supported in cyanobacteria over dehydrogenation mechanism of dehydrogenase-monooxygenase [see, Sandmann G and Kowalczyk S (1989) In vitro carotenogenesis and characterization of the phytoene desaturase reaction in Anacystis. Biochem Biophys Res Com 163: 916-921]. A conserved FAD-binding motif exists in all phytoene desaturases whose primary structures have been analyzed [see, Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J WO 98/18910 PCT/US97/17819 (1992) A single polypeptide catalyzing the conversion ofphytoene to p-carotene is transcriptionally regulated during tomato fruit 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: the phytoene desaturase gene in tomato. In: Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer, Dordrectht]. The phytoene desaturase enzyme in pepper was shown to contain a protein-bound FAD [see, Hugueney P.

Romer S, Kuntz M and Camara B (1992) Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and (-carotene in o1 Capsicum chromoplasts. Eur J Biochem 209: 399-407]. Since phytoene desaturase is located in the membrane, an additional, soluble redox component is predicted.

This hypothetical component could employ NAD(P)+, as suggested [see, Mayer MP, Nievelstein V and Beyer P (1992) Purification and characterization of a NADPH dependent oxidoreductase from chromoplasts of Narcissus pseudonarcissus a redox-mediator possibly involved in carotene desaturation.

Plant Physiol Biochem 30: 389-398] or another electron and hydrogen carrier, such as a quinone. The cellular location of phytoene desaturase in Synechocystis sp.

strain PCC 6714 and Anabaena variabilis strain ATCC 29413 was determined with specific antibodies to be mainly in the photosynthetic thylakoid membranes [see, Serrano A, Gimenez P, Schmidt A and Sandmann G (1990) Immunocytochemical localization and functional determination of phytoene desaturase in photoautotrophic prokaryotes. J Gen Microbiol 136: 2465-2469].

In cyanobacteria algae and plants c-carotene is converted to lycopene via neurosporene. Very little is known about the enzymatic mechanism, which is predicted to be carried out by a single enzyme [see, Linden H, Vioque A and Sandmann G (1993) Isolation of a carotenoid biosynthesis gene coding for tcarotene desaturase from Anabaena PCC 7120 by heterologous complementation.

FEMS Microbiol Lett 106: 99-104]. The deduced amino acid sequence of Ccarotene desaturase in Anabaena sp. strain- PCC 7120 contains a dinucleotidebinding motif that is similar to the one found in phytoene desaturase.

Two cyclization reactions convert lycopene to p-carotene. Evidence has been obtained that in Synechococcus sp. strain PCC 7942 [see, Cunningham FX Jr, Chamovitz D, Misawa N, Gantt E and Hirschberg J (1993) Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of p-carotene. FEBS Lett 328: 130-138], as well as in plants [see, Camara B and Dogbo O (1986) Demonstration and solubilization of lycopene cyclase from Capsicum chromoplast membranes. Plant Physiol 80: 172-184], these two cyclizations are catalyzed by a single enzyme.

WO 98/18910 PCT/US97/17819 11 lycopene cyclase. This membrane-bound enzyme is inhibited by the triethylamine compounds, CPTA and MPTA [see, Sandmann G and Boger P (1989) Inhibition of carotenoid biosynthesis by herbicides. In: Boger P and Sandmann G (eds) Target Sites of Herbicide Action, pp 25-44. CRC Press, Boca Raton, Florida].

Cyanobacteria carry out only the P-cyclization and therefore do not contain ecarotene, 6-carotene and a-carotene and their oxygenated derivatives. The p-ring is formed through the formation of a "carbonium ion" intermediate when the C-1,2 double bond at the end of the linear lycopene molecule is folded into the position of the C-5,6 double bond, followed by a loss of a proton from C-6. No cyclic carotene has been reported in which the 7,8 bond is not a double bond. Therefore, full desaturation as in lycopene, or desaturation of at least half-molecule as in neurosporene, is essential for the reaction. Cyclization of lycopene involves a dehydrogenation reaction that does not require oxygen. The cofactor for this reaction is unknown. A dinucleotide-binding domain was found in the lycopene cyclase polypeptide ofSynechococcus sp. strain PCC 7942, implicating NAD(P) or FAD as coenzymes with lycopene cyclase.

The addition of various oxygen-containing side groups, such as hydroxy-, methoxy-, oxo-, epoxy-, aldehyde or carboxylic acid moieties, form the various xanthophyll species. Little is known about the formation of xanthophylls.

Hydroxylation of p-carotene requires molecular oxygen in a mixed-function oxidase reaction.

Clusters of genes encoding the enzymes for the entire pathway have been cloned from the purple photosynthetic bacterium Rhodobacter capsulatus [see, Armstrong GA, Alberti M, Leach F and Hearst JE (1989) Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus. Mol Gen Genet 216: 254-268] and from the nonphotosynthetic bacteria Erwinia herbicola [see, Sandmann G, Woods WS and Tuveson RW (1990) Identification of carotenoids in Erwinia herbicola and in transformed Escherichia coli strain. FEMS Microbiol Lett 71: 77-82; Hundle BS, 3o Beyer P, Kleinig H, Englert H and Hearst JE (1991) Carotenoids of Erwinia herbicola and an Escherichia coli HB101 strain carrying the Erwinia herbicola carotenoid gene cluster. Photochem Photobiol 54: 89-93; and, Schnurr G, Schmidt A and Sandmann G (1991) Mapping of a carotenogenic gene cluster from Erwinia herbicola and functional identification of six genes. FEMS Microbiol Lett 78: 157- 162] and Erwinia uredovora [see, Misawa N, Nakagawa M, Kobayashi K, Yamano S, Izawa I, Nakamura K and Harashima K (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products in Escherichia coli. J Bacteriol 172: 6704-6712]. Two genes, al-3 WO 98/18910 PCT/US97/17819 12 for GGPP synthase [see,-Nelson MA, Morelli G, Carattoli A, Romano N and Macino G (1989) Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes. Mol Cell Biol 9: 1271-1276; and, Carattoli A, Romano N, Ballario P, Morelli G and Macino G (1991) The Neurospora crassa carotenoid biosynthetic gene (albino J Biol Chem 266: 5854-5859] and al-1 for phytoene desaturase [see, Schmidhauser TJ, Lauter FR, Russo VEA and Yanofsky C (1990) Cloning sequencing and photoregulation of al-1, a carotenoid biosynthetic gene of Neurospora crassa. Mol Cell Biol 10: 5064-5070] have been cloned from the o0 fungus Neurospora crassa. However, attempts at using these genes as heterologous molecular probes to clone the corresponding genes from cyanobacteria or plants were unsuccessful due to lack of sufficient sequence similarity.

The first "plant-type" genes for carotenoid synthesis enzyme were cloned 1i from cyanobacteria using a molecular-genetics approach. In the first step towards cloning the gene for phytoene desaturase, a number of mutants that are resistant to the phytoene-desaturase-specific inhibitor, norflurazon, were isolated in Synechococcus sp. strain PCC 7942 [see, Linden H, Sandmann G, Chamovitz D, Hirschberg J and Boger P (1990) Biochemical characterization of Synechococcus mutants selected against the bleaching herbicide norflurazon. Pestic Biochem Physiol 36: 46-51]. The gene conferring norflurazon-resistance was then cloned by transforming the wild-type strain to herbicide resistance [see, Chamovitz D, Pecker I and Hirschberg J (1991) The molecular basis of resistance to the herbicide norflurazon. Plant Mol Biol 16: 967-974; Chamovitz D, Pecker 1, Sandmann G, Boger P and Hirschberg J (1990) Cloning a gene for norflurazon resistance in cyanobacteria. Z Naturforsch 45c: 482-486]. Several lines of evidence indicated that the cloned gene, formerly called pds and now named crtP, codes for phytoene desaturase. The most definitive one was the functional expression of phytoene desaturase activity in transformed Escherichia coli cells [see, Linden H, Misawa N, Chamovitz D, Pecker I, Hirschberg J and Sandmann G (1991) Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes. Z Naturforsch 46c: 1045-1051; and, Pecker 1, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing the conversion of phytoene to c-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966]. The crtP gene was also cloned from Synechocystis sp. strain PCC 6803 by similar methods [see, Martinez-Ferez IM and Vioque A (1992) Nucleotide sequence of the phytoene desaturase gene from Synechocystis sp. PCC WO 98/18910 PCT/US97/17819 13 6803 and characterization of a new mutation which confers resistance to the herbicide norflurazon. Plant Mol Biol 18: 981-983].

The cyanobacterial crtP gene was subsequently used as a molecular probe for cloning the homologous gene from an alga [see, Pecker I, Chamovitz D, Mann V. Sandmann G, Boger P and Hirschberg J (1993) Molecular characterization of carotenoid biosynthesis in plants: the phytoene desaturase gene in tomato. In: Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18. Kluwer, Dordrectht] and higher plants [see, Bartley GE, Viitanen PV, Pecker I, Chamovitz D, Hirschberg J and Scolnik PA (1991) Molecular cloning and expression in o0 photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536; and, Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing the conversion of phytoene to c-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966]. The phytoene desaturases in Synechococcus sp. strain PCC 7942 and Synechocystis sp. strain PCC 6803 consist of 474 and 467 amino acid residues, respectively, whose sequences are highly conserved (74% identities and 86% similarities). The calculated molecular mass is 51 kDa and, although it is slightly hydrophobic (hydropathy index it does not include a hydrophobic region which is long enough to span a lipid bilayer membrane. The primary structure of the cyanobacterial phytoene desaturase is highly conserved with the enzyme from the green alga Dunalliela bardawil (61% identical and 81% similar; [see, Pecker I,_ Chamovitz D, Mann V, Sandmann G, Boger P and Hirschberg J (1993) Molecular characterization of carotenoid biosynthesis in plants: the phytoene desaturase gene in tomato. In: Murata N (ed) Research in Photosynthesis, Vol III, pp 11-18.

Kluwer, Dordrectht]) and from tomato [see, Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing the conversion of phytoene to -carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966], pepper [see, Hugueney P, Romer S, Kuntz M and Camara B (1992) Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and (-carotene in Capsicum chromoplasts. Eur J Biochem 209: 399-407] and soybean [see, Bartley GE, Viitanen PV, Pecker I, Chamovitz D, Hirschberg J and Scolnik PA (1991) Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536] (62-65% identical and -79% similar; [see, Chamovitz D (1993) Molecular analysis of the early steps of carotenoid biosynthesis in cyanobacteria: Phytoene synthase and phytoene WO 98/18910 PCTIUS97/17819 14 desaturase. Ph.D. Thesis, The Hebrew University of Jerusalem]). The eukaryotic phytoene desaturase polypeptides are larger (64 kDa); however, they are processed during import into the plastids to mature forms whose sizes are comparable to those of the cyanobacterial enzymes.

There is a high degree of structural similarity in carotenoid enzymes of Rhodobacter capsulatus, Erwinia sp. and Neurospora crassa [reviewed in Armstrong GA, Hundle BS and Hearst JE (1 9 9 3 )-Evolutionary conservation and structural similarities of carotenoid biosynthesis gene products from photosynthetic and nonphotosynthetic organisms. Meth Enzymol 214: 297-311], to including in the crtI gene-product, phytoene desaturase. As indicated above, a high degree of conservation of the primary structure of phytoene desaturases also exists among oxygenic photosynthetic organisms. However, there is little sequence similarity, except for the FAD binding sequences at the amino termini, between the "plant-type" crtP gene products and the "bacterial-type" phytoene desaturases (crtI gene products; 19-23% identities and 42-47% similarities). It has been hypothesized that crtP and crtI are not derived from the same ancestral gene and that they originated independently through convergent evolution [see, Pecker I, Chamovitz D, Linden H, Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing the conversion of phytoene to (-carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966]. This hypothesis is supported by the different dehydrogenation sequences that are catalyzed by the two types of enzymes and by their different sensitivities to inhibitors.

Although not as definite as in the case of phytoene desaturase, a similar distinction between cyanobacteria and plants on the one hand and other microorganisms is also seen in the structure of phytoene synthase. The crtB gene (formerly psy) encoding phytoene synthase was identified in the genome of Synechococcus sp. strain PCC 7942 adjacent to crtP and within the same operon [see, Bartley GE, Viitanen PV, Pecker I, Chamovitz D, Hirschberg J and Scolnik PA (1991) Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536]. This gene encodes a 36-kDa polypeptide of 307 amino acids with a hydrophobic index of 0.4. The deduced amino acid sequence of the cyanobacterial phytoene synthase is highly conserved with the tomato phytoene synthase (57% identical and similar; Ray JA, Bird CR, Maunders M, Grierson D and Schuch W (1987) Sequence of pTOM5, a ripening related cDNA from tomato. Nucl Acids Res 10587-10588]) but is less highly conserved with the crtB sequences from other WO 98/18910 PCT/US97/17819 bacteria (29-32% identical and 48-50% similar with ten gaps in the alignment).

Both types of enzymes contain two conserved sequence motifs also found in prenyl transferases from diverse organisms [see, Bartley GE, Viitanen PV, Pecker 1. Chamovitz D, Hirschberg J and Scolnik PA (1991) Molecular cloning and expression in photosynthetic bacteria of a soybean cDNA coding for phytoene desaturase, an enzyme of the carotenoid biosynthesis pathway. Proc Natl Acad Sci USA 88: 6532-6536; Carattoli A, Romano N, Ballario P, Morelli G and Macino G (1991) The Neurospora crassa carotenoid biosynthetic gene (albino J Biol Chem 266: 5854-5859; Armstrong GA, Hundle BS and Hearst JE (1993) Evolutionary conservation and structural similarities of carotenoid biosynthesis gene products from photosynthetic and nonphotosynthetic organisms. Meth Enzymol 214: 297-311; Math SK, Hearst JE and Poulter CD (1992) The crtE gene in Erwinia herbicola encodes geranylgeranyl diphosphate synthase. Proc Natl Acad Sci USA 89: 6761-6764; and, Chamovitz D (1993) Molecular analysis of the early steps of carotenoid biosynthesis in cyanobacteria: Phytoene synthase and phytoene desaturase. Ph.D. Thesis, The Hebrew University of Jerusalem]. It is conceivable that these regions in the polypeptide are involved in the binding and/or removal of the pyrophosphate during the condensation of two GGPP molecules.

The crtQ gene encoding c-carotene desaturase (formerly zds) was cloned from Anabaena sp. strain PCC 7120 by screening an expression library of cyanobacterial genomic DNA in cells of Escherichia coli carrying the Erwinia sp.

crtB and crtE genes and the cyanobacterial crtP gene [see, Linden H, Vioque A and Sandmann G (1993) Isolation of a carotenoid biosynthesis gene coding for carotene desaturase from Anabaena PCC 7120 by heterologous complementation.

FEMS Microbiol Lett 106: 99-104]. Since these Escherichia coli cells produce Ccarotene, brownish-red pigmented colonies that produced lycopene could be identified on the yellowish background of cells producing c-carotene. The predicted (-carotene desaturase from Anabaena sp. strain PCC 7120 is a 56-kDa polypeptide which consists of 499 amino acid residues. Surprisingly, its primary structure is not conserved with the "plant-type" (crtP gene product) phytoene desaturases, but it has considerable sequence similarity to the bacterial-type enzyme (crtI gene product) [see, Sandmann G (1993) Genes and enzymes involved in the desaturation reactions from phytoene to lycopene. (abstract), International Symposium on Carotenoids, Trondheim CL1-2]. It is possible that the cyanobacterial crtQ gene and crtI gene of other microorganisms originated in evolution from a common ancestor.

The crtL gene for lycopene cyclase (formerly Icy) was cloned from Synechococcus sp. strain PCC 7942 utilizing essentially the same cloning strategy WO 98/18910 PCT/US97/17819 16 as for crtP. By using an inhibitor of lycopene cyclase, 2 4 -methylphenoxy)triethylamine hydrochloride (MPTA), the gene was isolated by transformation of the wild-type to herbicide-resistance [see, Cunningham FX Jr, Chamovitz D, Misawa N, Gantt E and Hirschberg J (1993) Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of P-carotene. FEBS Lett 328: 130-138]. Lycopene cyclase is the product of a single gene product and catalyzes the double cyclization reaction of lycopene to p-carotene. The crtL gene product in Synechococcus sp.

strain PCC 7942 is a 46-kDa polypeptide of 411 amino acid residues. It has no o0 sequence similarity to the crtY gene product (lycopene cyclase) from Erwinia uredovora or Erwinia herbicola.

The gene for P-carotene hydroxylase (crtZ) and zeaxanthin glycosilase (crtX) have been cloned from Erwinia herbicola [see, Hundle B, Alberti M, Nievelstein V, Beyer P, Kleinig H, Armstrong GA, Burke DH and Hearst JE (1994) Functional assignment of Erwinia herbicola EholO carotenoid genes expressed in Escherichia coli. 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 a proposed diphosphate binding site. Proc Natl Acad Sci USA 89: 9321-9325] and from Erwinia uredovora [see, Misawa N, Nakagawa M, Kobayashi K, Yamano S, Izawa I, Nakamura K and Harashima K (1990) Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products in Escherichia coli. J Bacteriol 172: 6704-6712].

The ketocarotenoid astaxanthin (3,3'-dihydroxy-3,P-carotene-4,4'-dione) was first described in aquatic crustaceans as an oxidized form of P-carotene.

Astaxanthin was later found to be very common in many marine animals and algae. However, only few animals can synthesize astaxanthin de novo from other carotenoids and most of them obtain it in their food. In the plant kingdom, astaxanthin occurs mainly in some species of cyanobacteria, algae and lichens.

However, it is found rarely also in petals of higher plant species [see, Goodwin TW (1980) The Biochemistry of the carotenoids, Vol. 1. 2nd Ed, Chapman and Hall, London and New York].

The function of astaxanthin as a powerful antioxidant in animals has been demonstrated [see, Miki W (1991) Biological functions and activities of animal carotenoids. Pure Appl Chem 63: 141]. Astaxanthin is a strong inhibitor of lipid peroxidation and has been shown to play an active role in the protection of biological membranes from oxidative injury [see, Palozza P and Krinsky NI (1992) Antioxidant effects of carotenoids in vivo and in vitro an overview. Methods WO 98/18910 PCT/US97/17819 17 Enzymol 213: 403-420; and, Kurashige M, Okimasu E, Inove M and Utsumi K (1990) Inhibition of oxidative injury of biological membranes by astaxanthin.

Physiol Chem Phys Med NMR 22: 27]. The chemopreventive effects of astaxanthin have also been investigated in which astaxanthin was shown to significantly reduce the incidence of induced urinary bladder cancer in mice [see, Tanaka T, Morishita Y, Suzui M, Kojima T, Okumura A. and Mori H (1994).

Chemoprevention of mouse urinary bladder carcinogenesis by the naturally occurring carotenoid astaxanthin. Carcinogenesis 15: 15]. It has also been demonstrated that astaxanthin exerts immunomodulating effects by enhancing to antibody production [see, Jyonouchi H, Zhang L and Tomita Y (1993) Studies of immunomodulating actions of carotenoids. II. Astaxanthin enhances in vitro antibody production to T-dependent antigens without facilitating polyclonal B-cell activation. Nutr Cancer 19: 269; and, Jyonouchi H, Hill JR, Yoshifumi T and Good RA (1991) Studies of immunomodulating actions of carotenoids. I. Effects of f3-carotene and astaxanthin on murine lymphocyte functions and cell surface marker expression in-vitro culture system. Nutr Cancer 16: 93]. The complete biomedical properties of astaxanthin remain to be elucidated, but initial results suggest that it could play an important role in cancer and tumor prevention, as well as eliciting a positive response from the immune system.

Astaxanthin is the principal carotenoid pigment of salmonids and shrimps and imparts attractive pigmentation in the eggs, flesh and skin [see, Torrisen OJ, Hardy RW, Shearer KD (1989) Pigmentation of salmonid-carotenoid deposition and metabolism in salmonids. Crit Rev Aquatic Sci 1: 209]. The world-wide harvest of salmon in 1991 was approximately 720,000 MT.. of which 25-30% were produced in a variety of aquaculture facilities [see, Meyers SP (1994) Developments in world aquaculture, feed formulations, and role of carotenoids.

Pure Appl Chem 66: 1069]. This is set to increase up to 460,000 MT. by the year 2000 [see, Bjorndahl T (1990) The Economics of Salmon Aquaculture. Blackwell Scientific, Oxford. pp. The red coloration of the salmonid flesh contributes to consumer appeal and therefore affects the price of the final product. Animals cannot synthesize carotenoids and they acquire the pigments through the food chain from the primary producers marine algae and phytoplankton. Those grown in intensive culture usually suffer from suboptimal color. Consequently, carotenoid-containing nourishment is artificially added in aquaculture, at considerable cost to the producer.

Astaxanthin is the most expensive commercially used carotenoid compound (todays-1995 market value is of 2,500-3,500 It is utilized mainly as nutritional supplement which provides pigmentation in a wide variety of aquatic WO 98/18910 PCT/US97/17819 18 animals. In the Far-East it is used also for feeding poultry to yield a typical pigmentation of chickens. It is also a desirable and effective nontoxic coloring for the food industry and is valuable in cosmetics. Recently it was reported that astaxanthin is a potent antioxidant in humans and thus is a desirable food additive.

Natural (3S,3'S) astaxanthin is limited in availability. It is commercially extracted from some crustacea species [see, Torrisen OJ, Hardy RW, Shearer KD (1989) Pigmentation of salmonid-carotenoid deposition and metabolism in salmonids. Crit Rev Aquatic Sci 1: 209]. The (3R,3'R) stereoisomer of astaxanthin is produced from Phaffia [a yeast specie, see, Andrewes AG, Phaff HJ and Starr MP (1976) Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochemistry Vol. 15, pp. 1003-1007]. Synthetic astaxanthin, comprising a 1:2:1 mixture of the and (3R,3'R)-isomers is now manufactured by Hoffman-La Roche and sold at a high price (ca. $2,500/Kg) under the name "CAROPHYLL Pink" [see, Mayer H (1994) Reflections on carotenoid synthesis. Pure Appl Chem, Vol. 66, pp. 931-938]. Recently a novel gene involved in ketocompound biosynthesis, designated crtW was isolated ifom the marine bacteria Agrobacterium auranticacum and Alcaligenes PC-1 that produce ketocarotenoids such as astaxanthin. When the crtW gene was introduced into engineered Eschrichia coli that accumulated 3-carotene due to Erwinia carotenogenic genes, the Escherichia coli transformants synthesized canthaxanthin a precursor in the synthetic pathway of astaxanthin [see, Misawa N, Kajiwara S, Kondo K, Yokoyama A, Satomi Y, Saito T, Miki W and Ohtani T (1995) Canthaxanthin biosynthesis by the conversion of methylene to keto groups in a hydrocarbon -carotene by a single gene. Biochemical and biophysical research communications Vol. 209, pp. 867-876]. It is therefore desirable to find a relatively inexpensive source of (3S,3'S) astaxanthin to be used as a feed supplement in aquaculture and as a valuable chemical for various other industrial uses.

Although astaxanthin is synthesized in a variety of bacteria, fungi and algae, the key limitation to the use of biological systems for its production is the low yield of and costly extraction methods in these systems compared to chemical synthesis. One way to solve these problems is to increase the productivity of astaxanthin production in biological systems using recombinant DNA technology.

This allows for the production of astaxanthin in genetically engineered host which.

in the case of a higher plant, is easy to grow and simple to extract. Furthermore, production of astaxanthin in genetically engineered host enables by appropriate host selection to use thus produced astaxanthin in for example aquaculture applications, devoid of the need for extraction.

WO 98/18910 PCTIUS97/17819 19 There is thus a widely recognized need for, and it would be highly advantageous to have, a nucleic acid segment which encodes P-C- 4 -oxygenase, the enzyme that converts 3-carotene to canthaxanthin, as well as recombinant vector molecules comprising a nucleic acid sequence according to the invention, and host cells or transgenic organisms transformed or transfected with these vector molecules or DNA segment for the biotechnological production of (3S,3'S) astaxanthin.

Other features and advantages of the invention will be apparent from the following description and from the claims.

SUMMARYDE.THEI-INENTION

It is a general object of this invention to provide a biotechnological method for production of (3S,3'S) astaxanthin.

It is a specific object of the invention to provide a peptide having a P-C-4oxygenase activity and a DNA segment coding for this peptide to enable a biotechnological production of astaxanthin and other xanthophylls.

It is a further object of the invention to provide an RNA segments coding for a polypeptide comprising an amino acid sequence corresponding to above described peptide.

It is yet a further object of the invention to provide a recombinant DNA molecule comprising a vector and the DNA segment as described above.

It is still a further object of the invention to provide a host cell containing the above described recombinant DNA molecule.

It is another object of the invention to provide a host transgenic organism containing the above described recombinant DNA molecule or the above described DNA segment in its cells.

It is still another object of the invention to provide a host transgenic organism which expresses P-C-4-oxygenase activity in chloroplasts and/or chromoplasts-containing tissues.

It is yet another object of the invention to provide a food additive for animal or human consumption comprising the above described host cell or transgenic organism.

It is still another object of the invention to provide a method of producing.

astaxanthin using the above described host cell-or transgenic organism.

It is a further object of the invention to provide a method of producing canthaxanthin, echinenone, cryptoxanthin, isocryptoxanthin hydroxyechinenone, zeaxanthin, adonirubin, and/or adonixanthin using the above described host cell or transgenic organism.

WO 98/18910 PCT/US97/17819 Further objects and advantages of the present invention will be clear from the description that follows.

In one embodiment, the present invention relates to a DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to Haematococcus pluvialis crtO gene.

In a further embodiment, the present invention relates to an RNA segment coding for a polypeptide comprising an amino acid sequence corresponding to Haematococcus pluvialis crtO gene.

In yet another embodiment, the present invention relates to a polypeptide comprising an amino acid sequence corresponding to a Haematococcus pluvialis crtO gene.

In a further embodiment, the present invention relates to a recombinant DNA molecule comprising a vector and a DNA segment coding for a polypeptide, corresponding to a Haematococcus pluvialis crtO gene.

In another embodiment, the present invention relates to a host cell containing the above described recombinant DNA molecule or DNA segment.

In a further embodiment, the present invention relates to a host transgenic organism containing the above described recombinant DNA molecule or the above described DNA segment in its cells.

In another embodiment, the present invention relates to a method of producing astaxanthin using the above described host cell or transgenic organism.

In yet another embodiment, the present invention relates to a method of producing other xanthophylls.

In still another embodiment, the present invention relates to a method of obtaining high expression of a transgene in plants specifically in chromoplastscontaining cells.

In one further embodiment, the present invention relates to a method of importing a carotenoid-biosynthesis enzyme encoded by a transgene into chromoplasts.

BIUIEFDESCRIPTION OF THE DRAWNiGS The invention herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1 is a general biochemical pathway of p-carotene biosynthesis, in which pathway all molecules are depicted in an all-trans configuration, wherein 1PP is isopentenyl pyrophosphate, DMAPP is dimethylallyl pyrophosphate, GPP is geranyl pyrophosphate, FPP is farnesyl pyrophosphate, GGPP is geranylgeranyl pyrophosphate and, PPPP is prephytoene pyrophosphate; WO 98/18910 PCT/US97/17819 21 FIG. 2 is an identity map between the nucleotide sequence of the crtO cDNA of the present invention (CRTOA.SEQ) and the cDNA cloned by Kajiwara et al., (CRTOJ.SEQ) [see, 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], using a GCG software, wherein indicate identity, indicate a gap and nucleotides numbering is according to SEQ ID NO:4 for CRTOA.AMI and Kajiwara et al., for

CRTOJ.AMI;

FIG. 3 is an identity map between the amino acid sequence encoded by the crtO cDNA of the present invention (CRTOA.AMI) and the amino acid sequence encoded by the cDNA cloned by Kajiwara et al., (CRTOJ.AMI) [see, 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], using a GCG software, wherein indicate identity, indicate a gap and amino acids numbering is according to SEQ ID NO:4 for CRTOA.AMI and Kajiwara et al., for CRTOJ.AMI; FIG. 4 is a schematic depiction of a pACYC 184 derived plasmid designated pBCAR and includes the genes crtE, crtB, crtI and crtY of Erwinia herbicola, which genes are required for production of P-carotene in Escherichia coli cells; FIG. 5 is a schematic depiction of a pACYC 184 derived plasmid designated pZEAX and includes the genes crtE, crtB, crtl, crtY and crtZ from Erwinia herbicola, which genes are required for production of zeaxanthin in Escherichia coli cells; FIG. 6 is a schematic depiction of a pBluescriptSK- derived plasmid designated pHPK, containing a full length cDNA insert encoding a p-carotene C- 4-oxygenase enzyme from Haematococcus pluvialis, designated crtO and set forth in SEQ ID NO:1, which cDNA was identified by color complementation of Escherichia coli cells; FIG. 7 is a schematic depiction of a pACYC 184 derived plasmid designated pCANTHA which was derived by inserting a 1.2 kb PstI-PstI DNA fragment, containing the cDNA encoding the P-C-4-oxygenase from Haematococcus pluvialis isolated from the plasmid pHPK of Figure 6 and inserted into a PstI site in the coding sequence of the crtZ gene in the plasmid pZEAX of Figure 5; this recombinant plasmid carries the genes crtE, crtB, crtl, crtY-of Erwinia herbicola and the crtO gene of Haematococcus pluvialis, all required for production of canthaxanthin in Escherichia coli cells; WO 98/18910 PCT/US97/17819 22 FIG. 8 is a schematic depiction of a pACYC 184 derived plasmid designated pASTA which was derived by inserting the 1.2 kb PstI-PstI DNA fragment, containing the cDNA of the P-C-4-oxygenase from Haematococcus pluvialis isolated from the plasmid pHPK of Figure 6 and inserted into a PstI site which exists 600 bp downstream of the crtE gene in the plasmid pZEAX of Figure 5; this recombinant plasmid carries the genes crtE, crtB, crtl, crtY, crtZ of Erwinia herbicola and the crtO gene of Haematococcus pluvialis, all required for production of astaxanthin in Escherichia coli cells; FIG. 9 is a schematic depiction of a pBR328 derived plasmid designated to PAN3.5-KETO which was derived by inserting the 1.2 kb PstI-PstI DNA fragment, containing the cDNA of the P-C-4-oxygenase from Haematococcus pluvialis isolated from the plasmid pHPK of Figure 6 and inserted into a PstI site which exists in a P-lactamase gene in a plasmid designated pPAN35D5 [described in Hirschberg J, Ohad N, Pecker I and Rahat A (1987) Isolation and characterization of herbicide resistant mutants in the cyanobacterium Synechococcus R2. Z. Naturforsch 42c: 102-112], which carries the psbAI gene from the cyanobacterium Synechococcus PCC7942 in the plasmid vector pBR328 [see, Hirschberg J, Ohad N, Pecker I and Rahat A (1987) Isolation and characterization of herbicide resistant mutants in the cyanobacterium Synechococcus R2. Z. Naturforsch 42c: 102-112]; this recombinant plasmid carries the crtO gene of Haematococcus pluvialis, required for production of astaxanthin in Synechococcus PCC7942 cells; FIG. 10 is a schematic depiction of the T-DNA region of a Ti binary plasmid coli, Agrobacterium) designated pBIB [described by Becker D (1990) Binary vectors which allow the exchange of plant selectable markers and reporter genes. Nucleic Acids Research 18:230] which is a derivative of the Ti plasmid pBI101 [described by Jeffesrson AR, Kavanagh TA and Bevan WM (1987) GUS fusions: p-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO J. 6: 3901-3907], wherein BR and BL are the right and left borders, respectively, of the T-DNA region, pAg7 is the polyadenylation site of gene 7 ofAgrobacterium Ti-plasmid, pAnos is a 250 bp long DNA fragment containing the poly adenylation site of the nopaline synthase gene of Agrobacterium, NPT II is a 1,800 bp long DNA fragment coding for kanamycin resistance, pnos is a-300 bp long DNA fragment containing the promoter sequence of the nopaline synthase gene of Agrobacterium, whereas pAnos is a 300 bp long DNA fragment containing the poly adenylation site of the nopaline synthase gene of Agrobacterium; WO 98/18910 PCT/US97/17819 23 FIG. 11 is a schematic depiction of the T-DNA region of a Ti binary plasmid coli, Agrobacterium) designated pPTBIB which was prepared by cloning a genomic DNA sequence of a tomato species Lycopersicon esculentum marked PT (nucleotides 1 to 1448 of the Pds gene as published in Mann V, Pecker I and Hirschberg J (1994) cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum).

Plant Molecular Biology 24: 429-434), which contains the promoter of the Pds gene and the coding sequence for the amino terminus region of the polypeptide PDS that serve as a transit peptide for import into chloroplasts and chromoplasts, into a HindIII-SmaI site of the binary plasmid vector pBIB of Figure 10, wherein BR and BL, pAg7, pAnos, NPT II, pnos and pAnos are as defined above; FIG. 12 is a schematic depiction of the T-DNA region of a Ti binary plasmid coli, Agrobacterium) designated pPTCRTOBIB which was prepared by cloning a 1,110 nucleotide long Eco47III-NcoI fragment of the cDNA of crtO from H. pluvialis (nucleotides 211 to 1321 of SEQ ID NO:1) into the SmaI site of the plasmid pPTBIB of Figure 11, such that the coding nucleotide sequence of the amino terminus of PDS is in the same reading frame of crtO, wherein BR and BL, pAg7, pAnos, NPT II, pnos, and pAnos are as defined above, PT is the promoter and transit peptide coding sequences of Pds from tomato and CRTO is the nucleotide sequence of crtO from H. pluvialis (nucleotides 211 to 1321 of SEQ ID NO:1); FIG. 13 shows a Southern DNA blot analysis of HindIII-digested genomic DNA extracted from wild type (WT) and crtO tobacco transgenic plants, designated 2, 3, 4, 6, 9 and 10, according to the present invention, using the crtO cDNA as a radioactive probe essentially as described in Sambrook et al., Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989, wherein the size of marker DNA fragments in kilobase pairs (kb) is indicated on the left as well as the expected position (arrow) of an internal T-DNA HindIII fragment as was deduced from the sequence of pPTPDSBIB shown in Figure 12 which contain the crtO cDNA sequence; FIG. 14 shows a biosynthesis pathway of astaxanthin; FIG. 15 shows a flower from a wild type tobacco plant and a flower from a transgenic tobacco plant according to the present invention.

DESCRIPTION OF THE PREFERRED MBODIMENTS The present invention is, in general, of a biotechnological method for production of (3S,3'S) astaxanthin. In particular, the present invention is of a WO 98/18910 PCT/US97/17819 24 peptide having a P-C-4-oxygenase activity; a DNA segment coding for this peptide; an RNA segments coding for this peptide; a recombinant DNA molecule comprising a vector and the DNA segment; a host cell or organism containing the above described recombinant DNA molecule or DNA segment; and of a method for biotechnologically producing (3S,3'S) astaxanthin or a food additive containing (3S,3'S) astaxanthin, using the host.

The unicellular fresh-water green alga Haematococcus pluvialis accumulates large amounts of (3S,3'S) astaxanthin when exposed to unfavorable growth conditions, or following different environmental stresses such as phosphate to or nitrogen starvation, high concentration of salt in the growth medium or high light intensity [see, Yong YYR and Lee YK (1991) Phycologia 30 257-261; Droop MR (1954) Arch Microbiol 20: 391-397; and, Andrewes A.G, Borch G, Liaaen- Jensen S and Snatzke G.(1974) Acta Chem Scand B28: 730-736]. During this process, the vegetative cells of the alga form cysts and change their color from green to red. The present invention discloses the cloning of a cDNA from Haematococcus pluvialis, designated crtO, which encodes a p-C-4-oxygenase, the enzyme that converts P-carotene to canthaxanthin, and its expression in a heterologous systems expressing p-carotene hydroxylase Erwinia herbicola crtZ gene product), leading to the production of (3S,3'S) astaxanthin.

The crtO cDNA and its encoded peptide having a p-C-4-oxygenase activity are novel nucleic and amino acid sequences, respectively. The cloning method of the crtO cDNA took advantage of a strain of Escherichia coli, which was genetically engineered to produce p-carotene, to which a cDNA library of Haematococcus pluvialis was transfected and expressed. Visual screening for brown-red pigmented Escherichia coli cells has identified a canthaxanthin producing transformant. Thus cloned cDNA has been expressed in two heterologous systems (Escherichia coli and Synechococcus PCC7942 cells) both able to produce p-carotene and further include an engineered (Erwinia herbicola crtZ gene product) or endogenous P-carotene hydroxylase activity, and was shown to enable the production of (3S,3'S) astaxanthin in both these systems.

The crtO cDNA or its protein product exhibit no meaningful nucleic- or amino acid sequence similarities to the nucleic- or amino acid sequence of crtW and its protein product isolated from the marine bacteria Agrobacterium auranticacum and Alcaligenes PC-1 that produce ketocarotenoids such as astaxanthin [see, Misawa N, Kajiwara S, Kondo K, Yokoyama A, Satomi Y, Saito T, Miki W and Ohtani T (1995) Canthaxanthin biosynthesis by the conversion of methylene to keto groups in a hydrocarbon p-carotene by a single gene.

Biochemical and biophysical research communications Vol. 209, pp. 867-876].

WO 98/18910 PCT/US97/17819 However, the crtO cDNA and its protein product exhibit substantial nucleic- and amino acid sequence identities with the nucleic- and amino acid sequence of a recently cloned cDNA encoding a 320 amino acids protein product having p-carotene oxygenase activity, isolated from Haematococcus pluvialis [see, 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]. Nevertheless, as presented in Figure 2 the degree of sequence identity between the crtO cDNA (CRTOA.SEQ in io Figure 2) and the cDNA described by Kajiwara et al. (CRTOJ.SEQ in Figure 2) [see reference above] is 75.7% and, as presented in Figure 3 the degree of sequence identity between the crtO cDNA protein product (CRTOA.AMI in Figure 3) and the protein described by Kajiwara et al. (CRTOJ.AMI in Figure 3) is 78%, as was determined using a GCG software.

As will be described in details hereinbelow, the crtO cDNA can thus be employed to biotechnologically produce (3S,3'S) astaxanthin in systems which are either easy to grow and can be used directly as an additive to fish food, or systems permitting a simple and low cost extraction procedure of astaxanthin.

In one embodiment, the present invention relates to a DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to Haematococcus pluvialis crtO gene and allelic and species variations and functional naturally occurring and/or man-induced variants thereof. The phrase 'allelic and species variations and functional naturally occurring and/or maninduced variants' as used herein and in the claims below refer to the source of the DNA (or RNA as described below) or means known in the art for obtaining it.

However the terms 'variation' and 'variants' indicate the presence of sequence dissimilarities variations). It is the intention herein and in the claims below that the sequence variations will be 77-80%, preferably 80-85%, more preferably 85-90%, most preferably 90-100% of identical nucleotides. In a preferred embodiment the DNA segment comprises the sequence set forth in SEQ ID NO: 1.

In another preferred embodiment, the DNA segment encodes the amino acid sequence set forth in SEQ ID NO:4.

The invention also includes a pure DNA segment characterized as including a sequence which hybridizes under high stringency conditions as described in Sambrook et al., Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989] to a nucleic acid probe which includes at least fifteen, preferably at least fifty, more preferably at least hundred, even more preferably at least two hundred, even more preferably at least five WO 98/18910 PCT/US97/17819 26 hundred successive nucleotides of SEQ ID NO:1 or SEQ ID NO:2. Alternatively.

the DNA segment of the invention may be characterized as being capable of hybridizing under low-stringent conditions to a nucleic acid probe which includes the coding sequence (nucleotides 166 through 1152) of SEQ ID NO:1 or SEQ ID NO:2. An example of such low-stringency conditions is as described in Sambrook et al., using a lower hybridization temperature, such as, for example, 20 0 C below the temperature employed for high-stringency hybridization conditions, as described above.

The DNA segment of the invention may also be characterized as being to capable of hybridizing under high-stringent conditions to a nucleic acid probe which includes the coding sequence (nucleotides 166 through 1152) of SEQ ID NO:1 or SEQ ID NO:2.

The invention also includes a synthetically produced oligonucleotide oligodeoxyribonucleotide or oligoribonucleotide and analogs thereof) capable of hybridizing with at least ten-nucleotide segments of SEQ ID NO:1 or SEQ ID NO:2.

In another embodiment, the present invention relates to an RNA segment coding for a polypeptide comprising an amino acid sequence corresponding to Haematococcus pluvialis crtO gene and allelic and species variations and functional naturally occurring and/or man-induced variants thereof. 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 invention also includes a pure RNA characterized as including a sequence which hybridizes under high stringent conditions to a nucleic acid probe which includes at least at least fifteen, preferably at least fifty, more preferably at least hundred, even more preferably at least two hundred, even more preferably at least five hundred succsesive nucleotides of SEQ ID NO:1 or SEQ ID NO:2.

Alternatively, the RNA of the invention may be characterized as being capable of hybridizing under low-stringent conditions to a nucleic acid probe which includes the coding sequence (nucleotides 166 through 1152) of SEQ ID NO: or SEQ ID NO:2. Additionally, the RNA of the invention may be characterized as being capable of hybridizing under high-stringent conditions to a nucleic acid probe which includes the coding sequence (nucleotides 166 through 1152) of SEQ ID NO:1 or SEQ ID NO:2.

In another embodiment, the present invention relates to a polypeptide comprising an amino acid sequence corresponding to a Haematococcus pluvialis crtO gene and allelic, species variations and functional naturally occurring and/or WO 98/18910 PCTIUS97/17819 27 man-induced variants thereof. In a preferred embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.

It should be noted that the invention includes any peptide which is homologous 80-85%, preferably 85-90%, more preferably 90-100% of identical amino acids) to the above described polypeptide. The term 'homologous' as used herein and in the claims below, refers to the sequence identity between two peptides. When a position in both of the two compared sequences is occupied by identical amino acid monomeric subunits, it is homologous at that position. The homology between two sequences is a function of the number of homologous io positions shared by the two sequences. For example, if eight often of the positions in two sequences are occupied by identical amino acids then the two sequences are homologous.

Other polypeptides which are also included in the present invention are allelic variations, other species homologs, natural mutants, induced mutants and peptides encoded by DNA that hybridizes under high or low stringency conditions (see above) to the coding region (nucleotides 166 through 1152) of SEQ ID NO:l or SEQ ID NO:2.

In another embodiment, the present invention relates to a recombinant DNA molecule comprising a vector (for example plasmid or viral vector) and a DNA segment coding for a polypeptide, as described above. In a preferred embodiment, the DNA segment is present in the vector operably linked to a promoter.

In a further embodiment, the present invention relates to a host cell containing the above described recombinant DNA molecule or DNA segment.

Suitable host cells include prokaryotes (such as bacteria, including Escherichia coli) and both lower eukaryotes (for example yeast) and higher eukaryotes (for example, algae, plant or animal cells). Introduction of the recombinant molecule into the cell can be effected using methods known in the art such as, but not limited to, transfection, transformation, micro-injection, gene bombardment etc.

The cell thus made to contain the above described recombinant DNA molecules may be grown to form colonies or may be made to differentiate to form a differentiated organism. The recombinant DNA molecule may be transiently contained by a process known in the art as transient transfection) in the cell, nevertheless, it is preferred that the recombinant DNA molecule is stably contained by a process known in the art as stable transfection) in the cell. Yet in a preferred embodiment the cell is endogenously producing, or is made by genetic engineering means to produce, p-carotene, and the cell contains endogenous or genetically engineered p-carotene hydroxylase activity. Such a cell may be used as a food additive for animal salmon) and human consumption. Furthermore.

WO 98/18910 PCT/US97/17819 28 such a cell may be used for extracting astaxanthin and/or other xanthophylls, as described hereinbelow.

In a further embodiment, the present invention relates to a host transgenic organism a higher plant or animal) containing the above described recombinant DNA molecule or the above described DNA segment in its cells.

Introduction of the recombinant molecule or the DNA segment into the host transgenic organism can be effected using methods known in the art. Yet, in a preferred embodiment the host organism is endogenously producing, or is made by genetic engineering means to produce, p-carotene and, also preferably the host to organism contains endogenous or genetically engineered P-carotene hydroxylase activity. Such an organism may be used as a food additive for animal salmon) and human consumption. Furthermore, such an organism may be used for extracting astaxanthin and/or other xanthophylls, as described hereinbelow.

In another embodiment, the present invention relates to a method of producing astaxanthin using the above described host cell or transgenic organism.

In yet another embodiment, the present invention relates to a method of producing xanthophylls such as canthaxanthin, echinenone, cryptoxanthin, isocryptoxanthin, hydroxyechinenone, zeaxanthin, adonirubin, 3-hydroxyechinenone, 3'hydroxyechinenone and/or adonixanthin using the above described host cell or transgenic organism. For these purposes provided is a cell or a transgenic organism as described above. The host cell or organism are made to grow under conditions favorable of producing astaxanthin and the above listed additional xanthophylls which are than extracted by methods known in the art.

In yet another embodiment, the present invention relates to a transgenic plant expressing a transgene coding for a polypeptide including an amino acid sequence corresponding to Haematococcus pluvialis crtO gene, allelic and species variants or functional naturally occurring or man-induced variants thereof.

Preferably the expression is highest in chromoplasts-containing tissues.

In yet another embodiment, the present invention relates to a recombinant 3o DNA vector which includes a first DNA segment encoding a polypeptide for directing a protein into plant chloroplasts or chromoplasts derived from the Pds gene of tomato) and an in frame second DNA segment encoding a polypeptide including an amino acid sequence corresponding to Haematococcus pluvialis crtO gene, allelic and species variants or functional naturally occurring and maninduced variants thereof.

In yet another embodiment, the present invention relates to a recombinant DNA vector which includes a first DNA segment including a promoter highly expressible in plant chloroplasts or chromoplasts-containing tissues derived 21/02 '01 WED 15:44 FAX 61 3 9243 8333 GR1A3FITH HACK 0~009 613'19243 8333 -29 f rai the PdS gene off tomato) and a second DNA seginenL encoding a poly-peptide including an amino acid sequence corresponding to f-aematOCOCCUS pluviali5 c.rtO geile, al~lelic and species variants or functional. naturally occurring and man induced variant5 thereof.

All references, including any patents or patent.

applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, thi s reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country- For the purposes of this specification it will be clearly understood that the word "cormprising" mea-is "including but not limited to", anid that the word ucomprisesH has a corresponding meaning.

Reference is now made to the fol~lowing examples, which together with the above descriptions, ilutaethe ivention.

]EXAMPLES

0. 0:The following protocols and experimental details are referenced in the Examples that follow: Algae and growth conditions. Haematococcus poltvia1js (strain 34/7 from the Culture C ollection of Algae and Protozoa, Windermere, UK) was kindly provided by 0 0 Dr. Andrew Young from the Liverpool. John Moores Universtiy. Suspension cultures of the alga were grown in :0 a liquid medium as described by Nichols an~d Bol. [see, Nichols HW, Bold H(I (1964) 2 'rijchsazrcina polyrorpha gen et sp nov J Phycol 1: 34-39) For induction of astaxanthin biosynthesis cells were harvested, washed in water and RECEIVED TIME 21. FEB. 15 :3 8 PRINT TIME 22. FEB, 8 09 21/02 '01 WED 15:45 FAX 61 3 9243 8333 GR1VFITH HACK I010 g~61 39243 8333 -29a resuspended in a fitrogen.-depleted mediumn. The cultures were maintained in 250 Ml ErlenMeyer flasks under continluouis light (photon fluX of 75 p.E/mn Is) at 25 0 C, on a rotary shaker at 80 rpm.

Construct ion of cDNA library. The construction of a cDNA library form Haeznatococcus pluvialis was described in detail by Lotan and Hirschberg (1995) FEBs letters 364: 125-128. Briefly, total RNA was .extracted frorn algal cells grown for 5 days under nitrogen-depleted conditions (cell color brown-red). Cells from a 50 ml culture were harvested and their RNA content was extracted using Tni reagent (Molecular Research Center,

INC.).

Poly-An RNA was isolated by two cycles of fractiona~tion on oligo dT-cellulose (Boehringer). The final yield was of the total RNA. The cDNA library was constructed in a Uni-ZAP'~ XR vector, using a ZAP-cDNA synthesis kit (both fro Stratagene). Escherichia coli cells of strain XLl- Blue MRF' (Stratagene) were used for amplification of the cDNA library.

Plasmjds and Zschexichja coli strairLB. Plasrnid pPL376, which contains the genes necessazry for carotenoid biosynthesis ira eh bacterium Erwvinia herbi cola was obtained from Thveson. (for further detailsreadn plasmid pPL376 see, Tuvesoi RW, Larson RA Kagani L (1988) 25 Role of cloned carotenoid genes expressed in Escherich,a coli in protecting against inactivation by near-uJV light RECEIVED TIME 21. FEB. 15:3 8 PRINT TIME 22. FEB. 8 0 9 WO 98/18910 PCT/US97/17819 and specific phototoxic molecules. J Bacteriol 170: 4675-4680]. Cells of Escherichia coli strain JM109 that carry the plasmid pPL376 accumulate the bright yellow carotenoid, zeaxanthin glycoside. In a first step, a 1.1 kb Sall-Sall fragment was deleted from this plasmid to inactivate the gene crtX, coding for zeaxanthin glucosyl transferase. In a second step, partial BamHI cleavage of the plasmid DNA, followed by self ligation, deleted a 0.8 kb fragment which inactivated crtZ, encoding p-carotene hydroxylase. A partial BglII cleavage generated a fragment of 7.4 kb which was cloned in the BamH] site of the plasmid vector pACYC184. As shown in Figure 4, the resulting recombinant plasmid, which carried the genes crtE, crtB, crtI and crtY, was designated pBCAR [Lotan and Hirschberg (1995) FEBS letters 364: 125-128].

Plasmid pBCAR was transfected into SOLR strain cells of Escherichia coli (Stratagene). Colonies that appeared on chloramphenicol-containing Luria Broth (LB) medium [described in Sambrook et al., Molecular Cloning; A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989], carried this plasmid and developed a deep yellow-orange color due to the accumulation of P-carotene.

As shown in Figure 5, an additional plasmid, designated pZEAX, which allows for zeaxanthin synthesis and accumulation in Escherichia coli was constructed [this plasmid is described in details in Lotan and Hirschberg (1995) FEBS letters 364: 125-128]. SOLR strain Escherichia coli cells were used as a host for the pZEAX plasmid. Escherichia coli cells were grown on LB medium (see above), at 37 0 C in the dark on a rotary shaker at 225 rpm. Ampicillin (50 V g/ml) and/or chloramphenicol (30 pLg/ml) (both from Sigma) were added to the medium for selection of appropriate transformed cells.

As shown in Figure 6, a plasmid, pHPK, containing the full length cDNA of the p-carotene C-4-oxygenase enzyme was identified by color complementation as described by Lotan and Hirschberg (1995) FEBS letters 364: 125-128 (see description herein below). A 1.2 kb PstI-PstI DNA fragment, containing the cDNA of the p-C-4-oxygenase from Haematococcus pluvialis, was isolated from plasmid pHPK and inserted into a PstI site in the coding sequence of the crtZ gene in the plasmid pZEAX. This recombinant plasmid was designated pCANTHA and is shown in Figure 7.

The same 1.2 kb PstI-PstI fragment was also inserted into a PstI site which exists 600 bp downstream of the crtE gene in the plasmid pZEAX. The resulting recombinant plasmid was designated pASTA and is shown in Figure 8.

The same 1.2 kb Pstl-PstI fragment was also inserted into a PstI site which exists in the P-lactamase gene in the plasmid pPAN35D5 [Hirschberg J,.Ohad N.

WO 98/18910 PCT/US97/17819 31 Pecker I and Rahat A (1987) Isolation and characterization of herbicide resistant mutants in the cyanobacterium Synechococcus R2. Z. Naturforsch 42c: 102-112], which carries the psbAl gene from the cyanobacterium Synechococcus PCC7942 in the plasmid vector pBR328 [Hirschberg J, Ohad N, Pecker I and Rahat A (1987) Isolation and characterization of herbicide resistant mutants in the cyanobacterium Synechococcus R2. Z. Naturforsch 42c: 102-112]. This plasmid was designated and is shown in Figure 9. This plasmid was used in the transformation of Synechococcus PCC7942 cells following procedures described by Golden [Golden SS (1988) Mutagenesis of cyanobacteria by classical and io gene-transfer-based methods. Methods Enzymol 167: 714-727].

Excision of phage library and screening for a P3-carotene oxygenase gene. Mass excision of the cDNA library, which was prepared as described hereinabove, was carried out using the ExAssist helper phage (Stratagene) in cells of SOLR strain of Escherichia coli that carried the plasmid pBCAR. The excised library in phagemids form was transfected into Escherichia coli cells strain XL1- Blue and the cells were plated on LB plates containing 1 mM isopropylthio-P-Dgalactosidase (IPTG), 50 ig/ml ampicillin and 30 pg/ml chloramphenicol, in a density that yielded approximately 100-150 colonies per plate. The plates were incubated at 37 0 C overnight and further incubated for two more days at room temperature. The plates were then kept at 4 0 C until screened for changes in colony colors.

A plasmid for high expression of crtO in chromoplasts. As shown in Figures 10-11, a genomic DNA sequence of a tomato species Lycopersicon esculentum (nucleotides 1 to 1448 of the Pds gene [as published in Mann V, Pecker I and Hirschberg J (1994) cloning and characterization of the gene for phytoene desaturase (Pds) from tomato (Lycopersicon esculentum). Plant Molecular Biology 24: 429-434], which contains the promoter of the Pds gene and the coding sequence for the amino terminus region of the polypeptide PDS that serve as a transit peptide for import into chloroplasts and chromoplasts, was cloned into a HindIII-SmaI site of the binary plasmid vector pBIB, [described by Becker D (1990) Binary vectors which allow the exchange of plant selectable markers and reporter genes. Nucleic Acids Research 18:230], shown in Figure 10. The recombinant plasmid was designated pPTBIB and is shown in Figure 11.

As shown in Figure 12, a 1,110 nucleotide long Eco47III-NcoI fragment, containing the cDNA of crtO from H. pluvialis (nucleotides 211 to 1321 of SEQ ID NO:1) was sub-cloned into the Smal site of the plasmid pPTBIB (Figure 11) so that the coding nucleotide sequence of the amino terminus of WO 98/18910 PCT/US97/17819 32 Pds is in the same reading frame as crtO. The recombinant plasmid was designate pPTCRTOBIB.

Formation of transgenic higher plant. The DNA of pPTCRTOBIB was extracted from E. coli cells and was transferred into cells of Agrobacterium tumefaciens strain EHA105 [described by Hood EE, Gelvin SB, Melchers LS and Hoekema A (1993) Transgenic Research 2:208-218] using electroporation as described for E. coli [Dower JW, Miller FJ and Ragdsale WC (1988) High efficiency transformation of E. coli by high voltage electroporation. Nuc. Acids Res. 18: 6127-6145]. Agrobacterium cells were o0 grown at 28 OC in LB medium supplemented with 50 gig/ml streptomycin and pg/ml kanamycin as selective agents. Cells of Agrobacterium carrying pPTCRTOBIB were harvested from a suspension culture at the stationary phase of growth and used for transformation as described by Horsch RB, Fry JE, Hoffmann NL, Eicholtz D, Rogers SG and Fraley RT, A simple and general method for transferring genes into plants. Science (1985) 227:1229-1231; and Jeffesrson AR, Kavanagh TA and Bevan WM (1987) GUS fusions: 3glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

The EMBO J. 6: 3901-3907.

Leaf explants of Nicotiana tobaccum strain NN were infected with the transformed Agrobacterium cells and kanamycin-resistant transgenic plants were regenerated according to protocols described by Horsch et al. (1985) and Jefferson et al. (1987) cited above.

With reference now to Figure 13, the presence of the DNA sequence of the crtO gene-construct in the fully developed regenerated plants was determined by DNA Southern blot analysis. To this end DNA was extracted from the leaves [according to a protocol described by Kanazawa and Tsutsumi (1992) Extraction of restrictable DNA from plants of the genus Nelumbo. Plant Molecular Biology Reports 10: 316-318], digested with the endonuclease HindIII, the fragments were size separated by gel electrophoresis and hybridized with radioactively labeled crtO sequence (SEQ ID NO: 1).

It was determined that each transgenic plant that was examined contained at least one copy of the crtO DNA sequence, yielding a 1.75 kb band (arrow).

originating from an internal HindIII-HindIII fragment of the T-DNA of pPTCRTOBIB, additional bands originating from partial digestion, additional band/s whose sizes vary, depending on the position of insertion in the plant genome and a 1.0 kb band originating from the tobacco plant itself which therefore also appears in the negative control WT lane.

WO 98/18910 PCT/1S97/17819 Sequence analysis. DNA sequence analysis was carried out by the dideoxy method [see, Sanger F, Nicklen S Coulsen AR (1977) DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 74: 5463-5467].

Carotenoids analysis. Aliquots of Escherichia coli cells which were grown in liquid in LB medium were centrifuged at 13,000 g for 10 minutes, washed once in water and re-centrifuged. After removing the water the cells were resuspended in 70 jtl of acetone and incubated at 65°C for 15 minutes. The samples were centrifuged again at 13,000 g for 10 minutes and the carotenoidcontaining supernatant was placed in a clean tube. The carotenoid extract was to blown to dryness under a stream of nitrogen (N 2 gas and stored at -20 0 C until required for analysis. Carotenoids from plant tissues were extracted by mixing 0.5-1.0 gr of tissue with 100 pl of acetone followed by incubation at 65 0 C for minutes and then treating the samples as described above.

High-performance liquid chromatography (HPLC) of the carotenoid extracts was carried out using an acidified reverse-phase C18 column, Spherisorb ODS-2 (silica 5 um 4.6 mm x 250 mm) (Phenomenex®). The mobile phase was pumped by triphasic Merck-Hitachi L-6200A high pressure pumps at a flow rate of ml/min. The mobile phase consisted of an isocratic solvent system comprised of hexane/dichloromethane/isopropyl alcohol/triethylamine (88.5:10:1.5:0.1, v/v).

Peaks were detected at 470 nm using a Waters 996 photodiode-array detector.

Individual carotenoids were identified by their retention times and their typical absorption spectra, as compared to standard samples of chemically pure pcarotene, zeaxanthin, echinenone, canthaxanthin, adonirubin and astaxanthin (The latter four were kindly provided by Dr. Andrew Young from Liverpool John Moores University).

Thin layer chromatography (TLC) was carried out using silica gel 60 F254 plates (Merck), using ethyl acetate/benzene v/v) as an eluent. Visible absorption spectra were recorded with a Shimadzu UV-160A spectrophotometer.

All spectra were recorded in acetone. Spectral fine structure was expressed in terms of %III/II [Britton, G. (1995). UV/Visible Spectroscopy. In: Carotenoids; Vol IB, Spectroscopy. Eds. Britton G, Liaaen-Jensen S and Pfander H.

Birkhauser Verlag, Basel. pp. 13-62].

Isolation and identification of the carotenoids extractedfrom cells ofE. coli are treated in order of increasing adsorption (decreasing Rfvalues) on silica TLC plates. Carotenoids structure and the biosynthesis pathway of astaxanthin are given in Figure 14. The following details refer to the carotenoids numbered 1 through 9 in Figure 14.

WO98/18910 PCT/US97/17819 34 P-Carotene Rf0.92 inseparable from authentic

R

t .VIS max nm: (428), 452, 457, %III/II 0.

Echinenone Rf0.90 inseparable from authentic

R

t .VIS max nm: .455. %III/II 0.

Canthaxanthin Rf0.87. inseparable from authentic Rt .VIS Xmax nm: 470. %III/II 0.

P-Cryptoxanthin Rf0.83. Rt .VIS -max nm: (428), 451, 479, %III/II 0.

Adonirubin Rf0.82 inseparable from authentic Rt .VIS ,max nm: io 476, %II/II 0.

Astaxanthin Rf 0.79 inseparable from authentic Rt .VIS Xmax nm: 477, %III/II 0.

Adonixanthin Rf0.72. Rt .VIS Xmax nm: 464, %III/II 0.

Zeaxanthin Rf0.65 inseparable from authentic R, .VIS max nm: (428), 451, 483, %III/II= 27.

Hydroxyechinenone Rf0.80, Rt, 3.0. VIS Xmax nm: 464, %III/II 0.

Chirality configuration. Chirality configuration of astaxanthin was determined by HPLC of the derived diastereoisomeric camphanates of the astaxanthin [Renstrom B, Borch G, Skulberg M and Liaaen-Jensen S (1981) Optical purity of (3S,3S)-astaxanthin from Haematococcus pluvialis. Phytochem 2561-2565]. The analysis proved that the Escherichia coli cells synthesize pure (3S,3'S) astaxanthin.

EXAMPLE 1 Cloning the P-C-4-oxygenase gene A cDNA library was constructed in Lambda ZAP II vector from poly-An RNA of Haematococcus pluvialis cells that had been induced to synthesize astaxanthin by nitrogen deprivation as described hereinabove. The entire library was excised into 3 -carotene-accumulating cells of Escherichia coli, strain SOLR, which carried plasmid pBCAR (shown in Figure Screening for a p-carotene oxygenase gene was based on color visualization of colonies of size of 3 mm in diameter. Astaxanthin and other oxygenated forms of p-carotene xanthophylls) have distinct darker colors and thus can be detected from the yellow P-carotene background. The screening included approximately 100,000 colonies which were grown on LB medium plates containing ampicillin and chloramphenicol that selected-for both the Lambda ZAP II vector in its plasmid propagating form and the pBCAR plasmid. Several colonies showed different WO 98/18910 PCT/US97/17819 color tones but only one exhibited a conspicuous brown-red pigment. This colony presumed to contain a xanthophyll biosynthesis gene was selected for further analysis described hereinbelow in the following Examples.

EXAMPLE 2 Analysis of the -C-4-oxygenase activity in Escherichia coli The red-brown colony presumed to contain a xanthophyll biosynthesis gene (see Example 1 above) was streaked and further analyzed. First, the recombinant to ZAP II plasmid carrying the-cDNA clone that was responsible for xanthophyll synthesis in Escherichia coli was isolated by preparing plasmid DNA from the redbrown colony, transfecting it to Escherichia coli cells of the strain XL 1-Blue and selection on ampicillin-containing medium. This plasmid, designated pHPK (pHPK is a Lambda ZAP II vector containing an insert isolated from the red-brown colony), was used to transform 0-carotene-producing Escherichia coli cells (Escherichia coli SOLR strain that carry the plasmid pBCAR shown in Figure 4) resulting in the formation of red-brown colonies. Carotenoids from this transformant, as well as from the host cells (as control) were extracted by acetone and analyzed by HPLC.

HPLC analysis of carotenoids of the host bacteria which synthesized Pcarotene (Escherichia coli SOLR strain that carry the plasmid pBCAR shown in Figure as compared with a brown-red colony, revealed that only traces of 3carotene were observed in the transformant cells while a new major peak of canthaxanthin and another minor peak of echinenone appeared [described in detail by Lotan and Hirschberg (1995) FEBS letters 364: 125-128]. These results indicate that the cDNA in plasmid pHPK, designated crtO encodes an enzyme with P-C-4-oxygenase activity, which converts P-carotene to canthaxanthin via echinenone (see Figure 14). It is, therefore concluded that a single enzyme catalyzes this two-step ketonization conversion by acting symmetrically on the 4 and 4' carbons of the p- and p'-rings of 3-carotene, respectively.

EXAMPLE 3 Production of astaxanthin in Escherichia coli cells To determine whether p-carotene hydroxylase-(e.g., a product of the crtZ gene of Erwinia herbicola) can convert thus produced canthaxanthin to astaxanthin and/or whether zeaxanthin converted from p-carotene by P-carotene hydroxylase can be converted by P-C-4-oxygenase to astaxanthin, the crtO cDNA of WO 98/18910 PCT/US97/17819 36 Haematococcus pluvialis thus isolated, was expressed in Escherichia coli cells together with the crtZ gene of Erwinia herbicola. For this purpose, Escherichia coli cells of strain SOLR were transfected with either plasmid pASTA alone containing, as shown in Figure 8, both crtZ and crtO or, alternatively with both plasmids, pHPK containing, as shown in Figure 6, crtO, and pZEAX containing, as shown in Figure 5, crtZ. Carotenoids in the resulting transformed cells were extracted and analyzed by HPLC as described above. The results, given in Table 1, show the composition of carotenoids extracted from the cells containing the plasmid pASTA. Similar carotenoid composition is found in Escherichia coli cells io which carry both pHPK and pZEAX.

TABLE 1 Carotenoid of total carotenoid composition p-Carotene Echineone 1.7 P-Cryptoxanthin 4.2 Canthaxanthin 4.2 Zeaxanthin 57.8 Adonirubin Adonixanthin 17.9 Astaxanthin 5.2 The results presented in Table 1, prove that carotenoids possessing either a P-end group or a 4-keto--end group act .as substrates for the hydroxylation reactions catalyzed by crtZ gene product at carbons C-3 and The hydroxylation of P-carotene and canthaxanthin results in the production of zeaxanthin and astaxanthin, respectively. These hydroxylations result in the production of astaxanthin and the intermediate ketocarotenoids, 3hydroxyechinenone, adonixanthin and adonirubin. These results further demonstrate that astaxanthin can be produced in heterologous cells by expressing the gene crtO together with a gene that codes for a p-carotene hydroxylase.

EXAMPLE 4 Sequence analysis of the gene for 3--carotene C-4-oxygenase The full length, as was determined by the presence of a poly A tail, of the cDNA insert in plasmid pHPK (1771 base pairs) was subjected to nucleotide WO 98/18910 PCTIUS97/17819 37 sequence analysis. This sequence, set forth in SEQ ID NO:l, and its translation to an amino acid sequence set forth in SEQ ID NO:3 (329 amino acids), were deposited in EMBL database on May 1, 1995, and obtained the EMBL accession numbers X86782 and X86783, respectively.

An open reading frame (ORF) of 825 nucleotides (nucleotides 166 through 1152 in SEQ ID NO:3) was identified in this sequence. This ORF codes for the enzyme p-carotene C-4-oxygenase having 329 amino acids set forth in SEQ ID NO:4, as proven by its functional expression in Escherichia coli cells (see Example 3 above). The gene for this enzyme was designated crtO.

EXAMPLE Transformation of cyanobacteria with crtO The plasmid DNA of pPAN3.5-KETO, shown in Figure 9, was transfected into cells of the cyanobacterium Synechococcus PCC7942 according to the method described by Golden [Golden SS (1988) Mutagenesis of cyanobacteria by classical and gene-transfer-based methods. Methods Enzymol 167: 714-727]. The cyanobacterial cells were plated on BG11 medium-containing petri dishes that contained also chloramphenicol. Colonies of chloramphenicol-resistant Synechococcus PCC7942 which appeared after ten days were analyzed for their carotenoid content. As detailed in Table 2 below, HPLC analysis of these cells revealed that the major carotenoid components of the cells was p-carotene, echinenone, canthaxanthin, adonirubin and astaxanthin. A similar analysis of the wild type strain and of Synechococcus PCC7942 transfected with a plasmid in which the orientation of the crtO gene is reversed (not shown), which is therefore not capable of producing an active protein, did not revealed production of echinenone, canthaxanthin, adonirubin and astaxanthin.

These result prove that crtO of Haematococcus pluvialis can be expressed in cyanobacteria and that its expression provided a P-C-4-oxygenase enzymatic activity needed for the conversion of P-carotene to canthaxanthin. This result further demonstrates that the endogenous p-carotene hydroxylase of Synechococcus PCC7942 is able to convert thus produced canthaxanthin to astaxanthin. Since the carotenoid biosynthesis pathway is similar in all green photosynthetic organism [see Figures 1 and 10 and, Pecker I, Chamovitz D. Linden H, Sandmann G and Hirschberg J (1992) A single polypeptide catalyzing the conversion of phytoene to -carotene is transcriptionally regulated during tomato fruit ripening. Proc Natl Acad Sci USA 89: 4962-4966] it is deduced that astaxanthin can be produced in algae, and higher plants by expressing crtO in any WO 98/18910 PCTfUS97/1 7819.

38 tissue that express also the endogenous P-carotene hydroxylase. It is further deduced that astaxanthin can be produced by any organism provided it contains either endogenous or engineered p-carotene biosynthesis pathway, by expressing crtO in any tissue that express either endogenous or genetically engineered 3carotene hydroxylase.

TABLE 2 Carotenoid of total carotenoid composition 3-Carotene 31.5 Echinenone -18.5 Canthaxanthin 16.1 Zeaxanthin 22.3 Adonirubin Astaxanthin 5.6 EXAMPLE 6 Determining the chirality configuration of astaxanthin produced in heterologous systems The chirality configurations of astaxanthin produced by Escherichia coli cells, as described under Example 3 hereinabove, and by cyanobacterium Synechococcus PCC7942 cells, as described in Example 5 hereinabove, were determined by HPLC of the derived diastereoisomeric camphanates of the astaxanthin [Renstrom B, Borch G, Skulberg M and Liaaen-Jensen S (1981) Optical purity of (3S,3S')-astaxanthin from Haematococcus pluvialis. Phytochem 2561-2565]. The analysis proved that the Escherichia coli and Synechococcus PCC7942 cells described above, synthesize pure (3S,3'S) astaxanthin.

EXAMPLE 7 Transformation of a higher plant with crtO Producing natural astaxanthin in higher plants has two anticipated benefits. First, as a pure chemical, astaxanthin is widely used as feed additive for fish. It is a potential food colorant suitable for humans consumption and.

has potential applications in the cosmetic industry. Second, inducing astaxanthin biosynthesis in vivo in flowers and fruits will-provide attractive pink/red colors which will increase their appearance and/or nutritious worth.

WO 98/18910 PCTJUS97/17819 39 In flowers and fruits carotenoids are normally synthesized and accumulated to high concentration in chromoplasts, a typical pigmentcontaining plastids, thus providing typical intense colors to these organs.

Inducing synthesis of astaxanthin in chromoplasts enables the accumulation of high concentration of this ketocarotenoid. Over-expression of carotenoid biosynthesis genes which results in elevated concentrations of carotenoids in chloroplasts, or other alterations in carotenoid composition in chloroplasts may damage the thylakoid membranes, impair photosynthesis and thus is deleterious to the plants. In contrast, increase of carotenoid concentration or alteration in carotenoid composition in chromoplasts do not affect the viability of the plant nor the yield of fruits and flowers.

Thus, gene-transfer technology was used to implant the crtO gene isolated from the alga Haematococcus pluvialis, as described, into a higher plant, in such a way that its expression is up-regulated especially in chromoplast-containing cells.

To this end, a T-DNA containing binary plasmid vector as shown in Figure 12 was assembled in E. coli from the promoter and coding DNA sequences of the transit peptide encoded by the Pds gene from a tomato species Lycopersicon esculentum, linked to the coding DNA sequence of crtO from H.

pluvialis. Upon stable transfer of this DNA construct via Agrobacteriummediated transformation into a tobacco (Nicotiana tabacum NN) plant to form a transgenic plant, as described under methods above, the plant acquired the ability to produce ketocarotenoids especially in flower tissues (chromoplastcontaining cells). It should be noted that the Pds gene promoter is capable of directing transcription and therefore expression especially in chloroplasts and/or chromoplasts-containing tissues of plants. It should be further noted that the transit peptide encoded by part of the Pds coding sequence is capable of directing conjugated in frame) proteins into plant chromoplasts and/or chloroplasts.

As shown in Figure 15, in chromoplasts-containing cells, such as in the nectary tissue of the flower of tobacco, this DNA construct induces accumulation of astaxanthin and other ketocarotenoids to a higher level which alters the color from the normal yellow to red.

Concentration and composition of carotenoids in chloroplasts-containing tissues, such as leaves, and in chromoplast-containing tissues, such as flowers, were determined in the transgenic plants and compared to normal nontransformed plants.

WO 98/18910 PCT/US97/17819 Carotenoids compositions in leaves (chloroplasts-containing tissue) and in the nectary tissue of flowers (chromoplast containing tissue) of wild type and transgenic tobacco plants were determined by thin layer chromatography

(TLC)

and by high pressure liquid chromatography (HPLC) as described above.

Total carotenoids concentration in leaves (chloroplasts-containing tissue) and in the nectary tissue of flowers (chromoplast containing tissue) of wild type and transgenic tobacco plants are summarized in Tables 3 below.

Percents of carotenoids composition in leaves of wild-type and transgenic tobacco plants are summarized in Tables 4 below.

Percents of carotenoids composition in the nectary tissue of flowers of wild-type and transgenic tobacco plants are summarized in Tables 5 below.

TABLE 3 gtg carotenoids per gr fresh weight Wild-type Transgenic with crtO Leaf (Chloroplasts) 200 240 Nectary tissue (Chromoplasts) 280 360 TABLE 4 of total carotenoids composition in chloroplasts-containing tissue (leaf) Wild-type Transgenic p-carotene 29.9 26.7 neoxanthin 5.0 5.9 violaxanthin 11.6 18.1 antheraxanthin 4.9 2.6 lutein 43.9 41.4 zeaxanthin 4.7 4.3 astaxanthin adonirubin 0.0 WO 98/18910 PCT/US97/17819 41 TABLE of total carotenoid composition in chromoplasts-containing tissue (flower) Wild-type Transgenic beta-carotene 58.1 21.0 violaxanthin 40.3 lutein 0.0 1.1 zeaxanthin 1.6 hydroxyechinenone 0.0 13.7 3'hydroxyechinenone 0.0 4.1 adonirubin 0.0 22.4 adonixanthin 0.0 8.7 astaxanthin 0.0 26.5 Please note the elevated content of hydroxyechinenone, 3'hydroxyechinenone, adonirubin, adonixanthin and astaxanthin especially in the chromoplast containing tissue of the transgenic tobacco plants.

Thus, the present invention successfully addresses the shortcomings of the presently known configurations by enabling a relatively low cost biotechnological production of (3S,3'S) astaxanthin by providing a peptide having a p-C-4oxygenase activity; a DNA segment coding for this peptide; an RNA segments coding for this peptide; a recombinant DNA molecule comprising a vector and the DNA segment; a host containing the above described recombinant DNA molecule or DNA segment; and of a method for biotechnologically producing (3S,3'S) astaxanthin or a food additive containing (3S,3'S) astaxanthin, using the host.

While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made.

WO 98/18910 PCTJUS97/17819 GENERAL INFORMATION:

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42 SEQUENCE LISTING Joseph Hirschberg, Tamar Lotan and Mark Harker Polynucleotide molecule from Haematococcus pluvialis encoding a polypeptide having a P-C-4-oxygenase activity for biotechnological production of (3S,3'S) astaxanthin.

4 Mark M. Friedman c/o Robert Sheinbein 2940 Birchtree space Lane Silver Spring Maryland United 20906

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INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: States of America 1.44 megabyte, 3.5" microdisk Twinhead SLimnote-890TX MS DOS version 6.2, Windows version 3.11 Word for Windows version Friedmam, Mark M.

33,883 325/5 972-3-5625553 972-3-5625554

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

(xi) SEQUENCE DESCRIPTION: GGC ACG AGC TTG CAC GCA AGT CAG CGC GCG TCC ACA GCC TCA AAT AAT AAA GAG CTC AAG TGG CCA GTC TGC ACT GCC TTG AAC CCG CGA TGC CAT AGC ACA GCT AGA CGA ATG CAG CTA GAG CAG CTT ACC GGA AGC GCT GAG GCA CTC GTT GCA GGC AGC TCT GAC GTG TTG CGT ACA CTT CCG TCA GAA GAG TCA GAC GCG GCC CGC TAC AAG CCA CCA CCT TCC GAC ACA AAG GGC GTC ATC GGC TCC TGG GCC GCA GIG TTC CTC AAG CTT CCG ACC TCC TTG GAC CAG CTG CAC GCC ACA GCT CAG CTG GTT AGC GGC ACG Anr 1771 base pairs nucleic acid double linear SEQ ID NO:1: GTC AAC TTG TGC TCC CGC GCG ACA GAG AAG GCG ACC GGA CTG ACA ATG GCC ATT CTG CCC CTG CTC TGC CGG TCG ACG ACT GAC ATG TTG AAG GAG TAC TCG AAT GCC CTA CGT CAA ATC TCA GAT ATC GTC WO 98/18910 PCT/US97/17819 GTA GTA TTC TTT GTC CTG GAG TTC CTO ACG CAT GAT OCT ATG CAT GGC ACC ATC AAT GAC TTC TTG GGC AGA GTA TGC ATC TAC AAC ATG CTG CAC CGC AAG CAT TGG GAG GTG GOC AAG GAC CCC TGG TTT GCC AGC GCG CGC CTC GCA TGG ATG GCG AAC CTG CTG TTC CGC TTG TTC TAC GGC GCC GCG TCA GGC CGC ACT AGC CAG GCG TTC GAC CTG CAC TGG GAG CTG CCC AAC TGC TAG CTG GAC ACA CTG TGT GOC AGG ACT GGG ACG CTG CAT GGG CTA TTG TAG CTG TCG AGC OGA GTA CAC CCA CAG GAG GAG TOT TGG GCA GCC TTT AGO GGA GCG GTG CAG OCA CAA GCT AGA OGT GCG OGA GGG AAT OCT GGC GGT GTG ATT OTT TOA GCA GTC AGG TCA GGA GAG TGA TGC GCT TAT GAA GCT CCT GAC TTC CAC TTC ATG TCC AGC TGG ACG GTG GTC GTG TTC ATG GCG TTT GGC ACG TAC TCT TCA CCA GCC TCC GAC CTG GTC GAG CAC CAC CGC CGC COC CTG TCT CAG TGG GCC CTG TGA GOT GAA AAG CCC TGT GTA GCT TTG CCC CAT OGA GCC AAC ACC CTT GTG TAG ATG CTA ACA CTT AGT GCT AGG CTG GAC GAG TGG TGC CAC ACC GCA GTG AGA GCT TCA CTT ATT CTT GTA TGA ACA AOT GTA ACA ATA AAG ACA GGC CIT TTT ATG AGA AAC AGG TTG TAC GCC TGG CAC CAC AAC CAC OGA AAC CCT GGC ATG TCG ATG TGG CAG CTG CTG GGT GCG CCC ATC CTG CCC CAC AAG CCT ATG AAC TGG TGG TTT CTG ACC TGC CCC TTC GCC CCC CGA GGT CTG GTT CCA OCT GGG CAT CAG GCG CTO CTG GCC ACT AGG GGA AGC TGT GTA GTG GGA GAT GTC TTG TTG TAT CTT AAT CAG GCA ACG CCC TCG GTG GCA GGC TGG GCA AGA CCA TGA TTA ACT OGG ATC ACC CAG CTT TTT OAT ACT GGC ATT GTO CAG TTT GCG CCA TCC GCC GAG CCT AAO TCG TAC CAC TOO TGG CCT GCC GCA GGT CCG GAC GGG GGT GTG CAG CGT CGG GCT GAA TGC AAG AGO TGA TOC TGC CTA TGG 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1771 TGA TAT AGA TAC TGG TCA TGA GAG GTG GTG CGC TGC TGG TTC INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1771 bases TYPE: nucteic acid STRANOEDNESS: single TOPOLOGY: Linear (xi) SEQUENCE'DESCRIPTION: SEQ ID NO:2: GGC ACG AOC UUG CAC OCA AGU CAG UCC ACA 0CC UCA AAU AAU AAA GAG UGO CCA GUC UGC ACU 0CC UUO AAC UGC CAU AOC ACA GCU AGA COA AUG GAG CAG CUU ACC OGA AGC GCU GAG GUU GCA GGC AGC UCU GAC GUG UUG CUU CCG UCA GAA GAG UCA GAC GCO CGC GCG CAA GUC AAC ACC UGC CUC AAO CGU UUG UGC 0CC UCO CCG CGA GUC UCC COC COC ACU CAG CUA OCA GCG ACA OUA AUG GCA CUC AAO GAG AAO GAG AAG CGU ACA UGG GCG ACC CAG UAC 0CC COC CCG OGA CUG AAG AAU AAG GGC AUC ACA AUG GCG CUA UUC CUC CAC 0CC AUU UUU CAA UAC AAO GUC AUC AAG CUU 0CC ACA GUA GUA ACO CAU AAU GAC UAC AAC GAO GUG CCC UGO GCG CGC AUG GCG UUC CGC OGC 0CC CGC ACU CCA CCU UCC UCC UGG GCC ACC UCC UUG GAC ACA GCA GUG GAC CAG AGC GGC GAO UUC GGC ACC OUA UGC AAO CAU GAC UUC AUG UCC ACG GUG UUC AUG GGC ACO UCA CCA GAC CUG CUG CAC UGG ACO AOC AGC CUG UAC ACA AUC 0CC AUG AUC UCC UUG UGG GAG CAC CAC AGG GGA AGC UAC AUG GUC AUG CAG GCG GCC GCG UAC AUG CCC 0CC GUC AUG GUC AOC UUU CUG CCC GUG UCA CUG CUC GAC AUC GOC CUU UUU AUC AGA AAC AGO CAG UAC 0CC UGO UUU CAC AAC CAC ACU AAC CCU GOC AUU UCO AUG UGG CAG CUG CUG GGU GCG CCC AUC CUG UCC CAC AAO CCU GAG AAC UGO UGG AAO CUG ACC UGC UAC WO 98/18910 WO 9818910PCTIUS97/17819 GAG CAC CAC CGC CCC CGC CUG UCU CAG UGG CCC CUG UGA GGU GAA AAG CCC UGU GUA GCU UUG CCC CAU GGA GCC AAC ACC CUU GUG UAG AUG CUA ACA CUU AGU GCU AGG CUG GAC GAG UGG UGC CAC ACC GCA GUG AGA GCUI UCA CUU AUU CUU GUA UGA ACA AGU GUA ACA AUA AAG INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1771 ba TYPE: nucLeic STRANDEDNESS: double

TOPOLOGY:

(xi) SEQUENCE DESCRIPTION: SEQ ID se pa irs acid Si near NO:3: CAA GTC AAC CGT TTG IC CTC TCC CC GCA CC ACA Ala Ala Thr ACC GC CCC ICG CCC ACT GTA ATG Vat Met GAG CAG CII ACC GGA AGC GC GAG GLu Gtn Leu Ihr GLy Ser Ala GLu GCA CTC AAG GAG AAG Ala Leu Lys Glu Lys GAG AAG GAG Gtu Lys Glu GTI GCA GGC AGC TCI GAC GIG TTG CGT ACA TGC CC ACC CAG TAC TCG Vat Ala Gty Ser Sen Asp Vat Leu Arg Ihr Irp Ala Ihr G~n Tyr Ser 35 CIT CCC ICA GAA GAG ICA GAC CC CCC CCC CCC GGA CTC AAG AAT CC Leu Pro Sen Glu GLu Sen Asp Ala Ala Arg Pro Gly Leu Lys Asn Ala 50 TAC AAG CCA CCA CCT TCC CAC ACA AAG CCC AIC ACA ATG CC CTA CGI Tyr Lys Pro Pro Pro Sen Asp Ihr Lys Gly Ile Thr Met Ala Leu Arg 65 GTC AlT GGC TCC IGG CCC GCA GTG TTC CTC CAC CCC ATI TTT CAA ATC Vat Ile Gty Ser Irp Ala Ala Vat Phe Leu His ALa Ile Phe Gln Ile CCC ACC TCC TTG GAC CAG CTG CAC Pro Thr Sen Leu Asp G~n Leu His ACA GCT CAG CIC Ihr Ala Gln Leu 110 GTT AGC CCC ACC Vat Sen Gly Thr TGC CTC CCC GTG TCA CAT Irp Leu Pro Vat Sen Asp 100 105 AGC CIG CIC GAC ATC GTC Sen Leu Leu Asp Ile Vat 120 ACA CCC CTT ITT ATC ACC Ihr CLy Leu Phe Ile Thr 135 AIG AGA AAC AGG CAC CTT GTA CIA TTC TT Vat Vat Phe Phe 125 ACG CAT CAT CCT Thr His Asp Ala GIC CTG GAG TIC CTG TAC Vat Leu GLu Phe Leu Tyr AIC CAT CCC ACC Met His Gly Thr AIC GCC Ile Ala Met Ang Asn Ang CLn Leu 150 wo 98/18910 WO 9818910PCT/US97/17819 AAT GAC TIC Asn Asp Phe 155 TAC AAC ATG Tyr Asn Met 170 TTG GOC AGA GTA Leu OLy Arg Val 160 CTG CAC CGC AAG Leu His Arg Lys 175 TGC ATC TCC TTG TAC GCC TOG TTT OAT Cys Ile Ser Leu Tyr Ala Trp Phe Asp 165 CAT TOG GAG CAC CAC AAC CAC ACT GGC His Trp OWu His His Asn His Thr GLy GAG GTG GGC AAG Glu Vat Gly Lys CCC TOG TTT GCC Pro Trp Phe Ala 205 GCG COC CTC GCA Ala Arg Leu Ala 220 AT0 OCO AAC CTG Met Ala Asn Leu CCT GAC TTC CAC AGG OGA AAC CCT 0CC ATT Pro Asp Phe His Arg Gly Asn Pro Gly Ile AOC TTC ATG TCC AGC TAC ATG TCG ATO Ser Phe Met Ser Ser Tyr Met Ser Met 200 TOO CAG Trp Gin 215 TOG TOG ACO GTG GTC ATG CAG CTG CTG GGT GCG CCA Trp Trp Thr Vat Val Met Gin Leu LeU Gly Ala Pro 225 230 CTG GTG TTC ATO GCO GCC OCO CCC AIC CTG TCC 0CC Leu Vat Phe Met Ala Ala Ala Pro Ile Leu Ser Ala 240 TTG TTC TAC TTT Leu Phe Tyr Phe 255

GOC

Gly ACO TAC ATO CCC CAC AAG Thr Tyr Met Pro His Lys 260 CCA 0CC GTC ATG AAC TG CCT GAG CCT Pro Glu Pro 265 TOG AAG TCG 0CC GCO TCA Ala Ala Ser GOC TCT COC ACT AOC CAG Arg Thr Ser Gin 285 TIC GAC CIG CAC Phe Asp Leu His 300 Gly Ser Ser Pro Ala Vat Met Asn Trp Trp Lys Ser 270 275 280 GCG TCC GAC CTG GTC AGC TTT CTG ACC TGC TAC CAC Ala Ser Asp Leu Va t Ser Phe Leu Thr Cys Tyr His 290 295 TOO GAO CAC CAC C0C TOG CCC TTC 0CC CCC TGG TOG Trp GLu His His Arg Irp Pro Phe Ala Pro Trp Trp 305 310 TOC CGC COC CTG TCT GOC CGA GOT CTG OTT CCT 0CC Cys Arg Arg Leu Ser GLy Arg OLy Leu Vat Pro Ala

GAG

Glu CTG CCC Leu Pro 315 CTG GAC GOC AGO CTG CAT TAO CTG GTA CAC GAG TOT TTT AGO CAG OCA GOT OCO OCT GOC OTT TGA TCA OGA OCT TAT

AAC

Asn 320 CTG CAG TOO 000 TGA GOT CTA CCC TOT AOC TTG CCC CAG 0CC AAC OCA OTO TAG OCO ACA CTT OCT AOO CTG 000 TOO TOC GIG OCA GTG GTC TCA CTT TOA OTA TGA OCT GTA ACA 0CC CTG GAA AAG OTA OCT CAT OGA ACC CTI ATO CTA AGT OCT GAC GAG CAC ACC AGA OCT ATT CTT ACA AOT ATA AAG CTO CCA CTG CAO 0CC 0CC IGA AOC OCA OGA TOA TTG 000 CAG GAC TCG CAC TOO OCO TGA IGA TAT TOA GAO TOG TTC OCT 000 GCO CTG ACT AGO TOT OTA OAT GTC TAT CTT OCA ACO GTG OCA OCA AGA TTA ACT AGA TAC OTG OTG CAT GCA CTO CCG GGA 000 GTG OTO TTO COT AAT OCT CCC TOC GOC AGO CCA TOC 000 CTA TOO TCA COC TOC INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

TOPOLOGY:

(xi) SEQUENCE DESCRIPTION: 329 amino acids amino acid linear SEQ ID NO:4: Met Gin Leu Ala Ala Thr Vat Met Leu Glu Gin Leu Thr GLy Ser Ala GLu ALa Leu Lys Glu Lys Glu Lys Olu WO 98/18910 PCTIUS97/17819 Vat Ala GLy Ser Ser Asp Vat Leu Arg Thr Trp Ala Thr Gin Tyr Ser 35 Leu Pro Ser Giu GLu Ser Asp Ala Ala Arg Pro GLy Leu Lys Asn ALa 50 Tyr Lys Pro Pro Pro Ser Asp Thr Lys GLy lie Thr Met Ala Leu Arg 65 Vat lie Gty Ser Irp Ala ALa Vat Phe Leu His ALa lie Phe Gin lie 80 Lys Leu Pro Thr Ser Leu Asp Gin Leu His Trp Leu Pro Vat Ser Asp 95 100 105 Ala Thr Ala Gin Leu VaL Ser Gly Thr Ser Ser Leu Leu Asp lie Vat 110 115 120 Vat Vat Phe Phe Vat Leu Giu Phe Leu Tyr Thr Gly Leu Phe lie Thr 125 130 135 Thr His Asp Ala Met His GLy Thr IHe Ala Met Arg Asn Arg Gin Leu 140 145 150 Asn Asp Phe Leu Gty Arg Vat Cys lie Ser Leu Tyr Ala Trp Phe Asp 155 160 165 Tyr Asn Met Leu His Arg Lys His Trp Gtu His His Asn His Thr.Gty 170 175 180 185 Giu Vat GLy Lys Asp Pro Asp Phe His Arg GLy Asn *Pro Gly lie Vat 190 195 200 Pro Trp Phe Ala Ser Phe Met Ser Ser Tyr Met Ser Met Trp Gin Phe 205 210 215 Ala Arg Leu Ala Trp Trp Thr VaL Vat Met Gin Leu Leu Gly Ala Pro 220 225 230 Met Ala Asn Leu Leu Vat Phe Met Ala Ala Ala Pro lie Leu Ser Ala 235 240 245 Phe Arg Leu Phe Tyr Phe Gly Thr Tyr Met Pro His Lys Pro Giu Pro 250 255 260 265 GLy Ala Ala Ser GLy Ser Ser Pro Ala Vat Met Asn Trp Trp Lys Ser 270 275 280 Arg Thr Ser Gin Ala Ser'Asp Leu Vat Ser Phe Leu Thr Cys Tyr His 285 290 295 Phe Asp Leu His Trp GLu His His Arg Trp Pro Phe Ala Pro Trp, Trp 300 305 310 Giu Leu Pro Asn Cys Arg Arg Leu Ser GLy Arg Gly Leu Vat Pro Ala 315 320 325

Claims (12)

1. An isolated nucleic acid comprising a nucleotide sequence at least 95 t identical. to SEQ ID NO:1 or SEQ ID NO:2, said nlucleotide sequence encoding a polypeptide having a 0-carotene C- 4 -oxygenase activity.

2. The isolated nucleic acid according to claim 1, wherein said nucleotide sequence includes a sequence as set forth in SEQ IID NOs:1 or 2.

3. The isolated nucleic acid according to claim 1 or claim 2, wherein said nucleotide sequence incluides a sequence as set forth between and including nucleotides 166 and 1152 of SEQ ID N~s:l or 2.

4. A nucleic acid construct comprising the nucleic acid segment according to any one of claims 1 to 3.

5. A host comprising the nucleic acid cons-truct according to claim 4, said host 'is selected from the group consstin of a mcoraimand a pat

6. A host comprising the nucleic acid according Co 25 any one of claims 1 to 3, said host is selected from the group consi.sting of a microorganism and a plant.

7. A method of producing a xanthophyll, the method a comprising the steps of: 30 providing a host according to claim 6; providing said host: with growing conditions for produiction of said xanthophyll; and ()extracting sadxnhpylfo saidhot

8. A food additive comprising the host according to claim 5 or claimn 6. RECEIVED TIME

21. FEB. 15 :3 8 PRINT TIME

22. FEB. 8: 09 21/02 '01 WED 15:45 FAX 61 3 9243 8333 GR1AZFITH HACK I012 ~'61 39243 8333

48- 9. ~A transgenic_! plant expressing a tran-Sgene including a nucleotide sequence at least 95 identical t~o SEQ ID NQ:1, said nucleotide sequence encoding a Polypeptide having a !-carotene C- 4 -oxygenase activity. A transgenic plant. according to claim 9, wherein said transgene includes a sequence as set forth in SEQ ID NO: 1. 11. A transgenic plant according to claim 9 or claim wherein said transgene includes a sequence as set forth between and including nucleotides 166 and 1152 of SEQ ID NO:1. A nucleic acid construct, comprising a first polynuc.eotide encoding a polypeptide for directing a protein into plant chloroplasts or chromoplasts and an in- frame second polynucleotide at least 95 i dentical to SEQ ID N~s:l and encoding a poJlypeptide having 1-carotene C-4- oxygenase activity. The nucleic acid construct according to cl.aimt 12, :wherein said second polyniicleotide includes a sequierce as *:set, forth in SEQ ID NO:l. 14. The nucleic acid construct according to claim 12 or claim 13, wherein said second polynucleotide includes a sequence as set forth between and including nucleotides 166 and 1152 of SEQ ID NO:1. The nucle.c acid construct according to claim 12, wherein said first polyfluicleotide is derived~ from the Pds gene of tomaLo. 16. A polypeptide comprising an amino acid sequence corresponding toD Haernatococcus pluvialis crtc gene zind ~JRAZhaving P-carotene C-4-oxygenase activity. RECEIVED TIME 21. FEB. 15 38 PRINT TIME 22. FEB. 8 09 21/02 '01 WED 15:46 FAX 61 3 9243 8333 GR1&'FITH HACK 0~013 6 13 9 24 3 8 33 3 -49 17. A POlype ptide according to claim 16, wherein said amino acid secfuence is as set. for.11 in SEQ ID NO:4. 18. A Polypeptide comprising an amino acid sequence at least 95 11omologous to the sequence set forth in SEQ ID NO:4, said Polypeptide having P-carotene C- 4 -oxygenase act ivity. 19. A POlypeptide comprising anl amino acid sequence being encoded by a DNA segment at least 95 identical to nucleotides 166 through 1152 of SEQ ID N0O:1, the poiypeptide having a 1 -C-4-o>Wgenase activity. 20. A method according to claim 1, substantially .as herein described with reference to any one of the examples or figures. 21. A transgenic plant according to claim 9, substantially as herein described with reference to any one of the examples or figures. 22. A nucleic acid construct according to claim 12, .substantially as herein described with reference to any one of the examples or figures. 23. A polypeptide according to claim 16, substantially as herein described with reference to any one of the examples or figures. .*:Dated this 2 1st day of February

2003. YTSSUM RESEARCH AND DEVELOPMENT COMPANY OF THE HEBREW *e UNIVERSITY OF JERUSALEM By their Patent Attorney~s GRIFFITH HACK Fellows Inlstitute of Patent and Trade Mark Attorneys of Australia RECEIVED TIME 21, FEB. 15 38 PRINT TIME 22. FEB. 8:0 9