EP0951534A1 - Sequence d'acide nucleique codant la beta-c-4-oxygenase et provenant de haemotococcus pluvialis, utilisee pour la biosynthese de l'astaxanthine - Google Patents

Sequence d'acide nucleique codant la beta-c-4-oxygenase et provenant de haemotococcus pluvialis, utilisee pour la biosynthese de l'astaxanthine

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Publication number
EP0951534A1
EP0951534A1 EP97909944A EP97909944A EP0951534A1 EP 0951534 A1 EP0951534 A1 EP 0951534A1 EP 97909944 A EP97909944 A EP 97909944A EP 97909944 A EP97909944 A EP 97909944A EP 0951534 A1 EP0951534 A1 EP 0951534A1
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Prior art keywords
seq
host
astaxanthin
gene
dna segment
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German (de)
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EP0951534A4 (fr
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Varda Mann
Joseph Hirschberg
Tamar Lotan
Mark Harker
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Yissum Research Development Co of Hebrew University of Jerusalem
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Yissum Research Development Co of Hebrew University of Jerusalem
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Publication of EP0951534A1 publication Critical patent/EP0951534A1/fr
Publication of EP0951534A4 publication Critical patent/EP0951534A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis

Definitions

  • NUCLEIC ACID SEQUENCE ENCODING BETA-C-4-OXYGENASE FROM HAEMATOCOCCUS PLUVIALIS FOR THE BIOSYNTHESIS OF ASTAXANTHIN
  • the present invention relates, in general, to a biotechnological method for production of (3S,3'S) astaxanthin.
  • the present invention relates to a 0 peptide having a ⁇ -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 5 (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 0 photosynthesis, and they protect against photooxidative damage. These and additional biological functions of carotenoids, their important industrial role, and their biosynthesis are discussed hereinbelow.
  • carotenoids can absorb photons and transfer the energy to chlorophyll, thus assisting in the harvesting of light in the 5 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 0 photosynthesis: structure and photochemistry.
  • thermophilic cyanobacterium Synechococcus sp. 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 ⁇ -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: 1-8].
  • ⁇ -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 carotenoids in the assembly of an active PS II. Planta 179: 242-250].
  • 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 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 ⁇ - carotene molecules per P700 [see, Takahashi Y, Hirota K and Katoh S (1985) Multiple forms of P700-chlorophyll ⁇ -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 ⁇ -carotene serves as an efficient antenna for PS I.
  • 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 ⁇ -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.
  • the triplet state of carotenoids was monitored in closed PS II centers and its rise kinetics of approximately 25 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.
  • 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 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.
  • Carotenoids have important commercial uses as coloring agents in the food industry since they are non-toxic [see, Bauernfeind JC (1981) Carotenoids as
  • 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.
  • the flesh, feathers or eggs of fish and birds assume the i o 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
  • carotenoids are composed of a C40 hydrocarbon backbone, constructed from eight C5 isoprenoid units and contain a series of conjugated
  • 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 C50 carotenoids, or shortened yielding apocarotenoids.
  • 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
  • Carotenoids are synthesized de novo from isoprenoid precursors only in photosynthetic organisms and some microorganisms, they typically accumulate in protein complexes in the photosynthetic membrane, in the cell membrane and in the cell wall.
  • 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].
  • 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- sopentenyl pyrophosphate (IPP) a C5 molecule, is formed from mevalonate and is the building block for all long-chain isoprenoids.
  • IPP dimethylallyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • 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 ⁇ 5-cis- phytoene, a colorless C40 hydrocarbon molecule.
  • PPPP prephytoene pyrophosphate
  • 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.
  • phytoene desaturases from Rhodobacter capsulatus, Erwinia sp. or fungi convert phytoene to neurosporene, lycopene, or 3,4-dehydrolycopene, respectively.
  • Biochem Biophys Res Com 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].
  • 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 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 (85%) 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].
  • the ⁇ -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 of Synechococcus sp. strain PCC 7942, implicating NAD(P) or FAD as coenzymes with lycopene cyclase.
  • Rhodobacter capsulatus 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, Beyer P, Kleinig H, Englert H and Hearst JE (1991) Carotenoids o ⁇ Erwinia herbicola and an Escherichia coli HB 101 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.
  • Two genes, al-3 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.
  • the first "plant-type" genes for carotenoid synthesis enzyme were cloned from cyanobacteria using a molecular-genetics approach.
  • 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 o ⁇ Synechococcus mutants selected against the bleaching herbicide norflurazon. Pestic Biochem Physiol 36: 46-51 ].
  • 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 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 1 1-18.
  • 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 -0.2), it does not include a hydrophobic region which is long enough to span a lipid bilayer membrane.
  • 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 (1993) Evolutionary conservation and structural similarities of carotenoid biosynthesis gene products from photosynthetic and nonphotosynthetic organisms. Meth Enzymol 214: 297-31 1 ], including in the crtl gene-product, phytoene desaturase. As indicated above, a high degree of conservation of the primary structure of phytoene desaturases also exists among oxy genie photosynthetic organisms.
  • the crtQ gene encoding ⁇ -carotene desaturase was cloned from Anabaena sp. strain PCC 7120 by screening an expression library of cyanobacterial genomic DNA in cells o ⁇ 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].
  • the crtL gene for lycopene cyclase (formerly ley) was cloned from Synechococcus sp. strain PCC 7942 utilizing essentially the same cloning strategy as for crtP.
  • an inhibitor of lycopene cyclase 2-(4-methylphenoxy)- triethylamine hydrochloride (MPTA)
  • MPTA 2-(4-methylphenoxy)- triethylamine hydrochloride
  • Lycopene cyclase is the product of a single gene product and catalyzes the double cyclization reaction of lycopene to ⁇ -carotene.
  • the crtL gene product in Synechococcus sp. strain PCC 7942 is a 46-kDa polypeptide of 411 amino acid residues. It has no sequence similarity to the crtY gene product (lycopene cyclase) from Erwinia uredovora or Erwinia herbicola.
  • ketocarotenoid astaxanthin (3,3'-dihydroxy- ⁇ , ⁇ -carotene-4,4'-dione) was first described in aquatic crustaceans as an oxidized form of ⁇ -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].
  • 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 Enzymol 213 : 403-420; and, Kurashige M, Okimasu E, Inove M and Utsumi K (1990) Inhibition of oxidative injury of biological membranes by astaxanthin.
  • Astaxanthin enhances in vitro antibody production to T-dependent antigens without facilitating polyclonal B-cell activation.
  • 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.
  • Astaxanthin is the most expensive commercially used carotenoid compound (todays- 1995 market value is of 2,500-3,500 $/kg). It is utilized mainly as nutritional supplement which provides pigmentation in a wide variety of aquatic 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.
  • Synthetic astaxanthin comprising a 1 :2: 1 mixture of the (3S,3'S)-, (3S,37?)- 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].
  • crtW was isolated from the marine bacteria Agrobacterium auranticacum and Alcaligenes PC-1 that produce ketocarotenoids such as astaxanthin.
  • 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.
  • 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.
  • nucleic acid segment which encodes ⁇ -C-4-oxygenase, the enzyme that converts ⁇ -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.
  • 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.
  • the present invention relates to a DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to Haematococcus pluvialis crtO gene.
  • the present invention relates to an RNA segment coding for a polypeptide comprising an amino acid sequence corresponding to Haematococcus pluvialis crtO gene.
  • the present invention relates to a polypeptide comprising an amino acid sequence corresponding to a Haematococcus pluvialis crtO gene.
  • 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.
  • the present invention relates to a host cell containing the above described recombinant DNA molecule or DNA segment.
  • 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.
  • the present invention relates to a method of producing other xanthophylls.
  • the present invention relates to a method of obtaining high expression of a transgene in plants specifically in chromoplasts- containing cells.
  • the present invention relates to a method of importing a carotenoid-biosynthesis enzyme encoded by a transgene into chromoplasts.
  • FIG. 1 is a general biochemical pathway of ⁇ -carotene biosynthesis, in which pathway all molecules are depicted in an all-trans configuration, wherein
  • IPP is isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • GPP is geranyl pyrophosphate
  • FPP is farnesyl pyrophosphate
  • GGPP is geranylgeranyl pyrophosphate
  • PPPP is prephytoene pyrophosphate
  • 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.
  • 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, crtl and crtY of Erwinia herbicola, which genes are required for production of ⁇ -carotene in Escherichia coli cells;
  • FIG. 5 is a schematic depiction of a pACYC184 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 ⁇ -carotene C- 4-oxygenase enzyme from Haematococcus pluvialis, designated crtO and set forth in SEQ ID NO: l, which cDNA was identified by color complementation of Escherichia coli cells;
  • FIG. 7 is a schematic depiction of a pACYC184 derived plasmid designated pCANTHA which was derived by inserting a 1.2 kb Pstl-Pstl DNA fragment, containing the cDNA encoding the ⁇ -C-4-oxygenase from Haematococcus pluvialis isolated from the plasmid pHPK of Figure 6 and inserted into a Pstl 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; FIG.
  • FIG. 8 is a schematic depiction of a pACYC184 derived plasmid designated pASTA which was derived by inserting the 1.2 kb Pstl-Pstl DNA fragment, containing the cDNA of the ⁇ -C-4-oxygenase from Haematococcus pluvialis isolated from the plasmid pHPK of Figure 6 and inserted into a Pstl 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 PAN3.5-KETO which was derived by inserting the 1.2 kb Pstl-Pstl DNA fragment, containing the cDNA of the ⁇ -C-4-oxygenase from Haematococcus pluvialis isolated from the plasmid pHPK of Figure 6 and inserted into a Pstl site which exists in a ⁇ -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.
  • FIG. 10 is a schematic depiction of the T-DNA region of a Ti binary plasmid (E. 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 pBHOl [described by Jeffesrson AR, Kavanagh TA and Bevan WM ( 1987) GUS fusions: ⁇ -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO J.
  • BR and BL are the right and left borders, respectively, of the T-DNA region
  • pAg7 is the polyadenylation site of gene 7 o ⁇ Agrobacterium Ti-plasmid
  • pAnos is a 250 bp long DNA fragment containing the poly adenylation site of the nopaline synthase gene o ⁇ 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
  • pAnos is a 300 bp long DNA fragment containing the poly adenylation site of the nopaline synthase gene o ⁇ Agrobacterium
  • 1 1 is a schematic depiction of the T-DNA region of a Ti binary plasmid (E. 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).
  • pPTBIB Ti binary plasmid
  • 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 Hindl ⁇ l-Smal site of the binary plasmid vector pBIB of Figure 10, wherein BR and Bj ⁇ , 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 (E. coli, Agrobacterium) designated pPTCRTOBIB which was prepared by cloning a 1,110 nucleotide long £co47IH-NcoI fragment of the cD ⁇ A o ⁇ crtO from H.
  • pPTCRTOBIB Ti binary plasmid
  • pluvialis (nucleotides 211 to 1321 of SEQ ID ⁇ O: l) into the Smal site of the plasmid pPTBIB of Figure 11 , such that the coding nucleotide sequence of the amino terminus o ⁇ 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 H t/III-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 (M) DNA fragments in kilobase pairs (kb) is indicated on the left as well as the expected position (arrow) of an internal T-DNA HindUl 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.
  • the present invention is, in general, of a biotechnological method for production of (3S,3'S) astaxanthin.
  • the present invention is of a peptide having a ⁇ -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
  • 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 l o 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
  • the present invention discloses the cloning of a cDNA from Haematococcus pluvialis, designated crtO, which encodes a ⁇ -C-4-oxygenase, the enzyme that converts ⁇ -carotene to canthaxanthin, and its expression in a heterologous systems expressing ⁇ -carotene hydroxylase (e.g., Erwinia herbicola crtZ gene product), leading to the production of (3S,3'S) astaxanthin.
  • crtO which encodes a ⁇ -C-4-oxygenase
  • the enzyme that converts ⁇ -carotene to canthaxanthin and its expression in a heterologous systems expressing ⁇ -carotene hydroxylase (e.g., Erwinia herbicola crtZ gene product), leading to the production of (3S,3'S) astaxanthin.
  • the crtO cDNA and its encoded peptide having a ⁇ -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 ⁇ -carotene, to which a cDNA library of Haematococcus pluvialis was transfected and expressed. Visual screening for
  • 25 brown-red pigmented Escherichia coli cells has identified a canthaxanthin producing transformant.
  • cloned cDNA has been expressed in two heterologous systems (Escherichia coli and Synechococcus PCC7942 cells) both able to produce ⁇ -carotene and further include an engineered (Erwinia herbicola crtZ gene product) or endogenous ⁇ -carotene hydroxylase activity, and was shown
  • 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
  • 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 ⁇ -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].
  • 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.
  • 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 man- induced 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 (i.e., variations).
  • the sequence variations will be 77-80%o, preferably 80-85%o, more preferably 85-90%), most preferably 90-100%) of identical nucleotides.
  • the DNA segment comprises the sequence set forth in SEQ ID NO: l .
  • 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 [e.g., 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 hundred successive nucleotides of SEQ ID N0: 1 or SEQ ID NO:2.
  • 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: l or SEQ ID NO:2.
  • a nucleic acid probe which includes the coding sequence (nucleotides 166 through 1152) of SEQ ID NO: l 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°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 capable of hybridizing under high-stringent conditions to a nucleic acid probe which includes the coding sequence (nucleotides 166 through 1 152) of SEQ ID NO: l or SEQ ID NO:2.
  • the invention also includes a synthetically produced oligonucleotide (e.g.. oligodeoxyribonucleotide or oligoribonucleotide and analogs thereof) capable of hybridizing with at least ten-nucleotide segments of SEQ ID NO: l or SEQ ID NO:2.
  • oligonucleotide e.g.. oligodeoxyribonucleotide or oligoribonucleotide and analogs thereof
  • 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.
  • the RNA segment comprises the sequence set forth in SEQ ID NO:2.
  • 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: l or SEQ ID NO:2.
  • 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: l or SEQ ID NO:2.
  • 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: l or SEQ ID NO:2.
  • 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 man-induced variants thereof.
  • the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the invention includes any peptide which is homologous (i.e., 80-85%, preferably 85-90%, more preferably 90-100% of identical amino acids) to the above described polypeptide.
  • homologous 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 positions shared by the two sequences. For example, if eight of ten of the positions in two sequences are occupied by identical amino acids then the two sequences are 80%) homologous.
  • 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
  • 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.
  • a vector for example plasmid or viral vector
  • the DNA segment is present in the vector operably linked to a promoter.
  • 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 (e.g., 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 (e.g., by a process known in the art as stable transfection) in the cell.
  • the cell is endogenously producing, or is made by genetic engineering means to produce, ⁇ -carotene, and the cell contains endogenous or genetically engineered ⁇ -carotene hydroxylase activity.
  • Such a cell may be used as a food additive for animal (e.g., salmon) and human consumption.
  • such a cell may be used for extracting astaxanthin and/or other xanthophylls, as described hereinbelow.
  • the present invention relates to a host transgenic organism (e.g., a higher plant or animal) containing the above described recombinant DNA molecule or the above described DNA segment in its cells.
  • a host transgenic organism e.g., a higher plant or animal
  • the host organism is endogenously producing, or is made by genetic engineering means to produce, ⁇ -carotene and, also preferably the host organism contains endogenous or genetically engineered ⁇ -carotene hydroxylase activity.
  • Such an organism may be used as a food additive for animal (e.g., salmon) and human consumption.
  • animal e.g., salmon
  • such an organism may be used for extracting astaxanthin and/or other xanthophylls, as described hereinbelow.
  • the present invention relates to a method of producing astaxanthin using the above described host cell or transgenic organism.
  • the present invention relates to a method of producing xanthophylls such as canthaxanthin, echinenone, cryptoxanthin, isocryptoxanthin, hydro xyechinenone, zeaxanthin, adonirubin, 3 -hydroxy echinenone, 3'- hydroxyechinenone and/or adonixanthin using the above described host cell or transgenic organism.
  • xanthophylls such as canthaxanthin, echinenone, cryptoxanthin, isocryptoxanthin, hydro xyechinenone, zeaxanthin, adonirubin, 3 -hydroxy echinenone, 3'- hydroxyechinenone and/or adonixanthin using the above described host cell or transgenic organism.
  • xanthophylls such as canthaxant
  • 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.
  • the expression is highest in chromoplasts-containing tissues.
  • the present invention relates to a recombinant DNA vector which includes a first DNA segment encoding a polypeptide for directing a protein into plant chloroplasts or chromoplasts (e.g., 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 man- induced variants thereof.
  • a recombinant DNA vector which includes a first DNA segment encoding a polypeptide for directing a protein into plant chloroplasts or chromoplasts (e.g., 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 man- induced variants thereof.
  • 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 (e.g., derived from the Pds gene of tomato) and a 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 man- induced variants thereof.
  • a recombinant DNA vector which includes a first DNA segment including a promoter highly expressible in plant chloroplasts or chromoplasts-containing tissues (e.g., derived from the Pds gene of tomato) and a 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 man- induced variants thereof.
  • the cDNA library was constructed in a Uni-ZAPT XR vector, using a ZAP-cDNA synthesis kit (both from Stratagene). Escherichia coli cells of strain XL 1 -Blue MRF' (Stratagene) were used for amplification of the cDNA library.
  • Plasmids and Escherichia coli strains Plasmids and Escherichia coli strains. Plasmid pPL376, which contains the genes necessary for carotenoid biosynthesis in the bacterium Erwinia herbicola was obtained from Tuveson [for further details regarding plasmid pPL376 see. Tuveson RW, Larson RA & Kagan J (1988) Role of cloned carotenoid genes expressed in Escherichia coli in protecting against inactivation by near-UV light 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.
  • a 1.1 kb Sall-Sal fragment was deleted from this plasmid to inactivate the gene crtX, coding for zeaxanthin glucosyl transferase.
  • partial BamRl cleavage of the plasmid DNA, followed by self ligation deleted a 0.8 kb fragment which inactivated crtZ, encoding ⁇ -carotene hydroxylase.
  • a partial Bgl l cleavage generated a fragment of 7.4 kb which was cloned in the BamHI site of the plasmid vector pACYC184.
  • the resulting recombinant plasmid which carried the genes crtE, crtB, crtl and crtY, was designated pBCAR [Lotan and Hirschberg (1995) FEBS letters 364: 125-128].
  • Plasmid pBCAR was transfected into SOLR strain cells o ⁇ 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 ⁇ -carotene.
  • LB chloramphenicol-containing Luria Broth
  • 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°C in the dark on a rotary shaker at 225 rpm. Ampicillin (50 ⁇ g/ml) and/or chloramphenicol (30 ⁇ g/ml) (both from Sigma) were added to the medium for selection of appropriate transformed cells.
  • a plasmid, pHPK containing the full length cDNA of the ⁇ -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).
  • the excised library in phagemids form was transfected into Escherichia coli cells strain XL l- Blue and the cells were plated on LB plates containing 1 mM isopropylthio- ⁇ -D- galactosidase (IPTG), 50 ⁇ g/ml ampicillin and 30 ⁇ g/ml chloramphenicol, in a density that yielded approximately 100-150 colonies per plate.
  • IPTG isopropylthio- ⁇ -D- galactosidase
  • the plates were incubated at 37°C overnight and further incubated for two more days at room temperature. The plates were then kept at 4°C until screened for changes in colony colors.
  • a plasmid for high expression of crtO in chromoplasts As shown in Figures 10- 1 1 , 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 Hindlll-Smal 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 1 1.
  • a 1, 1 10 nucleotide long £co47III-NcoI fragment, containing the cD ⁇ A of crtO from H pluvialis (nucleotides 21 1 to 1321 of SEQ ID ⁇ O: l ) was sub-cloned into the Smal site of the plasmid pPTBIB ( Figure 1 1 ) so that the coding nucleotide sequence of the amino terminus of Pds is in the same reading frame as crtO.
  • the recombinant plasmid was designate pPTCRTOBIB.
  • 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 electrop oration 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 grown at 28 °C in LB medium supplemented with 50 ⁇ g/ml streptomycin and 50 ⁇ g/ml kanamycin as selective agents.
  • 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.
  • 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 Hind ⁇ ll-Hindl ⁇ l 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.
  • 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 ⁇ l of acetone and incubated at 65°C for 15 minutes. The samples were centrifuged again at 13,000 g for 10 minutes and the carotenoid- containing supernatant was placed in a clean tube. The carotenoid extract was blown to dryness under a stream of nitrogen (N2) gas and stored at -20°C until required for analysis. Carotenoids from plant tissues were extracted by mixing 0.5-1.0 gr of tissue with 100 ⁇ l of acetone followed by incubation at 65°C for 15 minutes and then treating the samples as described above.
  • N2 nitrogen
  • High-performance liquid chromatography (HPLC) of the carotenoid extracts was carried out using an acidified reverse-phase C 18 column, Spherisorb ODS-2 (silica 5 ⁇ m 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 1.5 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.
  • Echinenone (2) R O.90 inseparable from authentic (2).
  • %III/II 0.
  • %III/II 0. ⁇ -Cryptoxanthin (4).
  • R t .VIS ⁇ max nm: (428), 451, 479, %III/II 0.
  • Astaxanthin (6) R ⁇ 0.79 inseparable from authentic (6).
  • R t .VIS ⁇ max nm: 477, %III/II 0.
  • 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 20: 2561-2565]. The analysis proved that the Escherichia coli cells synthesize pure (3S,3'S) astaxanthin.
  • 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 ⁇ -carotene-accumulating cells o ⁇ Escherichia coli, strain SOLR. which carried plasmid pBCAR (shown in Figure 4). Screening for a ⁇ -carotene oxygenase gene was based on color visualization of colonies of size of 3 mm in diameter. Astaxanthin and other oxygenated forms of ⁇ -carotene (i.e..).
  • xanthophylls have distinct darker colors and thus can be detected from the yellow ⁇ -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 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.
  • the red-brown colony presumed to contain a xanthophyll biosynthesis gene was streaked and further analyzed.
  • the recombinant ZAP II plasmid carrying the cDNA clone that was responsible for xanthophyll synthesis in Escherichia coli was isolated by preparing plasmid DNA from the red- brown colony, transfecting it to Escherichia coli cells of the strain XL 1 -Blue and selection on ampicillin-containing medium.
  • pHPK pHPK
  • pHPK is a Lambda ZAP II vector containing an insert isolated from the red-brown colony
  • ⁇ -carotene-producing Escherichia coli cells Escherichia coli SOLR strain that carry the plasmid pBCAR shown in Figure 4
  • Carotenoids from this transformant, as well as from the host cells (as control) were extracted by acetone and analyzed by HPLC.
  • crtO encodes an enzyme with ⁇ -C-4-oxygenase activity, which converts ⁇ -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 ⁇ - and ⁇ '-rings of ⁇ -carotene, respectively.
  • ⁇ -carotene hydroxylase e.g., a product of the crtZ gene o ⁇ Erwinia herbicola
  • canthaxanthin to astaxanthin
  • zeaxanthin converted from ⁇ -carotene by ⁇ -carotene hydroxylase can be converted by ⁇ -C-4-oxygenase to astaxanthin
  • the crtO cDNA of Haematococcus pluvialis thus isolated, was expressed in Escherichia coli cells together with the crtZ gene of Erwinia herbicola.
  • 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 which carry both pHPK and pZEAX.
  • ORF open reading frame
  • the plasmid DNA of pPAN3.5-KETO 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.
  • HPLC analysis of these cells revealed that the major carotenoid components of the cells was ⁇ -carotene, echinenone, canthaxanthin, adonirubin and astaxanthin.
  • Producing natural astaxanthin in higher plants has two anticipated benefits.
  • 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.
  • carotenoids are normally synthesized and accumulated to high concentration in chromoplasts, a typical pigment- containing plastids, thus providing typical intense colors to these organs. Inducing synthesis of astaxanthin in chromoplasts enables the accumulation of high concentration of this ketocarotenoid.
  • 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.
  • 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.
  • 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.
  • 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.
  • this DNA construct via Agrobacterium- mediated 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 (chromoplast- containing cells).
  • the Pds gene promoter is capable of directing transcription and therefore expression especially in chloroplasts and/or chromoplasts-containing tissues of plants.
  • the transit peptide encoded by part of the Pds coding sequence is capable of directing conjugated (i.e., in frame) proteins into plant chromoplasts and/or chloroplasts.
  • conjugated proteins i.e., in frame
  • this DNA construct induces accumulation of astaxanthin and other ketocarotenoids to a higher level which alters the color from the normal yellow to red.
  • chloroplasts-containing tissues such as leaves
  • chromoplast-containing tissues such as flowers
  • 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.
  • 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 ⁇ -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 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.
  • TITLE OF INVENTION Polynucleotide molecule from Haematococcus pluvialis encoding a polypeptide having a ⁇ -C-4-oxygenase activity for biotechnological production of
  • NAME F ⁇ edmam, Mark M.
  • TGT GGC AGG ACT GGG TGA GGT GAA AAG CTG CAG GCG CTG CTG CCG GAC 1248

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Abstract

La présente invention se rapporte, de manière générale, à un procédé de production, par biosynthèse, de (3S, 3'S) astaxanthine. Elle se rapporte en particulier à un peptide possédant une activité de β-C-4-oxygénase, à un segment d'ADN codant ce peptide, à des segments d'ARN codant ce peptide, à une molécule d'ADN recombiné comprenant un vecteur et le segment d'ADN, à une cellule hôte ou à un organisme contenant la molécule d'ADN recombiné ou le segment d'ADN décrit ci-dessus, et enfin à un procédé de production, par processus biotechnologique et au moyen de l'hôte, de (3S, 3'S) astaxanthine ou d'un additif alimentaire contenant de la (3S, 3'S) astaxanthine.
EP97909944A 1996-10-28 1997-10-03 Sequence d'acide nucleique codant la beta-c-4-oxygenase et provenant de haemotococcus pluvialis, utilisee pour la biosynthese de l'astaxanthine Withdrawn EP0951534A4 (fr)

Applications Claiming Priority (3)

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US742605 1996-10-28
US08/742,605 US5965795A (en) 1995-11-24 1996-10-28 Polynucleotide molecule from Haematococcus pluvialis encoding a polypeptide having a beta-C-4-oxygenase activity for biotechnological production of (3S, 3'S) astaxanthin and its specific expression in chromoplasts of higher plants
PCT/US1997/017819 WO1998018910A1 (fr) 1995-11-24 1997-10-03 Sequence d'acide nucleique codant la beta-c-4-oxygenase et provenant de haemotococcus pluvialis, utilisee pour la biosynthese de l'astaxanthine

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US5916791A (en) * 1995-11-24 1999-06-29 Hirschberg; Joseph Polynucleotide molecule from Haematococcus pluvialis encoding a polypeptide having a β--C--4--oxygenase activity for biotechnological production of (3S,3S)astaxanthin

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