US20070094749A1

US20070094749A1 - Method for producing ketocarotinoids in plant fruit

Info

Publication number: US20070094749A1
Application number: US10/524,829
Authority: US
Inventors: Christel Schopfer; Ralf Flachmann; Karin Herbers; Irene Kunze; Matt Sauer; Martin Klebsattel
Original assignee: SunGene GmbH
Current assignee: SunGene GmbH
Priority date: 2002-08-20
Filing date: 2003-08-18
Publication date: 2007-04-26
Also published as: MXPA05001811A; WO2004018695A2; MXPA05001948A; EP1542945A2; WO2004018694A2; US20050281909A1; CA2496133A1; MXPA05001659A; IL166507A0; US20060031963A1; EP1531683B1; ZA200602230B; WO2004018693A3; CA2495235A1; WO2004018693A2; EP1532266A2; AU2003260424A1; NO20050755L; AU2003253416A1; WO2004017749A3

Abstract

The invention relates to a method for the production of ketocarotenoids by culturing genetically modified plants which show a ketolase activity in fruits.

Description

The present invention relates to a method for the production of ketocarotenoids by culturing genetically modified plants which show a ketolase activity in fruits, to the genetically modified plants, and to their use as foods and feeds and for the production of ketocarotenoid extracts.
Carotenoids are synthesized de novo in bacteria, algae, fungi and plants. Ketocarotenoids, i.e. carotenoids comprising at least one keto group, such as, for example, astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin and adonixanthin are natural antioxidants and pigments which are produced by some algae and microorganisms as secondary metabolites.
Owing to their color-imparting properties, the ketocarotenoids, and in particular astaxanthin, are used as pigmenting auxiliaries in animal nutrition, in particular in trout, salmon and shrimp farming.
Currently, astaxanthin is largely produced synthetically by chemical methods. Natural ketocarotenoids, such as, for example, natural astaxanthin, are currently obtained in small amounts by biotechnological methods by culturing algae, for example Haematococcus pluvialis, or by fermenting microorganisms which have been optimized by genetic engineering, followed by isolation.
An economical biotechnological method for the production of natural ketocarotenoids is therefore of great importance.
WO 98/18910 describes the synthesis of ketocarotenoids in nectar glands of tobacco flowers by introducing a ketolase gene into tobacco.
WO 01/20011 describes a DNA construct for the production of ketocarotenoids, in particular astaxanthin, in the seeds of oilseed plants such as oilseed rape, sunflower, soybean and mustard, using a seed-specific promoter and a ketolase from Haematococcus.
While the methods disclosed in the prior art yield genetically modified plants with a ketocarotenoid content in the specific tissues, they have the disadvantage that the level of the ketocarotenoid content and the purity, in particular with regard to astaxanthin, is as yet unsatisfactory.
The invention was therefore based on the object of providing an alternative method for the production of ketocarotenoids by culturing plants, or of providing further transgenic plants which produce ketocarotenoids, which have the optimized characteristics, such as, for example, a higher ketocarotenoid content, and which do not suffer from the above-described disadvantage of the prior art.
Accordingly, there has been found a method for the production of ketocarotenoids by culturing genetically modified plants which show a ketolase activity in fruits.
Ketolase activity is understood as meaning the enzyme activity of a ketolase.
A ketolase is understood as meaning a protein with the enzymatic activity of introducing a keto group at the optionally substituted β-ionone ring of carotenoids.
In particular, a ketolase is understood as meaning a protein with the enzymatic activity of converting β-carotene into canthaxanthin.
Accordingly, ketolase activity is understood as meaning the amount of β-carotene converted, or the amount of canthaxanthin formed, by the protein ketolase within a certain period of time.
To show a ketolase activity in the fruits of the genetically modified plants, a preferred embodiment involves the use of genetically modified plants which express a ketolase in fruits.
Genetically modified plants which comprise, in fruits, at least one nucleic acid encoding a ketolase are therefore preferably used in the method according to the invention.
No plants are known which show a ketolase activity in fruits as the wild type. In particular, the preferred plants described hereinbelow show no ketolase activity in fruits as the wild type.
In the present invention, the ketolase activity in fruits of the genetically modified plants is caused by the genetic modification of the starting plant. Thus, the genetically modified plant according to the invention shows a ketolase activity in fruits in comparison with the genetically non-modified starting plant and is thus preferably capable of expressing a ketolase in fruits.
The term “starting plant” or “wild type” is understood as meaning the corresponding non-genetically-modified starting plant.
The term “genetically modified plant” is preferably understood as meaning a plant which is genetically modified in comparison with the starting plant.
Depending on the context, the term “plant” can be understood as meaning the starting plant (wild type), or a genetically modified plant according to the invention or both.
Generating the gene expression of a nucleic acid encoding a ketolase, in the fruits of the plants, takes place preferably by introducing, into the starting plant, nucleic acids which encode ketolases.
The invention therefore relates in particular to the above-described method, wherein genetically modified plants into which at least one nucleic acid encoding a ketolase has been introduced, starting from a starting plant.
To this end, it is possible, in principle, that any ketolase gene, that is to say any nucleic acid which encodes a ketolase, can be used.
All the nucleic acids mentioned in the description can be for example an RNA, DNA or cDNA sequence.
In the case of genomic ketolase sequences from eukaryotic sources, which comprise introns, nucleic acid sequences which are preferably to be used are, in the event that the host plant is not capable, or cannot be made capable, of expressing the ketolase in question, ready-processed nucleic acids such as the corresponding cDNAs.
Examples of nucleic acids encoding a ketolase, and the corresponding ketolases, which can be used in the method according to the invention or in the genetically modified plants according to the invention described below are, for example, sequences from
Haematoccus pluvialis, in particular from Haematoccus pluvialis Flotow em. Wille (Accession No. X86782; nucleic acid: SEQ ID No. 1, protein SEQ ID No. 2),
Haematoccus pluvialis, NIES-144 (Accession No. D45881; nucleic acid: SEQ ID No. 3, protein SEQ ID No. 4),
Agrobacterium aurantiacum (Accession No. D58420; nucleic acid: SEQ ID No. 5, protein SEQ ID No. 6),
Alicaligenes spec. (Accession No. D58422; nucleic acid: SEQ ID No. 7, protein SEQ ID No. 8),
Paracoccus marcusii (Accession No. Y15112; nucleic acid: SEQ ID No. 9, protein SEQ ID No. 10).
Synechocystis sp. strain PC6803 (Accession No. 576617, NP442491; nucleic acid: SEQ ID No. 11, protein SEQ ID No. 12).
Bradyrhizobium sp. (Accession No. AF218415, BAB 74888; nucleic acid: SEQ ID No. 13, protein SEQ ID No. 14).
Nostoc sp. strain PCC7120 (Accession No. AP003592; nucleic acid: SEQ ID No. 15, protein SEQ ID No. 16).
Haematococcus pluvialis (Accession NO: AF534876, AAN03484; nucleic acid: SEQ ID NO: 37, protein: SEQ ID NO: 38)
Paracoccus sp. MBIC1143 (Accession NO: D58420, P54972; nucleic acid: SEQ ID NO: 39, protein: SEQ ID NO: 40)
Brevundimonas aurantiaca (Accession NO: AY166610, AAN86030; nucleic acid: SEQ ID NO: 41, protein: SEQ ID NO: 42)
Nodularia spumigena NSOR10 (Accession NO: AY210783, AAO64399; nucleic acid: SEQ ID NO: 43, protein: SEQ ID NO: 44)
Nostoc punctiforme ATCC 29133 (Accession NO: NZ_AABC01000195, ZP_—00111258; nucleic acid: SEQ ID NO: 45, protein: SEQ ID NO: 46)
Nostoc punctiforme ATCC 29133 (Accession NO: NZ_AABC01000196; nucleic acid: SEQ ID NO: 47, protein: SEQ ID NO: 48)
Deinococcus radiodurans R1 (Accession NO: E75561, AE001872; nucleic acid: SEQ ID NO: 49, protein: SEQ ID NO: 50)
Further natural examples of ketolases and ketolase genes which can be used in the method according to the invention can be found readily for example from various organisms whose genomic sequence is known by carrying out alignments of the amino acid sequences or of the corresponding backtranslated nucleic acid sequences from databases with the above-described sequences, and in particular with the sequences SEQ ID NO. 2 and/or SEQ ID NO. 16.
Further natural examples of ketolases and ketolase genes can furthermore be found readily from different organisms whose genomic sequence is not known by using hybridization techniques in the manner known per se, starting from the above-described nucleic acid sequences, in particular starting from the sequences SEQ ID NO. 1 and/or SEQ ID NO. 15.
The hybridization can be carried out under moderate (low-stringency) or, preferably under stringent (high-stringency) conditions.
Such hybridization conditions are described, for example, in Sambrook, J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (A Laboratory Manual), 2^ndEdition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
For example, the conditions during the washing step can be selected from the range of conditions delimited by those with less stringency (with 2×SSC at 50° C.) and those with high stringency (with 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC: 0.3 M sodium citrate, 3 M sodium chloride, pH 7.0).
Moreover, the temperature during the washing step can be increased from moderate conditions at room temperature, 22° C., to stringent conditions at 65° C.
Both parameters, salt concentration and temperature can be varied simultaneously, or else one of the two parameters can be kept constant, while only the other one is varied. Also, denaturing agents such as, for example, formamide or SDS can be employed during the hybridization step. In the presence of 50% formamide, the hybridization is preferably carried out at 42° C.
Some examples of conditions for hybridization and washing step are shown hereinbelow:

(1) hybridization conditions with, for example,
- (i) 4×SSC at 65° C., or
- (ii) 6×SSC at 45° C., or
- (iii) 6×SSC at 68° C., 100 mg/ml denatured fish sperm DNA, or
- (iv) 6×SSC, 0.5% SDS, 100 mg/ml denatured, fragmented salmon sperm DNA at 68° C., or
- (v) 6×SSC, 0.5% SDS, 100 mg/ml denatured, fragmented salmon sperm DNA, 50% formamide at 42° C., or
- (vi) 50% formamide, 4×SSC at 42° C., or
- (vii) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42° C., or
- (viii) 2× or 4×SSC at 50° C. (moderate conditions), or
- (ix) 30 to 40% formamide, 2× or 4×SSC at 42° C. (moderate conditions).
(2) washing steps for in each case 10 minutes, with, for example,
- (i) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C., or
- (ii) 0.1×SSC at 65° C., or
- (iii) 0.1×SSC, 0.5% SDS at 68° C., or
- (iv) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C., or
- (v) 0.2×SSC, 0.1% SDS at 42° C., or
- (vi) 2×SSC at 65° C. (moderate conditions).

In a preferred embodiment of the methods according to the invention, nucleic acids are introduced which encode a protein comprising the amino acid sequence SEQ ID NO. 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has at least 20%, by preference at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, especially preferably at least 90% identity at the amino acid level with the sequence SEQ ID NO. 2 and which has the enzymatic characteristic of a ketolase.
This may take the form of a natural ketolase sequence which can be found from other organisms as described above by alignment of the sequences, or else an artificial ketolase sequence which has been modified starting from the sequence SEQ ID NO. 2 by artificial variation, for example by substitution, insertion or deletion of amino acids.
In a further, preferred embodiment of the methods according to the invention, nucleic acids are introduced which encode a protein comprising the amino acid sequence SEQ ID NO. 16 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has at least 20%, by preference at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, especially preferably at least 90% identity at the amino acid level with the sequence SEQ ID NO. 16 and which has the enzymatic characteristic of a ketolase.
This may take the form of a natural ketolase sequence which can be found from other organisms as described above by alignment of the sequences, or else an artificial ketolase sequence which has been modified starting from the sequence SEQ ID NO. 16 by artificial variation, for example by substitution, insertion or deletion of amino acids.
In the description, the term “substitution” is understood as meaning the replacement of one or more amino acids by one or more amino acids. Substitutions which are preferably carried out are what are known as conservative substitutions, where the replaced amino acid has a similar property to the original amino acid, for example substitution of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, Ser by Thr.
Deletion is the replacement of an amino acid by a direct bond. Preferred positions for deletion are the termini of the polypeptide and the linkages between the individual protein domains.
Insertions are introductions of amino acids into the polypeptide chain, where a direct bond is formally replaced by one or more amino acids.

Identity between two proteins is understood as meaning the identity of the amino acids over in each case the entire protein length, in particular the identity which is calculated by comparison with the aid of the Lasergene software from DNASTAR, inc. Madison, Wis. (USA) using the Clustal method (Higgins DG, Sharp PM. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. April 1989; 5(2):151-1), setting the following parameters:



	Multiple alignment parameter:
	Gap penalty	10
	Gap length penalty	10
	Pairwise alignment parameter:
	K-tuple	1
	Gap penalty	3
	Window	5
	Diagonals saved	5

Accordingly, a protein which has at least 20% identity at the amino acid level with the sequence SEQ ID NO. 2 or 16 is, accordingly, understood as meaning a protein which, upon comparison of its sequence with the sequence SEQ ID NO. 2 or 16, in particular by the above program algorithm with the above parameter set, has at least 20% identity.
Suitable nucleic acid sequences are obtainable, for example, by backtranslation of the polypeptide sequence according to the genetic code.
Codons which are preferably used for this purpose are those which are used frequently in accordance with the plant-specific codon usage. The codon usage can be determined readily with the aid of computer evaluations of other, known genes of the organisms in question.
In an especially preferred embodiment, a nucleic acid comprising the sequence SEQ ID NO. 1 is introduced into the plant.
In a further especially preferred embodiment, a nucleic acid comprising the sequence SEQ ID NO. 15 is introduced into the plant.
All the abovementioned ketolase genes can furthermore be generated in the known manner by chemical synthesis, starting with the nucleotide units, such as, for example, by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix. Oligonucleotides can be synthesized chemically in the known manner for example by the phosphoamidite method (Voet, Voet, 2^ndEdition, Wiley Press New York, pp. 896-897). The annealing of synthetic oligonucleotides and filling in of gaps by means of the Klenow fragment of the DNA polymerase and ligation reactions, and general cloning methods, are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
In an especially preferred embodiment of the method according to the invention, genetically modified plants which show the highest expression rate of a ketolase in fruits are used.
This is preferably achieved by the gene expression of the ketolase taking place under the control of a fruit-specific promoter. For example, the above-described nucleic acids as described hereinbelow in detail are introduced into the plant in a nucleic acid construct in functional linkage with a fruit-specific promoter.
In accordance with the invention, plants are preferably understood as meaning plants which, as the wild type, have chromoplasts in fruits.
Further preferred plants additionally have, as the wild type, carotenoids, in particular β-carotene, zeaxanthin, neoxanthin, violaxanthin or lutein, in the fruits.
Further preferred plants have, as the wild type, additionally a hydroxylase activity in the fruits.
Hydroxylase activity is understood as meaning the enzyme activity of a hydroxylase.
A hydroxylase is understood as meaning a protein with the enzymatic activity of introducing a hydroxyl group at the optionally substituted β-ionone ring of carotenoids.
In particular, a hydroxylase is understood as meaning a protein with the enzymatic activity of converting β-carotene into zeaxanthin or cantaxanthin into astaxanthin.
Accordingly, hydroxylase activity is understood as meaning the amount of β-carotene or cantaxanthin converted, or the amount of zeaxanthin or astaxanthin formed, by the protein hydroxylase within a certain period of time.
In a preferred embodiment, plants are cultured which additionally show an increased hydroxylase activity and/or β-cyclase activity in comparison with the wild type.
Hydroxylase activity is understood as meaning the enzyme activity of a hydroxylase.
A hydroxylase is understood as meaning a protein with the enzymatic activity of introducing a hydroxyl group at the optionally substituted β-ionone ring of carotenoids.
In particular, a hydroxylase is understood as meaning a protein with the enzymatic activity of converting β-carotene into zeaxanthin or cantaxanthin into astaxanthin.
Accordingly, hydroxylase activity is understood as meaning the amount of β-carotene or cantaxanthin converted, or the amount of zeaxanthin or astaxanthin formed, by the protein hydroxylase within a certain period of time.
Thus, in the case of a hydroxylase activity which is increased in comparison with the wild type, the converted amount of β-carotene or cantaxanthin, or the amount of zeaxanthin or astaxanthin formed, by the protein hydroxylase is increased within a certain period of time in comparison with the wild type.
This increase of the hydroxylase activity amounts by preference to at least 5%, furthermore preferably at least 20%, furthermore preferably at least 50%, furthermore preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the hydroxylase activity of the wild type.
β-Cyclase activity is understood as meaning the enzyme activity of a β-cyclase.
A β-cyclase is understood as meaning a protein with the enzymatic activity of converting a terminal, linear residue of lycopene into a β-ionone ring.
In particular, a β-cyclase is understood as meaning a protein with the enzymatic activity of converting γ-carotene into β-carotene.
Accordingly, β-cyclase activity is understood as meaning the amount of γ-carotene converted, or the amount of β-carotene formed, by the protein β-cyclase within a certain period of time.
Thus, in the case of an increased β-cyclase activity in comparison with the wild type, the amount of γ-carotene converted, or the amount of β-carotene formed, by the protein β-cyclase within a certain period of time is increased in comparison with the wild type.
This increase of the β-cyclase activity amounts by preference to at least 5%, furthermore preferably at least 20%, furthermore preferably at least 50%, furthermore preferably at least 100%, more preferably at least 300%, even more preferably at least 500%, in particular at least 600% of the β-cyclase activity of the wild type.
In accordance with the invention, the term “wild type” is understood as meaning the corresponding non-genetically-modified starting plant.
Preferably, and in particular in cases where the plant or the wild type cannot be assigned unambiguously, “wild type” for increasing the hydroxylase activity, for increasing the β-cyclase activity and for increasing the ketocarotenoid content is understood as meaning in each case one reference plant.
This reference plant is preferably Lycopersicon esculentum.
The hydroxylase activity in genetically modified plants according to the invention and in wild-type, or reference, plants is preferably determined under the following conditions:
The hydroxylase activity is determined in vitro by the method of Bouvier et al. (Biochim. Biophys. Acta 1391 (1998), 320-328). Ferredoxin, ferredoxin-NADP oxidoreductase, catalase, NADPH and beta-carotene together with mono- and digalactosylglycerides are added to a certain amount of plant extract.
The hydroxylase activity is especially preferably determined by the method of Bouvier, Keller, d'Harlingue and Camara (Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L.; Biochim. Biophys. Acta 1391 (1998), 320-328) under the following conditions:
The in-vitro assay is carried out in a volume of 0.250 ml. The mixture comprises 50 mM potassium phosphate (pH 7.6), 0.025 mg spinach ferredoxin, 0.5 units spinach ferredoxin-NADP+ oxidoreductase, 0.25 mM NADPH, 0.010 mg beta-carotene (emulsified in 0.1 mg Tween 80), 0.05 mM of a mixture of mono- and digalactosylglycerides (1:1), 1 unit catalase, 200 mono- and digalactosylglycerides (1:1), 0.2 mg bovine serum albumin and plant extract in different volumes. The reaction mixture is incubated for 2 hours at 30° C. The reaction products are extracted with organic solvents such as acetone or chloroform/methanol (2:1) and determined by means of HPLC.
The β-cyclase activity in genetically modified plants according to the invention and in wild-type, or reference, plants is preferably determined under the following conditions:
The β-cyclase activity is determined in vitro by the method of Fraser and Sandmann (Biochem. Biophys. Res. Comm. 185(1) (1992) 9-15). The following are added to a certain amount of plant extract: potassium phosphate to act as buffer (pH 7.6), lycopene to act as substrate, stromaprotein from Capsicum, NADP+, NADPH and ATP.
The hydroxylase activity is especially preferably carried out by the method of Bouvier, d'Harlingue and Camara (Molecular Analysis of carotenoid cyclae inhibition; Arch. Biochem. Biophys. 346(1) (1997) 53-64) under the following conditions:
The in-vitro assay is carried out in a volume of 250 μl. The mixture comprises 50 mM potassium phosphate (pH 7.6), different amounts of plant extract, 20 nM lycopene, 250 μg of chromoplastidial stromaprotein from Capsicum, 0.2 mM NADP+, 0.2 mM NADPH and 1 mM ATP. NADP/NADPH and ATP are dissolved in 10 ml of ethanol together with 1 mg of Tween 80 immediately prior to addition to the incubation medium. After a reaction time of 60 minutes at 30° C., the reaction is quenched by addition of chloroform/methanol (2:1). The reaction products which are extracted in chloroform are analyzed by means of HPLC.
An alternative assay in which radioactive substrate is used is described in Fraser and Sandmann (Biochem. Biophys. Res. Comm. 185(1) (1992) 9-15).
Increasing the hydroxylase activity and/or β-cyclase activity can be effected in various ways, for example by eliminating inhibiting regulatory mechanisms at the expression and protein level, or by increasing the gene expression of nucleic acids encoding a hydroxylase and/or nucleic acids encoding a β-cyclase in comparison with the wild type.
Increasing the gene expression of the nucleic acids encoding a hydroxylase and/or increasing the gene expression of the nucleic acid encoding a β-cyclase in comparison with the wild type can likewise be effected in various ways, for example by inducing the hydroxylase gene and/or β-cyclase gene by activators, or by introducing one or more hydroxylase gene copies and/or β-cyclase gene copies, i.e. by introducing, into the plant, at least one nucleic acid encoding a hydroxylase and/or at least one nucleic acid encoding an ε-cyclase.
Increasing the gene expression of a nucleic acid encoding a hydroxylase and/or β-cyclase is also understood as meaning, in accordance with the invention, the manipulation of the expression of the plants' homologous, endogenous hydroxylase and/or β-cyclase.
This can be achieved for example by modifying the promoter DNA sequence of genes encoding hydroxylases and/or β-cyclases. Such a modification, which results in an increased expression rate of the gene, can be effected for example by the deletion or insertion of DNA sequences.
As described above, it is possible to modify the expression of the endogenous hydroxylase and/or β-cyclase by applying exogenous stimuli. This can be effected by specific physiological conditions, i.e. by the application of foreign substances.
Moreover, a modified, or increased, expression of an endogenous hydroxylase and/or β-cyclase gene can be achieved by a regulator protein which does not occur in the untransformed plant interacting with the promoter of this gene.
Such a regulator can be a chimeric protein which consists of a DNA binding domain and a transcription activator domain, as described, for example, in WO 96/06166.
In a preferred embodiment, the gene expression of a nucleic acid encoding a hydroxylase and/or increasing the gene expression of a nucleic acid encoding a β-cyclase is effected by introducing, into the plant, at least one nucleic acid encoding a hydroxylase and/or by introducing, into the plant, at least one nucleic acid encoding a β-cyclase.
In principle, any hydroxylase gene, or any β-cyclase gene, i.e. any nucleic acid which encodes a hydroxylase and any nucleic acid which encodes a β-cyclase can be used for this purpose.
In the case of genomic hydroxylase or β-cyclase nucleic acid sequences from eukaryotic sources, which comprise introns, it is preferred to use ready-processed nucleic acid sequences, such as the corresponding cDNAs, in the event that the host plant is not capable, or cannot be made capable, of expressing the hydroxylase or β-cyclase in question.
Examples of hydroxylase genes are nucleic acids encoding a hydroxylase from Haematococcus pluvialis, Accession AX038729, WO 0061764); (nucleic acid: SEQ ID NO: 51, protein: SEQ ID NO: 52),
and hydroxylases of the following Accession numbers:
|emb|CAB55626.1, CAA70427.1, CAA70888.1, CAB55625.1, AF499108 _—1, AF315289 _—1, AF296158 _—1, AAC49443.1, NP_—194300.1, NP_—200070.1, AAG10430.1, CAC06712.1, AAM88619.1, CAC95130.1, AAL80006.1, AF162276 _—1, AA053295.1, AAN85601.1, CRTZ_ERWHE, CRTZ_PANAN, BAB79605.1, CRTZ_ALCSP, CRTZ_AGRAU, CAB56060.1, ZP_—00094836.1, AAC44852.1, BAC77670.1, NP_—745389.1, NP_—344225.1, NP_—849490.1, ZP_—00087019.1, NP_—503072.1, NP_—852012.1, NP_—115929.1, ZP_—00013255.1
Moreover, an especially preferred hydroxylase is the hydroxylase from tomato (nucleic acid: SEQ ID No. 55; protein: SEQ ID No. 56).

Examples of b-cyclase genes are nucleic acids encoding a b-cyclase from tomato (Accession X86452). (Nucleic acid: SEQ ID NO: 53, protein: SEQ ID NO: 54), and b-cyclase genes of the following accession numbers:



S66350	lycopene beta-cyclase (EC 5.5.1.—) - tomato
CAA60119	lycopene synthase [Capsicum annuum]
S66349	lycopene beta-cyclase (EC 5.5.1.—) - common tobacco
CAA57386	lycopene cyclase [Nicotiana tabacum]
AAM21152	lycopene beta-cyclase [Citrus sinensis]
AAD38049	lycopene cyclase [Citrus × paradisi]
AAN86060	lycopene cyclase [Citrus unshiu]
AAF44700	lycopene beta-cyclase [Citrus sinensis]
AAK07430	lycopene beta-cyclase [Adonis palaestina]
AAG10429	beta cyclase [Tagetes erecta]
AAA81880	lycopene cyclase
AAB53337	Lycopene beta cyclase
AAL92175	beta-lycopene cyclase [Sandersonia aurantiaca]
CAA67331	lycopene cyclase [Narcissus pseudonarcissus]
AAM45381	beta cyclase [Tagetes erecta]
	AAO18661 lycopene beta-cyclase [Zea mays]
AAG21133	chromoplast-specific lycopene beta-cyclase
	[Lycopersicon esculentum]
AAF18989	lycopene beta-cyclase [Daucus carota]
ZP_001140	hypothetical protein [Prochlorococcus marinus str.
	MIT9313]
ZP_001050	hypothetical protein [Prochlorococcus marinus subsp.
	pastoris str. CCMP1378]
ZP_001046	hypothetical protein [Prochlorococcus marinus subsp.
	pastoris str. CCMP1378]
ZP_001134	hypothetical protein [Prochlorococcus marinus str.
	MIT9313]
ZP_001150	hypothetical protein [Synechococcus sp. WH 8102]
AAF10377	lycopene cyclase [Deinococcus radiodurans]
BAA29250	393aa long hypothetical protein [Pyrococcus horikoshii]
BAC77673	lycopene beta-monocyclase [marine bacterium P99-3]
AAL01999	lycopene cyclase [Xanthobacter sp. Py2]
ZP_000190	hypothetical protein [Chloroflexus aurantiacus]
ZP_000941	hypothetical protein [Novosphingobium aromaticivorans]
AAF78200	lycopene cyclase [Bradyrhizobium sp. ORS278]
BAB79602	crtY [Pantoea agglomerans pv. milletiae]
CAA64855	lycopene cyclase [Streptomyces griseus]
AAA21262	dycopene cyclase [Pantoea agglomerans]
C37802	crtY protein - Erwinia uredovora
BAB79602	crtY [Pantoea agglomerans pv. milletiae]
AAA64980	lycopene cyclase [Pantoea agglomerans]
AAC44851	lycopene cyclase
BAA09593	Lycopene cyclase [Paracoccus sp. MBIC1143]
ZP_000941	hypothetical protein [Novosphingobium aromaticivorans]
CAB56061	lycopene beta-cyclase [Paracoccus marcusii]
BAA20275	lycopene cyclase [Erythrobacter longus]
ZP_000570	hypothetical protein [Thermobifida fusca]
ZP_000190	hypothetical protein [Chloroflexus aurantiacus]
AAK07430	lycopene beta-cyclase [Adonis palaestina]
CAA67331	lycopene cyclase [Narcissus pseudonarcissus]
AAB53337	Lycopene beta cyclase
BAC77673	lycopene beta-monocyclase [marine bacterium P99-3]

Furthermore, an especially preferred β-cyclase is the chromoplast-specific β-cyclase from tomato (AAG21133) (nucleic acid: SEQ ID No. 57; protein: SEQ ID No. 58)
Thus, in the transgenic plants which are preferred in accordance with the invention, at least one further hydroxylase gene and/or β-cyclase gene is present in this preferred embodiment in comparison with the wild type.
In this preferred embodiment, the genetically modified plant has, for example, at least one exogenous nucleic acid encoding a hydroxylase or at least two endogenous nucleic acids encoding a hydroxylase and/or at least one exogenous nucleic acid encoding a β-cyclase or at least two endogenous nucleic acids encoding a β-cyclase.
Hydroxylase genes which are preferably used in the above-described preferred embodiment are nucleic acids which encode proteins comprising the amino acid sequence SEQ ID NO: 52 or a sequence which is derived from this sequence by substitution, insertion or deletion of amino acids and which have at least 30%, by preference at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% identity at the amino acid level with the sequence SEQ ID NO: 52 and which have the enzymatic property of a hydroxylase.
Further examples of hydroxylases and hydroxylase genes can be found readily, as described above, for example from various organisms whose genomic sequence is known, by homology comparisons of the amino acid sequences or of the corresponding backtranslated nucleic acid sequences from databases with SEQ ID NO: 52.
Further examples of hydroxylases and hydroxylase genes can furthermore be found readily in the manner known per se from various organisms whose genomic sequence is not known, by hybridization and PCR techniques as described above, for example starting from the sequence SEQ ID NO: 51.
In a further especially preferred embodiment, nucleic acids which encode proteins comprising the amino acid sequence of the hydroxylase of the sequence SEQ ID NO: 52 are introduced into organisms in order to increase the hydroxylase activity.
For example, suitable nucleic acid sequences can be obtained by backtranslation of the polypeptide sequence in accordance with the genetic code.
Codons which are preferably used for this purpose are codons which are used frequently in accordance with the plant-specific codon usage. The codon usage can be determined readily with the aid of computer evaluations of other, known genes of the organisms in question.
In an especially preferred embodiment, a nucleic acid comprising the sequence SEQ ID NO: 51 is introduced into the organism.
β-Cyclase genes which are preferably used in the above-described preferred embodiment are nucleic acids which encode proteins comprising the amino acid sequence SEQ ID NO: 54 or a sequence which is derived from this sequence by substitution, insertion or deletion of amino acids and which have at least 30%, by preference at least 50%, more preferably at least 70%, even more preferably at least 90%, most preferably at least 95% identity at the amino acid level with the sequence SEQ ID NO: 54 and which have the enzymatic property of a β-cyclase.
Further examples of β-cyclases and β-cyclase genes can be found readily, as described above, for example from various organisms whose genomic sequence is known, by homology comparisons of the amino acid sequences or of the corresponding backtranslated nucleic acid sequences from databases with SEQ ID NO: 54.
Further examples of β-cyclases and β-cyclase genes can furthermore be found readily in the manner known per se from various organisms whose genomic sequence is not known, by hybridization and PCR techniques, for example starting from the sequence SEQ ID NO: 53.
In a further especially preferred embodiment, nucleic acids which encode proteins comprising the amino acid sequence of the β-cyclase of the sequence SEQ ID NO: 54 are introduced into organisms in order to increase the β-cyclase activity.
For example, nucleic acid sequences can be obtained by backtranslation of the polypeptide sequence in accordance with the genetic code.
Codons which are preferably used for this purpose are codons which are frequently used in accordance with the plant-specific codon usage. The codon usage can be determined readily with the aid of computer evaluations of other, known genes of the organisms in question.
In an especially preferred embodiment, a nucleic acid comprising the sequence SEQ ID NO: 53 is introduced into the organism.
All of the abovementioned hydroxylase genes or β-cyclase genes can furthermore be generated in a known manner by chemical synthesis, starting with the nucleotide units, such as, for example, by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix. Oligonucleotides can be synthesized chemically in a known manner for example by the phosphoamidite method (Voet, Voet, 2^ndEdition, Wiley Press New York, pp. 896-897). The annealing of synthetic oligonucleotides and filling in of gaps by means of the Klenow fragment of the DNA polymerase and ligation reactions, and general cloning methods, are described in Sambrook et al. (1989), Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.
Especially preferred plants are plants selected from the plant genera Actinophloeus, Aglaeonema, Ananas, Arbutus, Archontophoenix, Area, Aronia, Asparagus, Attalea, Berberis, Bixia, Brachychilum, Bryonia, Cliptocalix, Capsicum, Carica, Celastrus, Citrullus, Citrus, Convallaria, Cotoneaster, Crataegus, Cucumis, Cucurbita, Cuscuta, Cycas, Cyphomandra, Dioscorea, Diospyrus, Dura, Elaeagnus, Elaeis, Erythroxylon, Euonymus, Ficus, Fortunella, Fragaria, Gardinia, Gonocaryum, Gossypium, Guava, Guilielma, Hibiscus, Hippophaea, Iris, Lathyrus, Lonicera, Luffa, Lycium, Lycopersicum, Malpighia, Mangifera, Mormodica, Murraya, Musa, Nenga, Palisota, Pandanus, Passiflora, Persea, Physalis, Prunus, Ptychandra, Punica, Pyracantha, Pyrus, Ribes, Rosa, Rubus, Sabal, Sambucus, Seaforita, Shepherdia, Solanum, Sorbus, Synaspadix, Tabernae, Tamus, Taxus, Trichosanthes, Triphasia, Vaccinium, Viburnum, Vignia or Vitis.
The ketolase activity in genetically modified plants according to the invention is determined by a method similar to that of Frazer et al., (J. Biol. Chem. 272(10): 6128-6135, 1997). The ketolase activity in plant extracts is determined using the substrates beta-carotene and canthaxanthin in the presence of lipid (soya lecithin) and detergent (sodium cholate). Substrate/product ratios from the ketolase assays are determined by HPLC.
In the method according to the invention for the production of ketocarotenoids, the cultivation step of the genetically modified plants, hereinbelow also referred to as transgenic plants, is preferably followed by harvesting of the plants and isolating ketocarotenoids from the fruits of the plants.
The transgenic plants are grown in a manner known per se on substrates and harvested in a suitable manner.
Ketocarotenoids are isolated from the harvested fruits in a manner known per se, for example by drying followed by extraction and, if appropriate, further chemical or physical purification processes such as, for example, precipitation methods, crystallography, thermal separation methods such as rectification methods or physical separation methods such as, for example, chromatography. Preferably, for example, ketocarotenoids are isolated from the fruits with organic solvents such as acetone, hexane, ether or tert-methyl butyl ether.
Further isolation methods for ketocarotenoids are described, for example, in Egger and Kleinig (Phytochemistry (1967) 6, 437-440) and Egger (Phytochemistry (1965) 4, 609-618).
By preference, the ketocarotenoids are selected from the group astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin and adonixanthin.
An especially preferred ketocarotenoid is astaxanthin.
The transgenic plants are preferably generated by transforming the starting plants with a nucleic acid construct which comprises at least one, preferably also a plurality of the above-described nucleic acids which are functionally linked to one or more regulatory signals which ensure the transcription and translation in plants.
These nucleic acid constructs in which the coding nucleic acid sequence is functionally linked to one or more regulatory signals which ensure the transcription and translation in plants are hereinbelow also referred to as expression cassettes.
By preference, the regulatory signals comprise one or more promoters which ensure the transcription and translation in plants.
The expression cassettes comprise regulatory signals, i.e. regulatory nucleic acid sequences which regulate the expression of the coding sequence in the host cell. In accordance with a preferred embodiment, an expression cassette comprises upstream, i.e. at the 5′terminus of the coding sequence, a promoter and downstream, i.e. at the 3′terminus, a polyadenylation signal and, if appropriate, further regulatory elements which are linked operably with the interjacent coding sequence for at least one of the above-described genes. Operable linkage is understood as meaning the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfil its intended function when the coding sequence is expressed.
The preferred nucleic acid constructs, expression cassettes and vectors for plants and methods for producing transgenic plants, and the transgenic plants themselves, are described hereinbelow by way of example.
The sequences which are preferred for the operable linkage, but not limited thereto, are targeting sequences for ensuring the subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrium, in the endoplasmic reticulum (ER), in the nucleus, in oil bodies or other compartments, and translation enhancers such as the tobacco mosaic virus 5′-leader sequence (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).
In principle, any promoter which is capable of controlling the expression of foreign genes in plants is suitable as promoter of the expression cassette.
“Constitutive” promoter means those promoters which ensure expression in a large number of, preferably all, tissues over a substantial period of the plant's development, preferably at all points in time of the plant's development.
A promoter which is used by preference is, in particular, a plant promoter or a promoter derived from a plant virus. Especially preferred is the promoter of the CaMV cauliflower mosaic virus 35S transcript (Franck et al. (1980) Cell 21:285-294; Odell et al. (1985) Nature 313:810-812; Shewmaker et al. (1985) Virology 140:281-288; Gardner et al. (1986) Plant Mol Biol 6:221-228) or the 19S CaMV promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J 8:2195-2202).
A further suitable constitutive promoter is the pds promoter (Pecker et al. (1992) Proc. Natl. Acad. Sci USA 89: 4962-4966) or the “Rubisco small subunit (SSU)” promoter (U.S. Pat. No. 4,962,028), the legumin B promoter (GenBank Acc. No. X03677), the promoter of the Agrobacterium nopaline synthase, the TR dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649), the ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol 18:675-689; Bruce et al. (1989) Proc Natl Acad Sci USA 86:9692-9696), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich protein from wheat (WO 91/13991), the Pnit promoter (Y07648.L, Hillebrand et al. (1998), Plant. Mol. Biol. 36, 89-99, Hillebrand et al. (1996), Gene, 170, 197-200, the ferredoxin NADPH oxidoreductase promoter (database entry AB011474, position 70127 to 69493), the TPT promoter (WO 03006660), the “superpromoter” (U.S. Pat. No. 5,955,646), the 34S promoter (U.S. Pat. No. 6,051,753), and further promoters of genes whose constitutive expression in plants is known to the skilled worker.
The expression cassettes may also comprise a chemically inducible promoter (review paper: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol 48:89-108), by means of which the expression of the ketolase gene in the plant can be controlled at a particular point in time. Such promoters such as, for example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), salicylic-acid-inducible promoter (WO 95/19443), a benzene-sulfonamide-inducible promoter (EP 0 388 186), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J 2:397-404), an abscisic-acid-inducible promoter (EP 0 335 528) or an ethanol- or cyclohexanone-inducible promoter (WO 93/21334) can likewise be used.
Other preferred promoters are those which are induced by biotic or abiotic stress such as, for example, the pathogen-inducible promoter of the PRP1 gene (Ward et al. (1993) Plant Mol Biol 22:361-366), the heat-inducible hsp70 or hsp80 promoter from tomato (U.S. Pat. No. 5,187,267), the cold-inducible alpha-amylase promoter from potato (WO 96/12814), the light-inducible PPDK promoter or the wounding-induced pinII promoter (EP375091).
Pathogen-inducible promoters comprise the promoters of genes which are induced as the result of a pathogen attack such as, for example, genes of PR proteins, SAR proteins, β-1,3-glucanase, chitinase and the like (for example Redolfi et al. (1983) Neth J Plant Pathol 89:245-254; Uknes, et al. (1992) The Plant Cell 4:645-656; Van Loon (1985) Plant Mol Viral 4:111-116; Marineau et al. (1987) Plant Mol Biol 9:335-342; Matton et al. (1987) Molecular Plant-Microbe Interactions 2:325-342; Somssich et al. (1986) Proc Natl Acad Sci USA 83:2427-2430; Somssich et al. (1988) Mol Gen Genetics 2:93-98; Chen et al. (1996) Plant J 10:955-966; Zhang and Sing (1994) Proc Natl Acad Sci USA 91:2507-2511; Warner, et al. (1993) Plant J 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968(1989).
Also comprised are wounding-inducible promoters such as that of the promoter of the pinII gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996) Nat Biotech 14:494-498), of the wun1 and wun2 gene (U.S. Pat. No. 5,428,148), of the win1 and win2 gene (Stanford et al. (1989) Mol Gen Genet 215:200-208), of the systemin (McGurl et al. (1992) Science 225:1570-1573), of the WIP1 gene (Rohmeier et al. (1993) Plant Mol Biol 22:783-792; Ekelkamp et al. (1993) FEBS Letters 323:73-76), of the MPI gene (Corderok et al. (1994) The Plant J 6(2):141-150) and the like.
Further suitable promoters are, for example, fruit-maturation-specific promoters such as, for example, the fruit-maturation-specific promoter from tomato (WO 94/21794, EP 409 625). Some of the promoters which the development-promoters comprise are the tissue-specific promoters since, naturally, the individual tissues are formed as a function of the development.
Furthermore preferred are in particular those promoters which ensure the expression in tissues or plant parts in which, for example, the biosynthesis of ketocarotenoids or their precursors takes place. Examples of preferred promoters are promoters with specificities for the anthers, ovaries, petals, sepals, flowers, leaves, stems, roots and fruits and combinations hereof.
Tuber-specific, storage-root-specific or root-specific promoters are, for example, the patatin promoter class I (B33) or the promoter of the cathepsin D inhibitor from potato.
Examples of leaf-specific promoters are, for example, the promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of Rubisco (ribulose-1,5-bisphosphate carboxylase) or the ST-LSI promoter from potato (Stockhaus et al. (1989) EMBO J 8:2445-2451).
Examples of flower-specific promoters are the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).
Examples of anther-specific promoters are the 5126 promoter (U.S. Pat. No. 5,689,049, U.S. Pat. No. 5,689,051), the glob-1 promoter or the g-zein promoter.
Fruit-specific promoters are, for example,
the Pds promoter from tomato (Genbank ACCESSION U46919; Corona, V., Aracri, B., Kosturkova, G., Bartley, G. E., Pitto, L., Giorgetti, L., Scolnik, P. A. and Giuliano, G., Regulation of a carotenoid biosynthesis gene promoter during plant development Plant J. 9 (4), 505-512 (1996)), SEQ ID NO. 17, the 2A11 promoter from tomato (Pear, J. R., Ridge, N., Rasmussen, R., Rose, R. E. and Houck, C. M. Isolation and characterization of a fruit-specific cDNA and the corresponding genomic clone from tomato Plant Mol. Biol. 13 (6), 639-651 (1989), SEQ ID NO. 18,
the cucumisin promoter (Yamagata, H., Yonseu, K., Hirata, A. and Aizono, Y., TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved in Fruit-specific Expression of the cucumisin Gene J. Biol. Chem. 277 (13), 11582-11590 (2002), SEQ ID NO. 19,
the promoter of the endogalacturonase gene (Redondo-Nevado, J., Medina-Escobar, N., Caballero-Repullo, J. L. and Muonz-Blanco, J.
A fruit-specific and developmentally regulated endo-polygalacturonase gene from strawberry (Fragaria x ananassa c.v. Chandler), J Experimental Botany 52 (362) 1941-1945 (2001), SEQ ID NO. 20,
the polygalacturonase promoter from tomato (Nicholass, F. J., Smith, C. J., Schuch, W., Bird, C. R. and Grierson, D., High levels of ripening-specific reporter gene expression directed by tomato fruit polygalacturonase gene-flanking regions, Plant Mol. Biol. 28 (3), 423-435 (1995)), SEQ ID NO. 21,
the TMF7 and TMF9 promoters (U.S. Pat. No. 5,608,150),
the E4 promoter (Cordes A. Deikman J. Margossian L J. Fischer R L. Interaction of a developmentally regulated DNA-binding factor with sites flanking two different fruit-ripening genes from tomato (1989), Plant Cell 1, 1025-1034) and
the E8 promoter (Deikman and Fisher, Interaction of a DNA binding factor with the 5′-flanking region of an ethylene-responsive fruit ripening gene from tomato (1988), EMBO J. 7, 3315-3320). Further promoters which are suitable for expression in plants are described (Rogers et al. (1987) Meth. in Enzymol 153:253-277; Schardl et al. (1987) Gene 61:1-11; Berger et al. (1989) Proc Natl Acad Sci USA 86:8402-8406).
As a rule, all of the promoters described in the present application make possible the expression of ketolase in fruits of the plants according to the invention.
Especially preferred in the method according to the invention are constitutive and, in particular, fruit-specific promoters.
The present invention therefore relates in particular to a nucleic acid construct comprising, in functional linkage, a fruit-specific promoter, particularly preferably a fruit-specific promoter described above and a nucleic acid encoding a ketolase.
An expression cassette is prefereably prepared by fusing a suitable promoter with an above-described nucleic acid encoding a ketolase and preferably a nucleic acid which is inserted between promoter and nucleic acid sequence and which encodes a plastid-specific transit peptide, and with a polyadenylation signal, using customary recombination and cloning techniques as are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman und L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
The nucleic acids which encode a plastidic transit peptide and which are preferably inserted ensure the localization in plastids and in particular in chromoplasts.
It is also possible to use expression cassettes whose nucleic acid sequence encodes a ketolase fusion protein, where part of the fusion protein is a transit peptide which governs the translocation of the polypeptide. Preferred are chromoplast-specific transit peptides which are cleaved enzymatically from the ketolase moiety after translocation of the ketolase into the chromoplasts.
Especially preferred is the transit peptide which is derived from the plastidic Nicotiana tabacum transketolase or from another transit peptide (for example the transit peptide of the Rubisco small subunit (rbcS) or the transit peptide of the ferredoxin-NADP oxidoreductase and of the isopentenyl-pyrophosphate isomerase-2) or its functional equivalent.

Particularly preferred are nucleic acid sequences of three cassettes of the plastid transit peptide of the tobacco plastidic transketolase in three reading frames as KpnI/BamHI fragments with an ATG codon in the NcoI cleavage site:


pTP09
KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATC
CTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTC
CCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCA
CCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGG
TCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGA
GACTGCGGGATCC_BamHI

pTP10
KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATC
CTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTC
CCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCA
CCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGG
TCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGA
GACTGCGCTGGATCC_BamHI

pTP11
KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATC
CTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTC
CCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCA
CCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGG
TCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGA
GACTGCGGGGATCC_BamHI

Further examples of a plastidic transit peptide are the transit peptide of the plastidic isopentenyl-pyrophosphate isomerase-2 (IPP-2) from Arabidopsis thaliana and the transit peptide of the ribulose-bisphosphate carboxylase small subunit (rbcS) from pea (Guerineau, F, Woolston, S, Brooks, L, Mullineaux, P (1988) An expression cassette for targeting foreign proteins into the chloroplasts. Nucl. Acids Res. 16: 11380).
The nucleic acids according to the invention can be generated synthetically or obtained naturally or comprise a mixture of synthetic and natural nucleic acid constituents, and consist of various heterologous gene segments from a variety of organisms.
Preferrred are, as described above, synthetic nucleotide sequences with codons which are preferred by plants. These codons which are preferred by plants can be determined from codons with the highest protein frequency which are expressed in most of the plant species of interest.
When preparing an expression cassette, various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and is equipped with a correct reading frame. To link the DNA fragments to one another, adaptors or linkers may be added to the fragments.
Expediently, the promoter and the terminator regions can be provided, in the direction of transcription, with a linker or polylinker comprising one or more restriction sites for the insertion of this sequence. As a rule, the linker has 1 to 10, in most cases 1 to 8, preferably 2 to 6, restriction sites. In general, the linker has a size of less than 100 bp, frequently less than 60 bp, but at least 5 bp, within the regulatory regions. The promoter can either be native, or homologous, or else foreign, or heterologous, to the host plant. Preferably, the expression cassette comprises, in the 5′-3′ direction of transcription, the promoter, a coding nucleic acid sequence or a nucleic acid construct and a region for transcriptional termination. Various termination regions can be exchanged for one another as desired.
An example of a terminator is the 35S terminator (Guerineau et al. (1988) Nucl Acids Res. 16: 11380), the nos terminator (Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman H M. Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet. 1982; 1(6):561-73) or the ocs terminator (Gielen, J, de Beuckeleer, M, Seurinck, J, Debroek, H, de Greve, H, Lemmers, M, van Montagu, M, Schell, J (1984) The complete sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5. EMBO J. 3: 835-846).
Furthermore, it is possible to employ manipulations which provide suitable restriction cleavage sites or which remove superfluous DNA or restriction cleavage sites. Where insertions, deletions or substitutions such as, for example, transitions and transversions are suitable, it is possible to use in-vitro mutagenesis, primer repair, restriction or ligation.
In the case of suitable manipulations such as, for example, restriction, chewing-back or filling up overhangs for blunt ends, it is possible to provide complementary ends of the fragments for the ligation.
Preferred polyadenylation signals are plant polyadenylation signals, preferably those which correspond essentially to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular the gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 et seq.), or functional equivalents.
The transfer of foreign genes in the genome of a plant is referred to as transformation.
To this end, it is possible to exploit methods which are known per se for the transformation and regeneration of plants from plant tissues or plant cells in order to carry out a transient or stable transformation.
Suitable methods for the transformation of plants are the transformation of protoplasts by means of polyethylene-glycol-induced DNA uptake, the biolistic method using the gene gun—what is known as the particle bombardment method, electroporation, incubation of dry embryos in DNA-comprising solution, microinjection, and the above-described Agrobacterium-mediated gene transfer. The above methods are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press (1993), 128-143 and in Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225).
By preference, the construct to be expressed is cloned into a vector which is suitable for the transformation of Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711) or particularly preferably pSUN2, pSUN3, pSUN4 or pSUN5 (WO 02/00900).
Agrobacteria which have been transformed with an expression plasmid can be used in the known manner for the transformation of plants, for example by bathing scarified leaves or leaf segments in an agrobacterial solution and subsequently growing them in suitable media.
For the preferred generation of genetically modified plants, hereinbelow also referred to as transgenic plants, the fused expression cassette which expresses a ketolase is cloned into a vector, for example pBin19 or, in particular, pSUN2, which is suitable for being transformed into Agrobacterium tumefaciens.
Agrobacteria which have been transformed with such a vector can then be used in the known manner for the transformation of plants, in particular crop plants, for example by bathing scarified leaves or leaf segments in an agrobacterial solution and subsequently growing them in suitable media.
The transformation of plants by agrobacteria is known, inter alia, from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38. Transgenic plants can be regenerated in the known manner from the transformed cells of the scarified leaves or leaf segments, and such plants comprise a gene for the expression of a nucleic acid encoding a ketolase integrated into the expression cassette.
To transform a host plant with a nucleic acid which encodes a ketolase, an expression cassette is incorporated, as insertion, into a recombinant vector whose vector DNA comprises additional functional regulatory signals, for example sequences for replication or integration. Suitable vectors are described, inter alia, in “Methods in Plant Molecular Biology and Biotechnology” (CRC Press), chapter 6/7, pp. 71-119 (1993).
Using the above-cited recombination and cloning techniques, the expression cassettes can be cloned into suitable vectors which make possible their multiplication, for example in E. coli. Suitable cloning vectors are, inter alia, pJIT117 (Guerineau et al. (1988) Nucl. Acids Res. 16:11380), pBR332, pUC series, M13mp series, pACYC184, pMC1210, pMc1210 and pCL1920. Especially suitable are binary vectors, which are capable of replication both in E. coli and in agrobacteria.
In this context, expression can take place constitutively or, preferably, specifically in the fruits, depending on the choice of the promoter.
Accordingly, the invention furthermore relates to a method for the production of genetically modified plants, wherein a nucleic acid construct comprising, in functional linkage, a fruit-specific promoter and nucleic acids encoding a ketolase is introduced into the genome of the starting plant.
The invention furthermore relates to the genetically modified plants with a ketolase activity in fruits in comparison with the starting plant.
In a preferred embodiment, the ketolase activity is achieved by the fact that the genetically modified plant expresses a ketolase in the fruits.
The preferred genetically modified plants therefore comprise, in fruits, at least one nucleic acid encoding a ketolase.
In a further preferred embodiment, the gene expression of a nucleic acid encoding a ketolase is caused, as detailed above, by introducing, into the starting plant, nucleic acids encoding a ketolase.
The invention therefore especially preferably relates to an above-described genetically modified plant, wherein, starting from a starting plant, at least one nucleic acid encoding a ketolase has been introduced into the plant.
In particular, the invention relates to genetically modified plants selected from the plant genera Actinophloeus, Aglaeonema, Ananas, Arbutus, Archontophoenix, Area, Aronia, Asparagus, Attalea, Berberis, Bixia, Brachychilum, Bryonia, Cliptocalix, Capsicum, Carica, Celastrus, Citrullus, Citrus, Convallaria, Cotoneaster, Crataegus, Cucumis, Cucurbita, Cuscuta, Cycas, Cyphomandra, Dioscorea, Diospyrus, Dura, Elaeagnus, Elaeis, Erythroxylon, Euonymus, Ficus, Fortunella, Fragaria, Gardinia, Gonocaryum, Gossypium, Guava, Guilielma, Hibiscus, Hippophaea, Iris, Lathyrus, Lonicera, Luffa, Lycium, Lycopersicum, Malpighia, Mangifera, Mormodica, Murraya, Musa, Nenga, Palisota, Pandanus, Passiflora, Persea, Physalis, Prunus, Ptychandra, Punica, Pyracantha, Pyrus, Ribes, Rosa, Rubus, Sabal, Sambucus, Seaforita, Shepherdia, Solanum, Sorbus, Synaspadix, Tabernae, Tamus, Taxus, Trichosanthes, Triphasia, Vaccinium, Viburnum, Vignia or Vitis comprising at least one nucleic acid encoding a ketolase.
Very especially preferred plant genera are Ananas, Asparagus, Capsicum, Citrus, Cucumis, Cucurbita, Citrullus, Lycopersicum, Passiflora, Prunus, Physalis, Solanum, Vaccinium and Vitis, comprising at least one transgenic nucleic acid encoding a ketolase.
In preferred transgenic plants—as mentioned above—the ketolase is expressed in the fruits; especially preferably, the expression of the ketolase is highest in the fruits.
Especially preferred genetically modified plants have, as mentioned hereinabove, additionally an increased hydroxylase activity and/or β-cyclase activity in comparison with a wild plant. Further preferred embodiments are described above in the method according to the invention.
The present invention furthermore relates to the transgenic plants, their propagation material and their plant cells, tissues or parts, in particular their fruits.
As described above, the genetically modified plants can be used for the production of ketocarotenoids, in particular astaxanthin.
Genetically modified plants according to the invention which can be consumed by humans and animals and which have an increased ketocarotenoid content can also be used for example directly or after processing known per se as foodstuff or feedstuff, or else as food or feed supplement. Furthermore, the genetically modified plants can be used for the production of ketocarotenoid-comprising extracts of the plants and/or for the production of feed and food supplements.
The genetically modified plants have an increased ketocarotenoid content in comparison with the wild type.
An increased ketocarotenoid content is, as a rule, understood as meaning an increased total ketocarotenoid content.
However, an increased ketocarotenoid content is also understood as meaning, in particular, a modified content of the preferred ketocarotenoids without the total carotenoid content necessarily having to be increased.
In an especially preferred embodiment, the genetically modified plants according to the invention have an increased astaxanthin content in comparison with the wild type.
In this case, an increased content is in particular understood as meaning a generated content of ketocarotenoids or astaxanthin.
The invention is now illustrated by the examples which follow, but not limited thereto:
General Experimental Conditions:
Sequence Analysis of Recombinant DNA
Recombinant DNA molecules were sequenced using a laser fluorescence DNA sequencer from Licor (available from MWG Biotech, Ebersbach) following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).

EXAMPLE 1

Amplification of a cDNA Which Encodes the Entire Primary Sequence of the Ketolase from Haematococcus pluvialis Flotow em. Wille

The cDNA which encodes the ketolase from Haematococcus pluvialis was amplified from Haematococcus pluvialis (strain 192.80 of the “Sammlung von Algenkulturen der Universität Göttingen” [Collection of algal cultures of the university of Göttingen]) suspension culture by means of PCR.
To prepare total RNA from a suspension culture of Haematococcus pluvialis (strain 192.80) which had grown for 2 weeks with indirect daylight at room temperature in Haematococcus medium (1.2 g/l sodium acetate, 2 g/l yeast extract, 0.2 g/l MgCl₂×6H₂O, 0.02 CaCl₂×2H₂O; pH 6.8; after autoclaving addition of 400 mg/l L-asparagine, 10 mg/l FeSO₄×H₂O), the cells were harvested, frozen in liquid nitrogen and ground to a powder in a mortar. Thereafter, 100 mg of the frozen pulverized algal cells were transferred into a reaction vessel and taken up in 0.8 ml of Trizol buffer (LifeTechnologies). The suspension was extracted with 0.2 ml of chloroform. After centrifugation for 15 minutes at 12 000 g, the aqueous supernatant was removed, transferred into a fresh reaction vessel and extracted with one volume of ethanol. The RNA was precipitated with one volume of isopropanol, washed with 75% of ethanol, and the pellet was dissolved in DEPC water (overnight-incubation of water with 1/1000 volume diethyl pyrocarbonate at room temperature, then autoclaving). The RNA concentration was determined photometrically.
For the cDNA synthesis, 2.5 ug of total RNA were denatured for 10 min at 60° C., cooled on ice for 2 minutes and transcribed into cDNA by means of a cDNA kit (Ready-to-go-you-prime-beads, Pharmacia Biotech) following the manufacturer's instructions and using an antisense-specific primer (PRI SEQ ID No. 29).
The nucleic acid encoding a ketolase from Haematococcus pluvialis (strain 192.80) was amplified by means of polymerase chain reaction (PCR) from Haematococcus pluvialis using a sense-specific primer (PR2 SEQ ID No. 30) and an antisense-specific primer (PR1 SEQ ID No. 29).
The PCR conditions were as follows:
The PCR for the amplification of the cDNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 μl of reaction mixture comprising:

- 4 μl of a Haematococcus pluvialis cDNA (prepared as described above)
- 0.25 mM dNTPs
- 0.2 mM PR1 (SEQ ID No. 29)
- 0.2 mM PR2 (SEQ ID No. 30)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 25.8 μl distilled water.

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

53° C. 2 minutes

72° C. 3 minutes

1X 72° C. 10 minutes
The PCR amplification with SEQ ID No. 29 and SEQ ID No. 30 results in a 1155 bp fragment which encodes a protein consisting of the entire primary sequence (SEQ ID No. 22). Using standard methods, the amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega), giving rise to the clone pGKETO2.
Sequencing the clone pGKETO2 with the T7 and the SP6 primer confirmed a sequence which differs from the published sequence X86782 only in the three codons 73, 114 and 119 in, in each case, one base. These nucleotide substitutions were reproduced in an independent amplification experiment and thus represent the nucleotide sequence in the used Haematococcus pluvialis strain 192.80 (FIGS. 3 and 4, sequence alignments).
This clone was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380). Cloning was effected by isolating the 1027 bp SpHI fragment from pGKETO2 and ligation into the SpHI-cut vector pJIT117. The clone which comprises the Haematococcus pluvialis ketolase gene in the correct orientation as N-terminal translational fusion with the rbcs transit peptide sequence is named pJKETO2.

EXAMPLE 2

Amplification of a cDNA Which Encodes the Ketolase from Haematococcus pluvialis Flotow em. Wille Which is Truncated at the N-terminus by 14 Amino Acids

The cDNA which encodes the ketolase from Haematococcus pluvialis (strain 192.80) which is truncated at the N terminus by 14 amino acids was amplified by means of PCR from Haematococcus pluvialis suspension culture (strain 192.80 of the “Sammlung von Algenkulturen der Universität Göttingen”). The preparation of total RNA from a suspension culture of Haematococcus pluvialis (strain 192.80) was carried out as described in Example 1.
The cDNA synthesis was carried out as described in Example 1.
The nucleic acid encoding a ketolase from Haematococcus pluvialis (strain 192.80) which is truncated at the N-terminus by 14 amino acids was amplified by means of polymerase chain reaction (PCR) from Haematococcus pluvialis using a sense-specific primer (PR3 SEQ ID No. 31) and an antisense-specific primer (PR1 SEQ ID No. 29).
The PCR conditions were as follows:
The PCR for the amplification of the cDNA which encodes a ketolase protein which is truncated at the N-terminus by 14 amino acids was carried out in 50 μl of reaction mixture comprising:

- 4 μl of a Haematococcus pluvialis cDNA (prepared as described above)
- 0.25 mM dNTPs
- 0.2 mM PR1 (SEQ ID No. 29)
- 0.2 mM PR2 (SEQ ID No. 31)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 R1 R Taq polymerase (TAKARA)
- 25.8 R1 distilled water.

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

53° C. 2 minutes

72° C. 3 minutes

1X 72° C. 10 minutes
The PCR amplification with SEQ ID No. 29 and SEQ ID No. 31 resulted in a 1111 bp fragment which encodes a ketolase protein in which N-terminal amino acids (positions 2-16) are replaced by a single amino acid (leucin).
The amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega) using standard methods and the clone pGKETO3 was obtained. Sequencing reactions with the primers T7 and SP6 confirmed a sequence which is identical to the sequence SEQ ID No. 22, the 5′ region (positions 1-53) of SEQ ID No. 22 in the amplificate SEQ ID No. 24 having been replaced by a nonamer sequence whose sequence deviates. This clone was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
Cloning was carried out by isolating the 985 bp SpHI fragment from pGKETO3 and ligation with the SpHI-cut vector pJIT117. The clone which comprises the Haematococcus pluvialis ketolase which is truncated at the N terminus by 14 amino acids in the correct orientation as N-terminal translational fusion with the rbcs transit peptide is named pJKETO3.

EXAMPLE 3

Amplification of a cDNA Which Encodes the Ketolase from Haematococcus pluvialis Flotow em. Wille (Strain 192.80 of “Sammlung von Algenkulturen der Universität Göttingen”) Consisting of the Entire Primary Sequence and Fused C-terminal myc Tag

The cDNA which encodes the ketolase from Haematococcus pluvialis (strain 192.80) consisting of the entire primary sequence and fused C-terminal myc tag was prepared by means of PCR using the plasmid pGKETO2 (described in Example 1) and the primers PR15 (SEQ ID No. 32). The primer PR15 is compsed of an antisense-specific 3′ region (nucleotides 40-59) and a myc-tag encoding 5′ region (nucleotides 1-39).
Denaturing (5 min at 95° C.) and annealing (slow cooling at room temperature to 40° C.) of pGKETO2 and PR15 took place in an 11.5 μl reaction mixture comprising:

- 1 μg pGKETO2 PlasmidDNA
- 0.1 μg PR15 (SEQ ID No. 32)

The 3′ ends were filled in (30 min at 30° C.) in 20 μl of reaction mixture comprising:

- 11.5 μl pGKETO2/PR15 annealing reaction (prepared as described above)
- 40-50 μM dNTPs
- 2 μl 1× Klenow buffer
- 2U Klenow enzyme

The nucleic acid encoding a ketolase from Haematococcus pluvialis (strain 192.80) consisting of the entire primary sequence and fused C-terminal myc tag was amplified from Haematococcus pluvialis by means of polymerase chain reaction (PCR) using a sense-specific primer (PR2 SEQ ID No. 30) and an antisense-specific primer (PR15 SEQ ID No. 32).
The PCR conditions were as follows:
The PCR for the amplification of the cDNA which encodes a ketolase protein with fused C-terminal myc tag was carried out in 50 μl of reaction mixture comprising:

- 1 μl of an annealing reaction (prepared as described above)
- 0.25 mM dNTPs
- 0.2 μM PR15 (SEQ ID No. 32)
- 0.2 μM PR2 (SEQ ID No. 30)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 25.8 μl distilled water.

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

53° C. 1 minute

72° C. 1 minute

1X 72° C. 10 minutes
The PCR amplification with SEQ ID No. 32 and SEQ ID No. 30 results in a 1032 bp fragment which encodes a protein consisting of the entire primary sequence of the ketolase from Haematococcus pluvialis as double translational fusion with the rbcs transit peptide at the N terminus und the myc tag at the C terminus.
The amplificate was cloned into the PCR cloning vector pGEM-Teasy (Promega) using standard methods and the clone pGKETO4 was obtained. Sequencing reactions with the primers T7 and SP6 confirmed a sequence which was identical to the sequence SEQ ID No. 22, the 3′ region (positions 993-1155) of SEQ ID No. 22 in the amplificate SEQ ID No. 26 having been replaced by a 39 bp sequence which deviated. This clone was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
Cloning was effected by isolating the 1038 bp EcoRI/SpHI fragment from pGKETO4 and ligation with the EcoRI-SpHI-cut vector pJIT117. The ligation gives rise to a translational fusion between the C terminus of the rbcS transit peptide sequence and the N terminus of the ketolase sequence. The clone which comprises the Haematococcus pluvialis ketolase with fused C-terminal myc tag in correct orientation as translational N-terminal fusion with the rbcs transit peptide is named pJKET4.

EXAMPLE 4

Preparation of Expression Vectors for the Constitutive Expression of the Haematococcus pluvialis Ketolase in Lycopersicon esculentum

Expression of the ketolase from Haematococcus pluvialis in L. esculentum and in Tagetes erecta was under the control of the constitutive promoter d35S from CaMV (Franck et al. 1980, Cell 21: 285-294). The expression was carried out with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715).
An expression plasmid for the agrobacterium-mediated transformation of the ketolase from Haematococcus pluvialis in L. esculentum was prepared using the binary vector pSUN3 (WO02/00900).

- To prepare the expression vector pS3KETO2, the 2.8 kb SacI/XhoI fragment from pJKETO2 was ligated with the SacI-XhoI-cut vector pSUN3 (FIG. 5, construct map). In FIG. 5, fragment d35S comprises the duplicated 35S promoter (747 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KETO2 (1027 bp) the entire primary sequence encoding the Haematococcus pluvialis ketolase, fragment term (761 bp) the CaMV polyadenylation signal.
- To prepare the expression vector pS3KETO3, the 2.7 kb SacI/XhoI fragment from pJKETO3 was ligated with the SacI-XhoI-cut vector pSUN3 (FIG. 6, construct map). In FIG. 6, fragment d35S comprises the duplicated 35S promoter (747 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KETO3 (985 bp) the primary sequence encoding the Haematococcus pluvialis ketolase which has been truncated by 14 N-terminal amino acids, fragment term (761 bp) the CaMV polyadenylation signal.
- To prepare the expression vector pS3KETO4, the 2.8 kb SacI/XhoI fragment from pJKETO4 was ligated with the SacI-XhoI-cut vector pSUN3 (FIG. 7, construct map). In FIG. 7, fragment d35S comprises the duplicated 35S promoter (747 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KETO4 (1038 bp) the entire primary sequence encoding the Haematococcus pluvialis ketolase with C-terminal myc-tag, fragment term (761 bp) the CaMV polyadenylation signal.

EXAMPLE 5

Preparation of Expression Vectors for the Expression of the Haematococcus pluvialis Ketolase in Lycopersicon esculentum

The ketolase from Haematococcus pluvialis was expressed in L. esculentum using the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was under the control of a modified version AP3P of the promoter AP3 of Arabidopsis thaliana (AL132971: nucleotide region 9298-10200; Hill et al. (1998) Development 125: 1711-1721).
The DNA fragment which comprises the AP3 promoter region −902 to +15 from Arabidopsis thaliana was prepared by means of PCR using genomic DNA (isolated from Arabidopsis thaliana by standard methods) and the primers PR7 (SEQ ID No. 33) and PR10 (SEQ ID No. 36).
The PCR conditions were as follows:
The PCR for the amplification of the DNA which comprises the AP3 promoter fragment (−902 to +15) was carried out in 50 μl of reaction mixture comprising:

- 100 ng of genomic DNA from A. thaliana
- 0.25 mM dNTPs
- 0.2 mM PR7 (SEQ ID No. 33)
- 0.2 mM PR10 (SEQ ID No. 36)
- 5 μl 10× PCR buffer (Stratagene)
- 0.25 μl Pfu polymerase (Stratagene)
- 28.8 μl distilled water.

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

50° C. 1 minute

72° C. 1 minute

1X 72° C. 10 minutes
The 922 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods, giving rise to the plasmid pTAP3.
Sequencing the clone pTAP3 confirms a sequence which differs from the published AP3 sequence (AL132971, nucleotide region 9298-10200) only by one insertion (one G in position 9765 of the sequence AL132971) and one base substitution (one G instead of one A in position 9726 of the sequence AL132971). These nucleotide differences were reproduced in an independent amplification experiment and thus represent the actual nucleotide sequence in the Arabidopsis thaliana plants used.
The modified version AP3P was prepared by means of recombinant PCR using the plasmid pTAP3. The region 10200-9771 was amplified using the primers PR7 (SEQ ID No. 33) and PR9 (SEQ ID No. 35) (amplificate A7/9), and the region 9526-9285 was amplified using PR8 (SEQ ID No. 34) and PR10 (SEQ ID No. 36) (amplificate A8/10).
The PCR conditions were as follows:
The PCR reactions for the amplification of the DNA fragments which comprise the region 10200-9771 and the region 9526-9285 of the AP3 promoter were carried out in 50-μl batches of reaction mixture comprising:

- 100 ng AP3 amplificate (described above)
- 0.25 mM dNTPs
- 0.2 mM sense primer (PR7 SEQ ID No. 33 and PR8 SEQ ID No. 35, respectively)
- 0.2 mM antisense primer (PR9 SEQ ID No. 35 and PR10 SEQ ID No. 36, respectively)
- 5 μl 10× PCR buffer (Stratagene)
- 0.25 μl Pfu Taq polymerase (Stratagene)
- 28.8 μl distilled water.

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

50° C. 1 minute

72° C. 1 minute

1X 72° C. 10 minutes
The recombinant PCR comprises annealing of the amplificates A7/9 and A8/10, which overlap over a sequence of 25 nucleotides, complementation to give a double strand, and subsequent amplification. This gives rise to a modified version of the AP3 promoter, viz. AP3P, in which the positions 9670-9526 are deleted. Denaturation (5 minutes at 95° C.) and annealing (slow cooling at room temperature to 40° C.) of the two amplificates A7/9 and A8/10 were carried out in 17.6 ∝l of reaction mixture comprising:

- 0.5 μg A7/9 amplificate
- 0.25 μg A8/10 amplificate

Filling in the 3′ ends (30 minutes at 30° C.) was carried out in 20 ∝l of reaction mixture comprising:

- 17.6 μl A7/9 and A8/10 annealing reaction (prepared as described above)
- 50 μM dNTPs
- 2 μl 1× Klenow buffer
- 2U Klenow enzyme

The nucleic acid encoding the modified promoter version AP3P was amplified by means of PCR using a sense-specific primer (PR7 SEQ ID No. 28) and an antisense-specific primer (PR10 SEQ ID No. 36).
The PCR conditions were as follows:
The PCR for the amplification of the AP3P fragment was carried out in 50 μl of reaction mixture comprising:

- 1 μl annealing reaction (prepared as described above)
- 0.25 mM dNTPs
- 0.2 mM PR7 (SEQ ID No. 33)
- 0.2 mM PR10 (SEQ ID No. 36)
- 5 μl 10× PCR buffer (Stratagene)
- 0.25 μl Pfu Taq polymerase (Stratagene)
- 28.8 μl distilled water.

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

50° C. 1 minute

72° C. 1 minute

1X 72° C. 10 minutes
The PCR amplification with SEQ ID No. 33 and SEQ ID No. 36 resulted in a 778 bp fragment which encodes the modified promoter version AP3P. The amplificate was cloned into the cloning vector pCR2.1 (Invitrogen) and the clone pTAP3P was obtained. Sequencing reactions with the primers T7 and M13 confirmed a sequence with identity to the sequence AL132971, region 10200-9298, with the internal region 9285-9526 having been deleted. This clone was therefore used for cloning into the expression vector pJIT117 (Guerineau et al. 1988, Nucl. Acids Res. 16: 11380).
Cloning was carried out by isolating the 771 bp SacI/HindIII fragment from pTAP3P and ligation into the SacI/HindIII-cut vector pJIT117. The clone which comprises the promoter AP3P instead of the original promoter d35S is named pJAP3P.
To prepare an expression cassette pJAP3PKETO2, the 1027 bp SpHI fragment KETO2 (described in Example 1) was cloned into the SpHI-cut vector pJAP3P. The clone which comprises the fragment KETO2 in the correct orientation as N-terminal fusion with the rbcS transit peptide is named pJAP3PKETO2.
To prepare an expression cassette pJAP3PKETO4, the 1032 bp SpHI/EcoRI fragment KETO4 (described in Example 3) was cloned into the SpHI/EcoRI-cut vector pJAP3P. The clone which comprises the fragment KETO4 in the correct orientation as N-terminal fusion with the rbcS transit peptide is named pJAP3PKETO4.
An expression vector for the agrobacterium-mediated transformation of the AP3P-controlled ketolase from Haematococcus pluvialis in L. esculentum was prepared using the binary vector pSUN3 (WO02/00900).

- To prepare the expression vector pS3AP3PKETO2, the 2.8 kb SacI/XhoI fragment from pJAP3KETO2 was ligated with the SacI/XhoI-cut vector pSUN3 (FIG. 8, construct map). In FIG. 8, fragment AP3P comprises the modified AP3P promoter (771 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KETO2 (1027 bp) the entire primary sequence encoding the Haematococcus pluvialis ketolase, fragment term (761 bp) the CaMV polyadenylation signal.
- To prepare the expression vector pS3AP3PKETO4, the 2.8 kb SacI/XhoI fragment from pJAP3PKETO4 was ligated with the SacI/XhoI-cut vector pSUN3 (FIG. 9, construct map). In FIG. 9, fragment AP3P comprises the modified AP3P promoter (771 bp), fragment rbcS the rbcS transit peptide from pea (204 bp), fragment KETO4 (1038 bp) the entire primary sequence encoding the Haematococcus pluvialis ketolase with C-terminal myc-tag, fragment term (761 bp) the CaMV polyadenylation signal.

EXAMPLE 6

Generation of Transgenic Lycopersicon esculentum Plants

Tomato plants were transformed and regenerated by the published method of Ling and coworkers (Plant Cell Reports (1998), 17:843-847). A higher kanamycine concentration (100 mg/l) was used for the selection for the variety Microtom.
The starting explants for the transformation were cotyledons and hypocotyls of seven- to ten-day old seedlings of the line Microtom. The culture medium of Murashige and Skoog (1962: Murashige and Skoog, 1962, Physiol. Plant 15, 473-) supplemented with 2% sucrose, pH 6.1, was used for the germination. Germination took place at 21° C. at a low light level (20 to 100 μE). After seven to ten days, the cotyledons were divided horizontally and the hypocotyls were cut into segments 5 to 10 mm in length and placed on the medium MSBN (MS, pH 6.1, 3% sucrose+1 mg/l BAP, 0.1 mg/l NAA) which had been charged on the day before with tobacco cells grown in suspension culture. The tobacco cells were covered with sterile paper filters in such a way that there were no air bubbles. The explants were precultured on the above-described medium for three to five days. Cells of the strain Agrobacterium tumefaciens LBA4404 were transformed individually with the plasmids pS3KETO2, pS3KETO3 and pS3AP3KETO2, respectively. In each case one overnight culture of the individual Agrobacterium strains which had been transformed with the binary vectors pS3KETO2 and pS3KETO3, respectively, was grown in YEB medium with kanamycine (20 mg/l) at 28 degrees Celsius, and the cells were centrifuged. The bacterial pellet was resuspended in liquid MS medium (3% sucrose, pH 6.1) and brought to an optical density of 0.3 (at 600 nm). The precultured explants were transferred into the suspension and incubated for 30 minutes at room temperature with gentle shaking. Thereafter, the explants were dried with sterile paper filters and returned to their preculture medium for three days of coculture (21° C.).
After the coculture, the explants were transferred to MSZ2 medium (MS pH 6.1+3% sucrose, 2 mg/l zeatin, 100 mg/l kanamycin, 160 mg/l Timentin) and stored under low light conditions (20 to 100 ∝E, ohotoperiod 16 h/8 h) at 21° C. for the selective regeneration. The explants were transferred every two to three weeks until shoots formed. Small shoots were separated from the explants and rooted on MS (pH 6.1+3% sucrose), 160 mg/l Timentin, 30 mg/l kanamycine, 0.1 mg/l IAA. Rooted plants were transferred to the greenhouse.
In accordance with the above-described transformation method, the following lines were obtained with the following expression constructs:

the following were obtained with pS3KETO2: cs13-24, cs13-30, cs13-40.
the following were obtained with pS3KETO3: cs14-2, cs14-3, cs14-9, cs14-19.
the following were obtained with pS3AP3PKETO2: cs16-15, cs16-34, cs16-35, cs16-40.

EXAMPLE 8

Characterization of the Transgenic Fruits

The fruit material of the transgenic plants was crushed in liquid nitrogen and the powder (approximately 250 to 500 mg) was extracted with 100% acetone (three portions of 500 μl each). The solvent was evaporated and the carotenoids were resuspended in 100 μl of acetone.
It was possible to distinguish between mono- and diesters of the carotenoids by means of a C30 reversed-phase column. The HPLC running conditions were modified following a published method (Frazer et al. (2000), Plant Journal 24(4): 551-558). The following HPLC conditions were established:

separation column: Prontosil C30 column, 250×4.6 mm (Bischoff, Leonberg, Germany)
flow rate: 1.0 ml/min
eluents: eluent A—100% methanol
- eluent B—80% methanol, 0.2% ammonium acetate
- eluent C—100% t-butyl methyl ether

Gradient profile:

Flow % % %

Time rate eluent A eluent B eluent C

1.00 1.0 95.0 5.0 0

12.00 1.0 95.0 5.0 0

12.10 1.0 80.0 5.0 15.0

22.00 1.0 76.0 5.0 19.0

22.10 1.0 66.5 5.0 28.5

38.00 1.0 15.0 5.0 80.0

45.00 1.0 95.0 5.0 0

46.0 1.0 95.0 5.0 0

Detection: 300-530 nm

The spectra were recorded using a photodiode array detector. The carotenoids were identified via their absorption spectra and their retention times in comparison with standard samples.

Table 1 shows the carotenoid profile in tomato fruits of the transgenic tomatoes produced in accordance with the above-described examples and of control tomato plants. In comparison with the genetically non-modified control plant, the genetically modified plants show a ketocarotenoid content, in particular an astaxanthin content.

TABLE 1


			beta-
Plant	Lutein	Lycopene	Carotene	Cryptoxanthin	Canthaxanthin	Adonirubin	Astaxanthin

Control	+	+	+	(+)	−	−	−
Control	+	+	+	(+)	−	−	−
CS13-24	−	+	+	(+)	+	+	+
CS13-30	−	+	+	(+)	+	+	+
CS13-40	−	+	+	(+)	+	+	+
CS14-2	−	+	+	(+)	+	+	+
CS14-3	−	+	+	−	+	+	+
CS14-9	−	+	+	(+)	+	+	+
CS14-19	−	+	+	−	+	+	+
CS16-15	−	+	+	(+)	+	+	+
CS16-34	−	+	+	(+)	+	+	+
CS16-35	−	+	+	−	+	+	+
CS16-40	−	+	(+)	(+)	+	+	+

+ means that carotenoid is detectable
− means that carotenoid is not detected
(+) means that the carotenoid concentration is at the detection limit

Table 2a shows the amount of carotenoid in mature fruits of transgenic tomatoes and control plants. The data are means of different lines and shown as a percentage of the total carotenoid content.

Promoter Beta-

used Lycopene carotene Lutein Canthaxanthin Adonirubin Astaxanthin Zeaxanthin

Control 80.5 14.4 2.8 0.2

plants

CS16 84 9.4 0.3 0.5 0.2 5.0 0.3

CS13 78 16.5 2.8 0.3 0.2 6.1
Table 2b shows the amount of carotenoid in mature fruits of transgenic tomatoes and control plants. The data are means of different lines and shown as a percentage of the total carotenoid content.

Promoter Beta-

used Lycopene carotene Lutein Canthaxanthin Adonirubin Astaxanthin Zeaxanthin

Control 59 28.4 9 0.3

plants

CS16 61 22.3 5.2 1.6 3.1 3.9 2.5

CS13 52 19.5 5.4 1.2 4.7 6.1

EXAMPLE 9

Amplification of a DNA Which Encodes the Entire Primary Sequence of the NP196-ketolase from Nostoc punctiforme ATCC 29133

The DNA which encodes the NP196-ketolase from Nostoc punctiforme ATCC 29133 was amplified from Nostoc punctiforme ATCC 29133 (strain of the “American Type Culture Collection”) by means of PCR.
To prepare genomic DNA from a suspension culture of Nostoc punctiforme ATCC 29133 which had been grown for 1 week under continuous light with constant shaking (150 rpm) at 25° C. in BG 11 medium (1.5 g/l NaNO₃, 0.04 g/l K₂PO₄×3H₂O, 0.075 g/l MgSO₄×H₂O, 0.036 g/l CaCl₂×2H₂O, 0.006 g/l citric acid, 0.006 g/l ferric ammonium citrate, 0.001 g/l EDTA disodium magnesium, 0.04 g/l Na₂CO₃, 1 ml Trace Metal Mix “A5+Co”, 2.86 g/l H₃BO₃, 1.81 g/l MnCl₂×4H₂O, 0.222 g/l ZnSO₄×7H₂O, 0.39 g/l NaMoO₄×2H₂O, 0.079 g/l CuSO₄×5H₂O, 0.0494 g/l Co(NO₃)₂×6H₂O), the cells were harvested by centrifugation, frozen in liquid nitrogen and ground to a powder in a mortar.
Protocol for the DNA I1solation from Nostoc punctiforme ATCC 29133:
The bacterial cells were pelleted from a 10 ml liquid culture by centrifugation for 10 minutes at 8000 rpm. Thereafter, the bacterial cells were comminuted and ground in liquid nitrogen using a pestle and mortar. The cell material was resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (volume 2 ml). After addition of 100 μl of Proteinase K (concentration: 20 mg/ml), the cell suspension was incubated for 3 hours at 37° C. Thereafter, the suspension was extracted with 500 μl of phenol. After centrifugation for 5 minutes at 13 000 rpm, the aqueous top phase was transferred to a fresh 2 ml Eppendorf reaction vessel. The phenol extraction was repeated 3 times. The DNA was precipitated by addition of 1/10 volume 3 M sodium acetate (pH 5.2) and 0.6 volume isopropanol and subsequently washed with 70% ethanol. The DNA pellet was dried at room temperature, taken up in 25 μl of water and dissolved with heating at 65° C.
The nucleic acid encoding a ketolase from Nostoc punctiforme ATCC 29133 was amplified from Nostoc punctiforme ATCC 29133 by means of polymerase chain reaction (PCR) using a sense-specific primer (NP196-1, SEQ ID No. 59) and an antisense-specific primer (NP196-2 SEQ ID No. 60).
The PCR conditions were as follows:
The PCR for the amplification of the DNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 μl of reaction mixture comprising:

- 1 μl of a Nostoc punctiforme ATCC 29133 DNA (prepared as described above)
- 0.25 mM dNTPs
- 0.2 mM NP196-1 (SEQ ID No. 59)
- 0.2 mM NP196-2 (SEQ ID No. 60)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 25.8 μl distilled water

The PCR was carried out under the following cyclic conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

55° C. 1 minute

72° C. 3 minutes

1X 72° C. 10 minutes
The PCR amplification with SEQ ID No. 59 and SEQ ID No. 60 resulted in a 792 bp fragment which encodes a protein consisting of the entire primary sequence (NP196, SEQ ID No. 61). The amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard methods, giving rise to the clone pNP196.
Sequencing of the clone pNP196 with the M13F and the M13R primer verified a sequence which is identical to the DNA sequence of 140.571-139.810 of the database entry NZ_AABC01000196 (with inverse orientation relative to the published database entry), with the exception that G in position 140.571 was replaced by A in order to generate a standard ATG start codon. This nucleotide sequence was reproduced in an independent amplification experiment and thus represents the nucleotide sequence in the Nostoc punctiforme ATCC 29133 used.
This clone pNP196 was therefore used for cloning into the expression vector pJAP3P described in Example 5.
pJAP3P was modified by replacing the 35S terminator by the OCS terminator (octopine synthase) of the Ti plasmid pTi15955 of Agrobacterium tumefaciens (database entry X00493, position 12.541-12.350, Gielen et al. (1984) EMBO J. 3 835-846).
The DNA fragment which comprises the OCS terminator region was prepared by means of PCR using the plasmid pHELLSGATE (database entry AJ311874, Wesley et al. (2001) Plant J. 27 581-590, isolated from E. coli by standard methods) and the primers OCS-1 (SEQ ID No. 63) and OCS-2 (SEQ ID No. 64).
The PCR conditions were as follows:
The PCR for the amplification of the DNA which comprises the octopine synthase (OCS) terminator region (SEQ ID 65) was carried out in 50 μl of reaction mixture comprising:

- 100 ng pHELLSGATE plasmid DNA
- 0.25 mM dNTPs
- 0.2 mM OCS-1 (SEQ ID No. 63)
- 0.2 mM OCS-2 (SEQ ID No. 64)
- 5 μl 10× PCR buffer (Stratagene)
- 0.25 μl Pfu polymerase (Stratagene)
- 28.8 μl distilled water

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

50° C. 1 minute

72° C. 1 minute

1X 72° C. 10 minutes
The 210 bp amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) using standard conditions, giving rise to the plasmid pOCS.
Sequencing of the clone pOCS verified a sequence which agrees with a sequence segment on the Ti plasmid pTi15955 of Agrobacterium tumefaciens (database entry X00493) from position 12.541 to 12.350.
Cloning was carried out by isolating the 210 bp SalI/XhoI fragment from pOCS and ligation into the SalI/XhoI-cut vector pJAP3P.
This clone is named pJOAP and was therefore used for cloning into the expression vector pJOAP:NP196.
Cloning was effected by isolating the 782 bp SphI fragment from pNP196 and ligation into the SphI-cut vector pJOAP. The clone which comprises the NP196 ketolase of Nostoc punctiforme in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is named pJOAP:NP196.

EXAMPLE 10

Preparation of Expression Vectors for the Fruit-Specific Overexpression of the NP196-ketolase from Nostoc punctiforme ATCC 29133 (Strain of the “American Type Culture Collection”) in Lycopersicon esculentum

The NP196-ketolase from Nostoc punctiforme was expressed in L. esculentum using the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was effected under the control of the promoter AP3P from Arabidopsis thaliana described in Example 5.
An expression vector for the Agrobacterium-mediated transformation of the AP3P-controlled NP196-ketolase from Nostoc punctiforme ATCC 29133 into L. esculentum was prepared using the binary vector pSUN3 (WO02/00900).
To prepare the expression vector MSP20, the 1.958 kb SacI/XhoI fragment from pJOAP:NP196 was ligated with the SacI/XhoI-cut vector pSUN3 (FIG. 10, construct map). In FIG. 10, fragment AP3P PROM comprises the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP196 KETO CDS (761 bp), encoding the Nostoc punctiforme NP196-ketolase, fragment OCS Terminator (192 bp) the polyadenylation signal of octopine synthase.

EXAMPLE 11

Amplification of a DNA Which Encodes the Entire Primary Sequence of the NOST-ketolase from Nostoc spp. PCC 7120

The DNA which encodes the NOST-ketolase from Nostoc punctiforme PCC 7120 was amplified by means of PCR from Nostoc PCC 7120 (strain of the Pasteur Culture Collection of Cyanobacterium).
To prepare genomic DNA from a suspension culture of Nostoc spp. PCC 7120 which had been grown for 1 week under continuous light with constant shaking (150 rpm) at 25° C. in BG 11 medium (1.5 g/l NaNO₃, 0.04 g/l K₂PO₄×3H₂O, 0.075 g/l MgSO₄×H₂O, 0.036 g/l CaCl₂×2H₂O, 0.006 g/l citric acid, 0.006 g/l ferric ammonium citrate, 0.001 g/l EDTA disodium magnesium, 0.04 g/l Na₂CO₃, 1 ml Trace Metal Mix “A5+Co” (2.86 g/l H₃BO₃, 1.81 g/l MnCl₂×4H₂O, 0.222 g/l ZnSO₄×7H₂O, 0.39 g/l NaMoO₄×2H₂O, 0.079 g/l CuSO₄×5H₂O, 0.0494 g/l Co(NO₃)₂×6H₂O), the cells were harvested by centrifugation, frozen in liquid nitrogen and ground to a powder in a mortar.
Protocol for the DNA Isolation from Nostoc spp. PCC 7120:
The bacterial cells were pelleted from a 10 ml liquid culture by centrifugation for 10 minutes at 8000 rpm. Thereafter, the bacterial cells were crushed and ground in liquid nitrogen using a mortar. The cell material was resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (volume 2 ml). After addition of 100 μl of Proteinase K (concentration: 20 mg/ml), the cell suspension was incubated for 3 hours at 37° C. Thereafter, the suspension was extracted with 500 μl of phenol. After centrifugation for 5 minutes at 13 000 rpm, the aqueous top phase was transferred to a fresh 2 ml Eppendorf reaction vessel. The phenol extraction was repeated 3 times. The DNA was precipitated by addition of 1/10 volume 3 M sodium acetate (pH 5.2) and 0.6 volume isopropanol and subsequently washed with 70% ethanol. The DNA pellet was dried at room temperature, taken up in 25 μl of water and dissolved with heating at 65° C.
The nucleic acid encoding a ketolase from Nostoc PCC 7120 was amplified from Nostoc PCC 7120 by means of polymerase chain reaction (PCR) using a sense-specific primer (NOST-1, SEQ ID No. 66) and an antisense-specific primer (NOST-2, SEQ ID No. 67).
The PCR conditions were as follows:
The PCR for the amplification of the DNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 μl of reaction mixture comprising:

- 1 μl of a Nostoc PCC 7120 DNA (prepared as described in Example 9)
- 0.25 mM dNTPs
- 0.2 mM NOST-1 (SEQ ID No. 66)
- 0.2 mM NOST-2 (SEQ ID No. 67)
- 5 μl 1× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 25.8 μl distilled water

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

55° C. 1 minute

72° C. 3 minutes

1X 72° C. 10 minutes
The PCR amplification with SEQ ID No. 66 and SEQ ID No. 67 resulted in an 809 bp fragment which encodes a protein consisting of the entire primary sequence (SEQ ID No. 68). The amplificate was cloned into the PCR cloning vector PGEM-T (Promega) using standard methods, giving rise to the clone pNOST.
Sequencing of the clone pNOST with the M13F and the M13R primer verified a sequence which is identical to the DNA sequence of the database entry AP003592. This nucleotide sequence was reproduced in an independent amplification experiment and thus represents the nucleotide sequence in the Nostoc PCC 7120 used.
This clone pNOST was therefore used for cloning into the expression vector pJOAP (described in Example 9).
Cloning was effected by isolating the 799 bp SphI fragment from pNOST and ligation into the SphI-cut vector pJOAP. The clone which comprises the NOST-ketolase from Nostoc PCC 7120 in the correct orientation as N-terminal translational fusion with the rbcS transit peptide is named pJOAP:NOST.

EXAMPLE 12

Preparation of Expression Vectors for the Fruit-Specific Overexpression of the NOST-ketolase from Nostoc spp. PCC 7120 in Lycopersicon esculentum

The NOST-ketolase from Nostoc spp. PCC 7120 was expressed in L. esculentum with the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was effected under the control of the promoter AP3P from Arabidopsis thaliana (described in Example 5).
An expression vector for the Agrobacterium-mediated transformation of the AP3P-controlled NOST-ketolase from Nostoc spp. PCC 7120 into L. esculentum was generated using the binary vector pSUN3 (WO02/00900).
To generate the expression vector MSP121, the 1 982 KB SacI/XhoI fragment from pJOAP:NOST was ligated with the SacI/XhoI-cut vector pSUN3 (FIG. 11, construct map). In FIG. 11, fragment AP3P PROM comprises the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NOST KETO CDS (774 bp), encoding the Nostoc spp. PCC 7120 NOST-ketolase, fragment OCS terminator (192 bp) the polyadenylation signal of octopine synthase.

EXAMPLE 13

Amplification of a DNA Which Encodes the Entire Primary Sequence of the NP195-ketolase from Nostoc punctiforme ATCC 29133

The DNA which encodes the NP195-ketolase from Nostoc punctiforme ATCC 29133 was amplified from Nostoc punctiforme ATCC 29133 (strain of the American Type Culture Collection) by means of PCR. The preparation of genomic DNA from a suspension culture of Nostoc punctiforme ATCC 29133 was described in Example 9.
The nucleic acid encoding a ketolase from Nostoc punctiforme ATCC 29133 was amplified from Nostoc punctiforme ATCC 29133 by means of polymerase chain reaction (PCR) using a sense-specific primer (NP195-1, SEQ ID No. 70) and an antisense-specific primer (NP195-2, SEQ ID No. 71).
The PCR conditions were as follows:
The PCR for the amplification of the DNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 μl of reaction mixture comprising:

- 1 μl of a Nostoc punctiforme ATCC 29133 DNA (prepared as described in Example 9)
- 0.25 mM dNTPs
- 0.2 mM NP195-1 (SEQ ID No. 70)
- 0.2 mM NP195-2 (SEQ ID No. 71)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 25.8 μl distilled water

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

55° C. 1 minute

72° C. 3 minutes

1X 72° C. 10 minutes
The PCR amplification with SEQ ID No. 70 and SEQ ID No. 71 resulted in an 819 bp fragment which encodes a protein consisting of the entire primary sequence (SEQ ID No. 72). Using standard methods, the amplificate was cloned into the PCR cloning vector pGEM-T (Promega) and the clone pNP195 was obtained.
Sequencing the clone pNP195 with the M13F and the M13R primers confirmed a sequence which is identical to the DNA sequence of 55,604-56,392 of the database entry NZ_AABC010001965, with the exception that A in position 55 604 was substituted for T in order to generate a standard ATG start codon. This nucleotide sequence was reproduced in an independent amplification experiment and thus represents the nucleotide sequence in the Nostoc punctiforme ATCC 29133 used.
This clone pNP195 was therefore used for cloning into the expression vector pJOAP (described in Example 9).
Cloning was effected by isolating the 709 bp SphI fragment from pNP195 and ligation into the SphI-cut vector pJOAP. The clone which comprises the NP195-ketolase from Nostoc punctiforme ATCC 29133 in correct orientation as N-terminal translational fusion with the rbcS transit peptide is named pJOAP:NP195.

EXAMPLE 14

Preparation of Expression Vectors for the Fruit-Specific Overexpression of the NP195-ketolase from Nostoc punctiforme ATCC 29133 in Lycopersicon esculentum

The NP195-ketolase from Nostoc punctiforme ATCC 29133 (strain of the American Type Culture Collection) was expressed in L. esculentum using the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was effected under the control of the promoter AP3P from Arabidopsis thaliana (described in Example 5).
An expression vector for the Agrobacterium-mediated transformation of the AP3P-controlled NP195-ketolase from Nostoc punctiforme ATCC 29133 into L. esculentum was prepared using the binary vector pSUN3 (WO02/00900).
To prepare the expression vector MPS122, the 1992 KB bp SacI/XhoI fragment from pJOAP:NP195 was ligated with the SacI/XhoI-cut vector pSUN3 (FIG. 12, construct map). In FIG. 12, fragment AP3P PROM comprises the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NP195 KETO CDS (789 bp), encoding the Nostoc punctiforme ATCC 29133 NP195-ketolase, fragment OCS Terminator (192 bp) the polyadenylation signal of octopine synthase.

EXAMPLE 15

Amplification of a DNA Which Encodes the Entire Primary Sequence of the NODK-ketolase from Nodularia spumignea NSOR10

The DNA which encodes the ketolase from Nodularia spumignea NSOR10 was amplified from Nodularia spumignea NSOR10 by means of PCR.
To prepare genomic DNA from a suspension culture of Nodularia spumignea NSOR10 which had been grown for 1 week under continuous light with constant shaking (150 rpm) at 25° C. in BG 11 medium (1.5 g/l NaNO₃, 0.04 g/l K₂PO₄×3H₂O, 0.075 g/l MgSO₄×H₂O, 0.036 g/l CaCl₂×2H₂O, 0.006 g/l citric acid, 0.006 g/l ferric ammonium citrate, 0.001 g/l EDTA disodium magnesium, 0.04 g/l Na₂CO₃, 1 ml Trace Metal Mix “A5+Co”, 2.86 g/l H₃BO₃, 1.81 g/l MnCl₂×4H₂O, 0.222 g/l ZnSO₄×7H₂O, 0.39 g/l NaMoO₄×2H₂O, 0.079 g/l CuSO₄×5H₂O, 0.0494 g/l Co(NO₃)₂×6H₂O), the cells were harvested by centrifugation, frozen in liquid nitrogen and ground to a powder in a mortar.
Protocol for the DNA Isolation from Nodularia spumignea NSOR10:
The bacterial cells were pelleted from a 10 ml liquid culture by centrifugation for 10 minutes at 8000 rpm. Thereafter, the bacterial cells were comminuted and ground in liquid nitrogen using a pestle and mortar. The cell material was resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5) and transferred to an Eppendorf reaction vessel (volume 2 ml). After addition of 100 μl of Proteinase K (concentration: 20 mg/ml), the cell suspension was incubated for 3 hours at 37° C. Thereafter, the suspension was extracted with 500 μl of phenol. After centrifugation for 5 minutes at 13 000 rpm, the aqueous top phase was transferred to a fresh 2 ml Eppendorf reaction vessel. The phenol extraction was repeated 3 times. The DNA was precipitated by addition of 1/10 volume 3 M sodium acetate (pH 5.2) and 0.6 volume isopropanol and subsequently washed with 70% ethanol. The DNA pellet was dried at room temperature, taken up in 25 μl of water and dissolved with heating at 65° C.
The nucleic acid encoding a ketolase from Nodularia spumignea NSOR10 was amplified from Nodularia spumignea NSOR10 by means of polymerase chain reaction (PCR) using a sense-specific primer (NODK-1, SEQ ID No. 74) and an antisense-specific primer (NODK-2 SEQ ID No. 75).
The PCR conditions were as follows:
The PCR for the amplification of the DNA which encodes a ketolase protein consisting of the entire primary sequence was carried out in 50 μl of reaction mixture comprising:

- 1 μl of a Nodularia spumignea NSOR10 DNA (prepared as described above)
- 0.25 mM dNTPs
- 0.2 mM NODK-1 (SEQ ID No. 74)
- 0.2 mM NODK-2 (SEQ ID No. 75)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 25.8 μl distilled water

The PCR was carried out under the following cycling conditions:

1X 94° C. 2 minutes

35X 94° C. 1 minute

55° C. 1 minute

72° C. 3 minutes

1X 72° C. 10 minutes
The PCR amplification with SEQ ID No. 74 and SEQ ID No. 75 resulted in an 720 bp fragment which encodes a protein consisting of the entire primary sequence (NODK, SEQ ID No. 76). Using standard methods, the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pNODK was obtained.
Sequencing the clone pNODK with the M13F and the M13R primers confirmed a sequence which is identical to the DNA sequence of 2130-2819 of the database entry AY210783 (inverse orientation to the published database entry). This nucleotide sequence was reproduced in an independent amplification experiment and thus represents the nucleotide sequence in the Nodularia spumignea NSOR10 used.
This clone pNODK was therefore used for cloning into the expression vector pJOAP (described in Example 9).
Cloning was effected by isolating the 710 bp SphI fragment from pNODK and ligation into the SphI-cut vector pJOAP. The clone which comprises the NODK-ketolase from Nodularia spumignea NSOR10 in correct orientation as N-terminal translational fusion with the rbcS transit peptide is named pJOAP:NODK.

EXAMPLE 16

Preparation of Expression Vectors for the Fruit-Specific Overexpression of the NODK-ketolase from Nodularia spumignea NSOR10 in Lycopersicon esculentum

The NODK-ketolase from Nodularia spumignea NSOR10 was expressed in L. esculentum using the transit peptide rbcS from pea (Anderson et al. 1986, Biochem J. 240:709-715). The expression was under the control of the promoter AP3P from Arabidopsis thaliana (described in Example 5).
An expression vector for the Agrobacterium-mediated transformation of the AP3P-controlled NP195-ketolase from Nostoc punctiforme ATCC 29133 into L. esculentum was prepared using the binary vector pSUN3 (WO02/00900).
To prepare the expression vector MSP123, the 1893 KB bp SacI/XhoI fragment from pJOAP:NODK was ligated with the SacI/XhoI-cut vector pSUN3 (FIG. 13, construct map). In FIG. 13, fragment AP3P PROM comprises the AP3P promoter (765 bp), fragment rbcS TP FRAGMENT the rbcS transit peptide from pea (194 bp), fragment NODK KETO CDS (690 bp), encoding the Nodularia spumignea NSOR10 NODK-ketolase, fragment OCS Terminator (192 bp) the polyadenylation signal of octopine synthase.

EXAMPLE 17

Preparation of an Expression Cassette for the Fruit-Specific Overexpression of the Chromoplast-Specific b-hydroxylase from Lycopersicon esculentum

The chromoplast-specific β-hydroxylase from Lycopersicon esculentum is expressed in tomato under the control of the fruit-specific promoter AP3P from Arabidopsis (Example 2). The terminator element used is LB3 (database entry AX696005) from Vicia faba. The sequence of the chromoplast-specific β-hydroxylase (database entry Y14810 & BE354440) was prepared by RNA isolation, reverse transcription and PCR.
The DNA fragment which comprises the LB3 terminator region was isolated by means of PCR.
Genomic DNA is isolated from Vicia faba tissue by standard methods and used by genomic PCR, using the primers PR206 (SEQ ID No. 78) and PR207 (SEQ ID No. 79). The PCR for the amplification of this LB3 DNA fragment is carried out in 50 μl of reaction mixture comprising:

- 1 μl of genomic DNA (prepared as described above)
- 0.25 mM dNTPs
- 0.2 μM PR206 (SEQ ID No. 78)
- 0.2 μM PR207 (SEQ ID No. 79)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 28.8 μl distilled water

The PCR amplification with SEQ ID No. 78 and SEQ ID No. 79 results in a 307 bp fragment (SEQ ID No. 80) which comprises the LB terminator. Using standard methods, the amplificate was cloned into the PCR cloning vector pCR 2.1 (Invitrogen) and the clone pLB3 was obtained. Sequencing of the clone pLB3 with the M13F and the M13R primer confirmed a sequence which is identical to the DNA sequence of 3-298 of the database entry AX696005. This clone is named pLB3 and is therefore used for cloning into the vector pJAP3P (see Example 5).
The expression cassette pJAP3P was modified by replacing the 35S terminator by the legumin LB3 terminator of Vicia faba (database entry AX696005; WO03/008596) (see below).
To prepare the β-hydroxylase sequence, total RNA is prepared from tomato. To this end, 100 mg of the frozen pulverized flowers are transferred to a reaction vessel and taken up in 0.8 ml of Trizol buffer (LifeTechnologies). The suspension is extracted with 0.2 ml of chloroform. After centrifugation for 15 minutes at 12 000 g, the aqueous supernatant is removed and transferred to a fresh reaction vessel and extracted with one volume of ethanol. The RNA is precipitated with one volume of isopropanol, washed with 75% of ethanol, and the pellet is dissolved in DEPC water (overnight incubation of water with 1/1000 volume of diethyl pyrocarbonate at room temperature, followed by autoclaving). The RNA concentration is determined photometrically. For the cDNA synthesis, 2.5 μg of total RNA are denatured for 10 minutes at 60° C., cooled on ice for 2 minutes, and transcribed into cDNA using a cDNA kit (Ready-to-go-you-prime-beads, Pharmacia Biotech) following the manufacturer's instructions and using an antisense-specific primer (PR17 SEQ ID NO: 56).
The conditions for the subsequent PCR reactions are as follows:
The nucleic acid encoding the β-hydroxylase was amplified from tomato by means of polymerase chain reaction (PCR) using a sense-specific primer (VPR204, SEQ ID No. 81) and an antisense-specific primer (PR215 SEQ ID No. 82).
The PCR for the amplification of the DNA which encodes a β-hydroxylase protein consisting of the entire primary sequence was carried out in 50 μl of a reaction mixture comprising:

- 1 μl of cDNA (prepared as described above)
- 0.25 mM dNTPs
- 0.2 μM VPR204 (SEQ ID No. 81)
- 0.2 μM PR215 (SEQ ID No. 82)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 28.8 μl distilled water

The PCR amplification with VPR204 and PR215 results in a 1040 bp fragment (SEQ ID No. 83) which encodes the b-hydroxylase. The amplificate is cloned into the PCR cloning vector pCR 2.1 (Invitrogen). This clone is named pCrtR-b2.
Sequencing reactions of the clone pCrtR-b2 with the primers M13-R and M13-R confirmed a sequence which is identical to the DNA sequence of 33-558 of the database entry BE354440 and which is identical to the DNA sequence of 1-1009 of the database entry Y14810. The clone pCrtR-b2 is therefore used for cloning into the vector pCSP02 (see below).
The first cloning step was carried out by isolating the 1034 bp HindIII/EcoRI fragment from pCrtR-b2, derived from the cloning vector pCR-2.1 (Invitrogen), and ligation with the HindIII/EcoRI-cut vector pJPA3P (see Example 5). The clone which comprises the b-hydroxylase fragment CrtR-b2 is named pCSP02.
The second cloning step is carried out by isolating the 301 bp EcoRI/XhoI fragment from pLB3, derived from the cloning vector pCR-2.1 (Invitrogen), and ligation with the EcoRI/XhoI-cut vector pCSP02. The clone which comprises the 296 bp terminator LB3 is named pCSP03. Ligation gives rise to a transcriptional fusion between the terminator LB3 and the b-hydroxylase fragment CrtR-b2. Moreover, it gives rise to a transcriptional fusion between the AP3P promoter and the b-hydroxylase fragment.

EXAMPLE 18

Preparation of an Expression Cassette for the Fruit-Specific Overexpression of the B Gene from Lycopersicon esculentum

The expression of the B gene from Lycopersicon esculentum in tomato (lycopene b-cyclase; database entry AF254793) takes place under the control of the fruit-specific promoter PDS (phytoene desaturase; database entry U46919) from Lycopersicon esculentum. The terminator element used is 35S from CaMV. The sequence of the B gene was prepared from genomic DNA from Lycopersicon esculentum by means of PCR.
The oligonucleotide primers BGEN-1 (SEQ ID No. 85) and BGEN-2 (SEQ ID No. 86) were used for isolating the B gene by means of PCR with genomic DNA from Lycopersicon esculentum.
The genomic DNA was isolated from Lycopersicon esculentum as described (Galbiati M et al. Funct. Integr. Genomics 2000, 20 1:25-34).
The PCR amplification was carried out as follows:

- 80 ng of genomic DNA
- 1× Expand Long Template PCR buffer
- 2.5 mM MgCl₂
- 350 μM each of dATP, dCTP, dGTP, dTTp
- 0.3 μM BGEN-1 (SEQ ID No. 85)
- 0.3 μM BGEN-2 (SEQ ID No. 86)
- 2.5 units Expand Long Template polymerase in an end volume of 25 μl

The following temperature program was used:

- 1 cycle of 120 seconds at 94° C.
- 35 cycles of 10 seconds at 94° C., 30 seconds at 48° C. and 3 minutes at 68° C.
- 1 cycle for 10 minutes at 68° C.

The PCR amplification with BGEN-1 and BGEN-2 results in a 1505 kb fragment (SEQ ID No. 87) which encodes the b-hydroxylase. The amplificate is cloned into the PCR cloning vector pCR-2.1 (Invitrogen). This clone is named pBGEN.
Sequencing reactions of the clone pBGEN with the primers M13-R and M13-F confirmed a sequence which is identical to the DNA sequence of 1-1497 of the database entry AF254793. The clone pCrtR-b2 is therefore used for cloning into the vector pCSP02 (see below).
To prepare the PDS promoter sequence from Lycopersicon esculentum, genomic DNA is isolated from Lycopersicon esculentum tissue by standard methods and employed by genomic PCR using the primers PDS-1 and PDS-2. The PCR for the amplification of this PDS promoter fragment is carried out in 50 μl of reaction mixture comprising:

- 1 μl of genomic DNA (prepared as described above)
- 0.3 mM dNTPs
- 0.2 μM PDS-1 (SEQ ID No. 89)
- 0.2 μM PDS-2 (SEQ ID No. 90)
- 5 μl 10× Pfu-Turbo polymerase (Stratagene)
- 1 μl Pfu-Turbo polymerase (Stratagene)
- 28.8 μl distilled water

The following temperature program was used:

- 1 cycle of 120 seconds at 94° C.
- 36 cycles of 60 seconds at 94° C., 120 seconds at 55° C. and 4 minutes at 72° C.
- 1 cycle for 10 minutes at 72° C.

The PCR amplification with PDS-1 and PDS-2 results in a fragment which comprises the sequence for the PDS promoter. The amplificate is cloned into pCR4-BLUNT (Invitrogen). This clone is named pPDS.
Sequencing reactions with the primers M13-R and M13-F confirm a sequence which is identical to the sequence SEQ ID No. 91. This clone is named pPDS and is therefore used for cloning into the vector pJBGEN (see below).
The first cloning step is carried out by isolating the 1499 bp NcoI/EcoRI fragment from pBGEN, derived from the cloning vector pCR2.1 (Invitrogen). First, pBGEN is cut with BamHI, and the 3′ ends are filled in by standard methods (30 minutes at 30° C.) (Klenew fill-in) and then a partial digest with NcoI is carried out in which the resulting 1499 kb fragment isolates. This fragment was subsequently cloned into pCSP02 which, previously cut with EcoRI, the 3′ ends filled in by standard methods (30 minutes at 30° C.) (Klenow fill-in), and is then cut with NcoI. The clone which comprises the 1497 bp B gene fragment BGEN is named pJAP:BGEN. The ligation gives rise to a transcriptional fusion between the 35S terminator and the B gene.
The second cloning step is carried out by isolating the 2078 bp PDS PROM fragment from pPDS. First, pPDS is cut with SmaI and then a partial digest with SacI is carried out in which the resulting 2088 bp fragment isolates. This fragment was subsequently cloned into pJAP:BGEN which previously cut with BamHI, the 3′ ends filled in by standard methods (30 minutes at 30° C.) (Klenow fill-in), and is then cut with SacI. The ligation gives rise to transcriptional fusion between the promoter PDS and the B gene. The clone which comprises the 2078 bp PDS promoters BGEN is named pJPDS:BGEN.

EXAMPLE 19

Preparation of a Triple Expression Vector for Overexpressing the B gene, for Expressing the Nostoc punctiforme Ketolase NP196 and for Overexpressing the Chromoplast-Specific B-hydroxylase from Lycopersicon esculentum in a Fruit-Specific Manner in Lycopersicon esculentum

First, a double construct which comprises expression cassettes for overexpressing the Nostoc punctiforme ATCC 29133 NP196 ketolase and for overexpressing the B-hydroxylase is prepared.
First, the fragment AP3P:b-hydroxylase:LB3, which comprises the B-hydroxylase expression cassette, is isolated from pCSP03 as a 2104 bp Ecl136II/XhoI fragment (described in Example 18). The 3′ ends are filled in by standard methods (Klenow fill-in; 30 minutes at 30° C.). Thereafter, this fragment was cut with Ecl136II and EcoRI in the vector MSP120 (described in Example 10), and the 3′ ends were filled in by standard methods (30 minutes at 30° C.; Klenow fill-in). The ligation gives rise to T-DNA which comprises two expression cassettes: firstly, a cassette for the chromoplast-specific overexpression of the B-hydroxylase from Lycopersicon esculentum and, secondly, a cassette for overexpressing the ketolase NP196 from Nostoc punctiforme. The B-hydroxylase downregulation cassette can ligate into the vector in two orientations. It is preferred to use the version in which the two expression cassettes have the same orientation (see FIG. 14). This version can be identified by PCR as described:
The PCR for the amplification of the PR206-PR010 plasmid fragment which comprises the ligation of LB3 terminator of the B-hydroxylase cassette and the AP3P promoter of the ketolase cassette is carried out in 50 μl of reaction mixture comprising:

- 1 μl of plasmid DNA (prepared by standard methods)
- 0.25 mM dNTPs
- 0.2 μM PR010 (SEQ ID No. 92)
- 0.2 μM PR206 (SEQ ID No. 93)
- 5 μl 10× PCR buffer (TAKARA)
- 0.25 μl R Taq polymerase (TAKARA)
- 28.8 μl distilled water

The PCR amplification with PR010 and PR206 results in a 1080 bp fragment which suggests the presence of the above-described ligation of LB3 terminator and AP3P promoter and thus the preferred orientation of the two expression cassettes. This clone is named pBHYX:NP196.
To clone this B gene overexpression cassette into expression vectors for the Agrobacterium-mediated transformation of tomato is carried out by isolating the 4362 bp EcoRV/XhoI fragment from pJPDS:BGEN (see Example 19) and ligation into the SmaI/XhoI-cut vector pBHYX:NP196 (described above). The ligation gives rise to a T-DNA which comprises three expression cassettes: firstly, a cassette for overexpressing the B gene, secondly, a cassette for overexpressing the NP196-1 ketolase from Nostoc punctiforme, and, thirdly, a cassette for the chromoplast-specific overexpression of the B-hydroxylase from Lycopersicon esculentum (FIG. 14, construct map). This clone is named MSP124. In FIG. 14, fragment AP3P PROM (765 bp) comprises the AP3P promoter, the fragment BHYX b2 CDS (2 bp) the B-hydroxylate CrtRb2, fragment LB3 TERM (296 bp) the LB3 terminator.
Furthermore, fragment AP3P PROM (765 bp) comprises the AP3P promoter, fragment rbcS TP FRAGMENT (194 bp) the transit peptide of the rbcS gene from pea, NP196 KETO CDS (761 bp) the ketolase from Nostoc punctiforme ATCC29133, and OCS TERM (192 bp) the polyadenylation signal of the octopin synthase gene.
Furthermore, fragment PDS PROM (2078 bp) comprises the PDS promoter, fragment BGEN CDS (1497 bp) the B gene sequence, and fragment 35S TERM (746 bp) the 35 S terminator.

EXAMPLE 20

Generation of Transgenic Lycopersicon esculentum Plants

The transformation and regeneration of tomato plants was described in Example 6.
In accordance with the transformation method described, the following lines were obtained with the following expression constructs:

the following were obtained with MSP120: MSP120-1, MSP120-2, MSP120-3
the following were obtained with MSP121: MSP121-1, MSP121-2, MSP121-3
the following were obtained with MSP122: MSP122-1, MSP122-2, MSP133-3
the following were obtained with MSP123: MSP123-1, MSP123-2, MSP123-3
the following were obtained with MSP124: MSP124-1, MSP124-2, MSP124-3

Claims

1. A method for the production of ketocarotenoids by culturing genetically modified plants which show a ketolase activity in fruits.

2. The method according to claim 1, wherein genetically modified plants are used which express a ketolase in fruits.

3. The method according to claim 1, wherein genetically modified plants are used which comprise, in fruits, at least one nucleic acid encoding a ketolase.

4. The method according to claim 3, wherein genetically modified plants are used into which, starting from a starting plant, at least one nucleic acid encoding a ketolase has been introduced.

5. The method according to claim 4, wherein nucleic acids are introduced which encode a protein comprising the amino acid sequence SEQ ID NO. 2 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has at least 20% identity at the amino acid level with the sequence SEQ ID NO. 2 and which has the enzymatic characteristic of a ketolase.

6. The method according to claim 4, wherein nucleic acids comprising the sequence SEQ ID NO. 1 are introduced.

7. The method according to claim 4, wherein nucleic acids are introduced which encode a protein comprising the amino acid sequence SEQ ID NO. 16 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has at least 20% identity at the amino acid level with the sequence SEQ ID NO. 16 and which has the enzymatic characteristic of a ketolase.

8. The method according to claim 6, wherein nucleic acids comprising the sequence SEQ ID NO. 15 are introduced.

9. The method according to claim 1, wherein genetically modified plants are used which show the highest expression rate of a ketolase in fruits.

10. The method according to claim 9, wherein the gene expression of the ketolase takes place under the control of a fruit-specific promoter.

11. The method according to claim 1, wherein the plants additionally show an increased activity of at least one of the activities selected from the group consisting of hydroxylase activity and β-cyclase activity in comparison with the wild type.

12. The method according to claim 11, wherein, to additionally increase at least one of the activities, the gene expression of at least one nucleic acid selected from the group consisting of nucleic acids encoding a hydroxylase and nucleic acids encoding a β-cyclase is increased in comparison with the wild type.

13. The method according to claim 12, wherein, to increase the gene expression of at least one of the nucleic acids, at least one nucleic acid selected from the group consisting of nucleic acids encoding a hydroxylase and nucleic acids encoding a β-cyclase is introduced into the plant.

14. The method according to claim 13, wherein nucleic acids encoding a hydroxylase comprising the amino acid sequence SEQ ID NO: 52 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has at least 20% identity at the amino acid level with the sequence SEQ ID NO: 52 are introduced as nucleic acid encoding a hydroxylase.

15. The method according to claim 14, wherein nucleic acids comprising the sequence SEQ ID NO: 51 are introduced.

16. The method according to claim 13, wherein nucleic acids encoding a cyclase comprising the amino acid sequence SEQ ID NO: 54 or a sequence derived from this sequence by substitution, insertion or deletion of amino acids which has at least 20% identity at the amino acid level with the sequence SEQ ID NO: 54 are introduced as nucleic acid encoding a β-cyclase.

17. The method according to claim 16, wherein nucleic acids comprising the sequence SEQ ID NO: 53 are introduced.

18. The method according to claim 11, wherein genetically modified plants are used which have the highest expression rate of a hydroxylase and/or β-cyclase in flowers.

19. The method according to claim 18, wherein the gene expression of the hydroxylase and/or β-cyclase is effected under the control of a flower-specific promoter.

20. The method according to claim 1, wherein the plant used is a plant which has chromoplasts in fruits.

21. The method according to claim 1, wherein the plant used is a plant selected from the plant genera consisting of Actinophloeus, Aglaeonema, Ananas, Arbutus, Archontophoenix, Area, Aronia, Asparagus, Attalea, Berberis, Bixia, Brachychilum, Bryonia, Caliptocalix, Capsicum, Carica, Celastrus, Citrullus, Citrus, Convallaria, Cotoneaster, Crataegus, Cucumis, Cucurbita, Cuscuta, Cycas, Cyphomandra, Dioscorea, Diospyrus, Dura, Elaeagnus, Elaeis, Erythroxylon, Euonymus, Ficus, Fortunella, Fragaria, Gardinia, Gonocaryum, Gossypium, Guava, Guilielma, Hibiscus, Hippophaea, Iris, Lathyrus, Lonicera, Luffa, Lycium, Lycopersicum, Malpighia, Mangifera, Mormodica, Murraya, Musa, Nenga, Palisota, Pandanus, Passiflora, Persea, Physalis, Prunus, Ptychandra, Punica, Pyracantha, Pyrus, Ribes, Rosa, Rubus, Sabal, Sambucus, Seaforita, Shepherdia, Solanum, Sorbus, Synaspadix, Tabemae, Tamus, Taxus, Trichosanthes, Triphasia, Vaccinium, Viburnum, Vignia or Vitis.

22. The method according to claim 1, wherein, after cultivation, the genetically modified plants are harvested and the ketocarotenoids are subsequently isolated from the plant's fruits.

23. The method according to claim 1, wherein the ketocarotenoids are selected from the group consisting of astaxanthin, canthaxanthin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin and adonixanthin.

24. A nucleic acid construct comprising, in functional linkage, a fruit-specific promoter and a nucleic acid encoding a ketolase.

25. A genetically modified plant, which shows a ketolase activity in fruits.

26. The genetically modified plant according to claim 25, wherein the genetically modified plant expresses a ketolase in the fruits.

27. The genetically modified plant according to claim 25, comprising, in fruits, at least one nucleic acid encoding a ketolase.

28. The genetically modified plant according to claim 25, wherein starting from a starting plant, at least one nucleic acid encoding a ketolase has been introduced into the plant.

29. The genetically modified plant according to claim 25, wherein the genetic modification additionally increases, in comparison with a wild-type plant, at least one of the activities selected from the group consisting of hydroxylase activity and b-cyclase activity.

30. A genetically modified plant selected from the plant genera Actinophloeus, Aglaeonema, Ananas, Arbutus, Archontophoenix, Area, Aronia, Asparagus, Attalea, Berberis, Bixia, Brachychilum, Bryonia, Caliptocalix, Capsicum, Carica, Celastrus, Citrullus, Citrus, Convallaria, Cotoneaster, Crataegus, Cucumis, Cucurbita, Cuscuta, Cycas, Cyphomandra, Dioscorea, Diospyrus, Dura, Elaeagnus, Elaeis, Erythroxylon, Euonymus, Ficus, Fortunella, Fragaria, Gardinia, Gonocaryum, Gossypium, Guava, Guilielma, Hibiscus, Hippophaea, Iris, Lathyrus, Lonicera, Luffa, Lycium, Lycopersicum, Malpighia, Mangifera, Mormodica, Murraya, Musa, Nenga, Palisota, Pandanus, Passiflora, Persea, Physalis, Prunus, Ptychandra, Punica, Pyracantha, Pyrus, Ribes, Rosa, Rubus, Sabal, Sambucus, Seaforita, Shepherdia, Solanum, Sorbus, Synaspadix, Tabernae, Tamus, Taxus, Trichosanthes, Triphasia, Vaccinium, Viburnum, Vignia or Vitis, comprising at least one nucleic acid encoding a ketolase.

31. The genetically modified plant according to claim 30, wherein the ketolase is expressed in fruits.

32. The genetically modified plant according to claim 25, wherein the expression rate of a ketolase is highest in fruits.

33. The genetically modified plants according to claim 25, wherein the plants are used as feeds or foods.

34. The genetically modified plants according to claim 25, wherein the fruits are used for the production of ketocarotenoid-comprising extracts or for the production of feed additives or food additives.

35. A method for the generation of genetically modified plants according to claim 32, wherein a nucleic acid construct comprising, in functional linkage, a fruit-specific promoter and nucleic acids encoding a ketolase is introduced into the genome of the starting plant.