AU778297B2 - Fatty acid desaturase gene from plants - Google Patents

Abstract

The invention relates to a method for producing unsaturated or saturated fatty acids and to a method for producing triglycerides with an increased unsaturated or saturated fatty acid content. The invention also relates to a nucleic acid sequence and a nucleic acid construct, a vector and organisms containing at least one nucleic acid sequence or a nucleic acid construct. Finally, the invention relates to saturated or unsaturated fatty acids and triglycerides with an increased unsaturated or saturated fatty acid content and to their use.

Description

0050/50669 Fatty acid desaturase gene from plants The present invention relates to a process for the preparation of unsaturated or saturated fatty acids and a process for the preparation of triglycerides with an increased content of unsaturated or saturated fatty acids.

Moreover, the invention relates to a nucleic acid sequence; a nucleic acid construct, a vector and organisms comprising at least one nucleic acid sequence or one nucleic acid construct.

Furthermore, the invention relates to saturated or unsaturated fatty acids and triglycerides with an increased content of unsaturated or saturated fatty acids and their use.

Fatty acids and triglycerides have a multiplicity of applications in the food industry, animal nutrition, cosmetics and in the pharmaceutical sector. Depending on whether they are free saturated or unsaturated fatty acids or triglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a very wide range of applications; thus, for example, polyunsaturated fatty acids are added to baby formula to increase the nutritional value. The various fatty acids and triglycerides are obtained mainly from microorganisms such as Mortierella or from oil-producing plants such as soya, oilseed rape, sunflowers and others, where they are usually obtained in the form of their triacyl glycerides. Alternatively, they are obtained advantageously from animals, such as fish. The free fatty acids are prepared advantageously by hydrolysis.

Whether oils with unsaturated or with saturated fatty acids are preferred depends on the intended purpose; thus, for example, lipids with unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred in human nutrition since they have a positive effect on the cholesterol level in the blood and thus on the possibility of heart disease. They are used in a variety of dietetic foodstuffs or medicaments.

Especially valuable and sought-after unsaturated fatty acids are the so-called conjugated unsaturated fatty acids, such as conjugated linoleic acid. A series of positive effects have been found for conjugated fatty acids; thus, the administration of conjugated linoleic acid reduces body fat in humans and animals, and increases the conversion of feed into body weight in the case of animals (WO 94/16690, WO 96/06605, WO 97/46230, WO 97/46118).

By administering conjugated linoleic acid, it is also possible to positively affect, for example, allergies (WO 97/32008) or cancer 0050/50669 2 (Banni et al., Carcinogenesis, Vol. 20, 1999: 1019 1024, Thompson et al., Cancer, Res., Vol. 57, 1997: 5067 5072).

The chemical preparation of conjugated fatty acids, for example calendulic acid or conjugated linoleic acid, is described in US 3,356,699 and US 4,164,505. Calendulic acid occurs naturally in Calendula officinalis (Ul'chenko et al., Chemistry of Natural Compounds, 34, 1998: 272 274). Conjugated linoleic acid is found, for example, in beef (Chin et al., Journal of Food Composition and Analysis, 5, 1992: 185 197). Biochemical studies into the synthesis of calendulic acid can be found in Crombie et al., J. Chem. Soc. Chem. Commun., 15, 1984: 953 955 and J. Chem. Soc. Perkin Trans., 1, 1985: 2425 2434.

Owing to their positive properties, there has been no lack of attempts in the past to make available genes which participate in the fatty acid or triglyceride synthesis for the production, in various organisms, of oils with an altered content of unsaturated fatty acids. Thus, WO 91/13972 and its US equivalent describe a A-9-desaturase. WO 93/11245 claims a A-15-desaturase, WO 94/11516 a A-12-desaturase. A-6-Desaturases are described in WO 93/06712 and WO 96/21022. Other desaturases are described, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144 20149, Wada et al., Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649 659. However, the biochemical characterization of the various desaturases is as yet only insufficient since the enzymes, being the membrane-bound proteins, can only be isolated and characterized with great difficulty (McKeon et al., Methods in Enzymol. 71, 1981: 12141 12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777 792).

In yeasts, both a shift of the fatty acid spectrum toward unsaturated fatty acids and an increase in productivity were found (see Huang et al., Lipids 34, 1999: 649 659, Napier et al., Biochem. Vol. 330, 1998: 611 614). However, the expression of the various desaturases in transgenic plants did not show the desired success. While it was possible to demonstrate a shift of the fatty acid spectrum toward unsaturated fatty acids, it emerged, simultaneously, that the synthetic productivity of the transgenic plants suffered greatly, viz.

lesser amounts of oils were isolated compared with the starting plants.

Thus, there remains a great need for new genes which encode enzymes which participate in the biosynthesis of unsaturated fatty acids and which allow the latter, specifically conjugated 0050/50669 3 unsaturated fatty acids, to be synthesized and produced on an industrial scale.

It is an object of the present invention to provide other desaturases for the synthesis of unsaturated conjugated fatty acids.

We have found that this object is achieved by an isolated nucleic acid sequence which encodes a polypeptide with desaturase activity, selected from the following group: a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1, b) nucleic acid sequences which, as a result of the degeneracy of the genetic code, are derived from the nucleic acid sequence shown in SEQ ID NO: 1, c) derivatives of the nucleic acid sequence shown in SEQ ID NO: 1 which encode polypeptides with the amino acid sequences shown in SEQ ID NO: 2 and which have at least 75% homology at amino acid level without substantially reducing the enzymatic activity of the polypeptides.

A derivative (or derivatives) is/are to be understood as meaning, for example, functional homologs of the enzyme encoded by SEQ ID NO: 1 or its enzymatic activity, viz. enzymes which catalyze the same enzymatic reactions as the enzyme encoded by SEQ ID NO:1.

These genes also allow an advantageous preparation of unsaturated conjugated fatty acids. Unsaturated fatty acids are to be understood, in the following text, as meaning mono- and polyunsaturated fatty acids whose double bonds may be conjugated or not conjugated. The sequence given in SEQ ID NO:1 encodes a novel, unknown desaturase which participates in the synthesis of calendulic acid in Calendula officinalis. The enzyme converts (9Z,12Z)octadecadienoic/linoleic acid to (8E,10E,12Z) octadecaconjutrienoic/calendulic acid. This is termed calendulic acid desaturase hereinbelow.

The nucleic acid sequence according to the invention or its fragments can be used advantageously for isolating further genomic sequences by means of homology screening.

0050/50669 4 The abovementioned derivatives can be isolated, for example, from other eukaryotic organisms such as plants like Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides, algae, protozoans such as dinoflagellates, or fungi.

Derivatives or functional derivatives of the sequence given in SEQ ID No.l are furthermore to be understood as meaning, for example, allelic variants which have at least 75% homology at the derived amino acid level, preferably at least 80% homology, especially preferably at least 85% homology, very especially preferably 90% homology. The homology was calculated over the entire amino acid range. The program used was PileUp Mol.

Evolution., 25 (1987), 351-360, Higgins et al., CABIOS, 5 1989: 151 153). The amino acid sequence derived from the abovementioned nucleic acid can be seen from the sequence SEQ ID No.2. Allelic variants encompass, in particular, functional variants which can be obtained from the sequence shown in SEQ ID No.l by means of deletion, insertion or substitution of nucleotides, the enzymatic activity of the derived synthetic proteins being retained.

Such DNA sequences can be isolated from other eukaryotes as mentioned above, starting from the DNA sequence described in SEQ ID No. 1 or parts of these sequences, for example using customary hybridization methods or the PCR technique. These DNA sequences hybridize with the sequences mentioned under standard conditions.

It is advantageous to use, for the hybridization, short oligonucleotides, for example from the conserved regions, which can be determined by the skilled worker by comparison with other desaturase genes.

Alternatively, it is possible to use longer fragments of the nucleic acids according to the invention or the full sequences for the hybridization. Depending on which nucleic acid: oligonucleotide, longer fragment or full sequence, or depending on which nucleic acid type, viz. DNA or RNA, is used for the hybridization, these standard conditions vary. Thus, for example, the melt temperatures for DNA:DNA hybrids are approximately lower than those of equally long DNA:RNA hybrids.

Depending on the nucleic acid, standard conditions are understood as meaning, for example, temperatures between 42 and 58 0 C in an aqueous buffer solution with a concentration of between 0.1 to [sic] 5 x SSC (1 X [sic] SSC 0.15 M NaC1, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide such as, for example, 42 0 C in 5 x SSC, 50% formamide. The hybridization conditions for DNA:DNA hybrids are advantageously 0.1 x SSC and temperatures between approximately 20 0 C to [sic] 45 0 C, preferably 0050/50669 between approximately 30 0 C to [sic] 45'C. The hybridization conditions for DNA:RNA hybrids are advantageously 0.1 x SSC and temperatures between approximately 30*C to [sic] 55°C, preferably between approximately 45°C to [sic] 55 0 C. These temperatures which are indicated for the hybridization are examples of calculated melting point data for a nucleic acid with a length of approx.

100 nucleotides and a G C content of 50% in the absence of formamide. The experimental conditions for the DNA hybridization are described in relevant genetics textbooks such as, for example, by Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989 and can be calculated using formulae known to the skilled worker, for example as a function of the length of the nucleic acids, the type of hybrid or the G C content. The skilled worker can find further information on hybridization in the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

Derivatives are furthermore to be understood as meaning homologs of the sequence SEQ ID No.1, for example eukaryotic homologs, 2 truncated sequences, simplex DNA of the coding and noncoding DNA sequence or RNA of the coding and noncoding DNA sequence.

Homologs of the sequence SEQ ID No.l are also to be understood as meaning derivatives such as, for example, promoter variants.

3 These variants can be altered by one or more nucleotide exchanges, by insertion(s) and/or deletion(s), without, however, adversely affecting the functionality or efficacy of the promoters. Moreover, it is possible to increase the efficacy of the promoters by altering their sequence or to exchange them completely by more efficient promoters from other organisms, including other species.

Derivatives are also advantageously to be understood as meaning variants whose nucleotide sequence in the region -1 to -2000 upstream of the start codon were [sic] altered in such a way that gene expression and/or protein expression is altered, preferably increased. Moreover, derivatives are also to be understood as meaning variants whose 3' end was altered.

4 To achieve optimal expression of heterologous genes in organisms, it is advantageous to alter the nucleic acid sequences in accordance with the specific codon usage used in the organism.

0050/50669 6 The codon usage can be determined readily by using computer evaluations of other, known genes of the organism in question.

The calendulic acid desaturase gene can be combined advantageously in the process according to the invention with other fatty acid biosynthesis genes.

The amino acid sequences according to the invention are to be understood as meaning proteins which contain an amino acid sequence shown in SEQ ID NO: 2 or a sequence obtainable therefrom by the substitution, inversion, insertion or deletion of one or more amino acid residues, the enzymatic activity of the protein shown in SEQ ID NO: 2 being retained or not reduced substantially. The term not reduced substantially is to be understood as meaning all enzymes which still have at least preferably 20%, especially preferably 30% of the enzymatic activity of the starting enzyme. For example, certain amino acids may be replaced by others with similar physico-chemical properties (spatial dimension, basicity, hydrophobicity and the like). For example, arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues. Alternatively, it is possible to exchange the sequence of, add or remove one or more amino acids, or two or more of these measures may be combined with each other.

The nucleic acid construct or nucleic acid fragment according to the invention is to be understood as meaning the sequence given in SEQ ID NO: 1, sequences which are the result of the genetic code and/or their functional or nonfunctional derivatives, all of which have been linked functionally to one or more regulatory signals, advantageously for increasing gene expression. These regulatory sequences are, for example, sequences to which inductors or repressors bind and thus regulate the expression of the nucleic acid. In addition to these novel regulatory sequences, or instead of these sequences, the natural regulation of these sequences upstream of the actual structural genes may still be present and, if desired, may have been genetically altered in such a way that the natural regulation has been switched off and the expression of the genes increased. However, the expression of the gene construct may also have a simpler structure, viz. no additional regulatory signals have been inserted upstream of the sequence or its derivatives and the natural promoter with its regulation has not been removed.

4 Instead, the natural regulatory sequence has been mutated in such a way that regulation no longer takes place and gene expression is increased. These altered promoters may also be placed upstream of the natural gene on their own, in order to increase activity.

0050/50669 7 In addition, the gene construct can also advantageously contain one or more so-called enhancer sequences functionally linked to the promoter, and these allow an increased expression of the nucleic acid sequence. It is also possible to insert, at the 3' end of the DNA sequences, additional advantageous sequences such as further regulatory elements or terminators. One or more copies of the calendulic acid desaturase gene may be contained in the gene construct.

Advantageous regulatory sequences for the process according to the invention are contained, for example, in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T3, gal, trc, ara, SP6, X-PR or in the X-PL promoter, all of which are advantageously used in Gram-negative bacteria. Other advantageous regulatory sequences are contained, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFa AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, or in the plant promoters such as CaMV/35S [Franck et al., Cell 21(1980) 285-294], PRP1 [Ward et al., Plant.Mol. Biol.22(1993)], SSU, OCS, lib4, STLS1, B33, nos or in the Ubiquitin promoter.

Other advantageous plant promoters are, for example, a benzenesulfonamide-inducible (EP 388186), a tetracyclin-inducible (Gatz et al., (1992) Plant J. 2,397-404), an abscisic-acid-inducible (EP335528) and an ethanol- or cyclohexanone-inducible (W09321334) promoter. Other plant promoters are, for example, the potato cytosolic FBPase promoter, the potato ST-LSI promoter (Stockhaus et al., EMBO J. 8 (1989) 2445-245), the Glycine max phosphoribosyl pyrophosphate amido transferase promoter (see also gene bank accession number U87999) or a node-specific promoter as described in EP 249676.

3Advantageous plant promoters are, in particular, those which ensure expression in tissues or parts of the plants in which the biosynthesis of fats or their precursors takes place. Promoters which must be mentioned in particular are those which ensure seed-specific expression such as, for example, the USP promoter, the LEB4 promoter, the phaseolin promoter or the napin promoter.

In principle, all natural promoters with their regulatory sequences as those mentioned above may be used for the process according to the invention. In addition, synthetic promoters may also advantageously be used.

The nucleic acid fragment gene construct, nucleic acid construct) may also contain further genes to be introduced into organisms, as this has been described above. These genes can be under separate regulation or under the same regulatory region as the desaturase gene according to the invention. These genes are, for example, other biosynthesis genes, advantageously of the 0050/50669 8 fatty acid and lipid biosynthesis, which allow increased synthesis. Examples which may be mentioned are the genes for A12-, A9-, A6-, A5-desaturase, the various hydroxylases, acetylenase, the acyl-ACP thioesterases, the P-ketoacyl-ACP synthases, the acyltransferases such as diacylglycerol acyltransferase, glycerol-3-phosphate acyltransferase or lysophosphatidic acid acyltransferase or P-ketoacyl-ACP reductases. It is advantageous to use the desaturase genes in the nucleic acid construct, especially the A12-desaturase gene.

For expression in a host organism, for example a microorganism such as fungus or a plant, the nucleic acid fragment is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which vector allows optimal expression of the genes in the host. Examples of suitable plasmids are, in E. coli, pLG338, pACYC184, pBR322, pUC18, pUC19, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III13-Bl, Xgtll or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL2 or pBB116, in yeasts 2pM, pAG-1, YEp6, YEpl3 or pEMBLYe23, or, in plants, pLGV23, pGHlac*, pBIN19, pAK2004, pVKH or pDH51, or derivatives of the abovementioned plasmids. The plasmids mentioned represent a small selection of the plasmids which are possible. Other plasmids are well known to the skilled worker and can be found, for example, in the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985 ISBN 0 444 904018). Suitable plant vectors are described, inter alia, in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chapter 6/7, pp.71-119.

In addition to plasmids, vectors are also to be understood as meaning all the other vectors which are known to the skilled worker, such as, for example, phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally. Chromosomal replication is preferred.

The vector advantageously contains at least one copy of the nucleic acid sequence according to the invention and/or of the nucleic acid fragment according to the invention.

To increase the gene copy number, the nucleic acid sequences or homologous genes can be introduced, for example, into a nucleic acid fragment or into a vector which preferably contains the regulatory gene sequences assigned to the genes in question, or 0050/50669 9 analogously acting promoter activity. Regulatory sequences which are used in particular are those which increase gene expression.

To express the other genes contained, the nucleic acid fragment advantageously additionally contains and/or regulatory sequences to increase expression, these sequences being selected for optimal expression, depending on the host organism chosen and the gene or genes.

These regulatory sequences should allow the targeted expression of the genes and protein expression. Depending on the host organism, this may mean, for example, that the gene is expressed and/or overexpressed only after induction, or that it is expressed and/or overexpressed immediately.

The regulatory sequences or factors can preferably have a positive effect on, and thus increase, the gene expression of the genes introduced. Thus, strengthening of the regulatory elements can advantageously take place at the transcriptional level by using strong transcription signals such as promoters and/or enhancers. In addition, however, strengthening of translation is also possible, for example by improving mRNA stability.

In a further embodiment of the vector, the gene construct according to the invention can advantageously also be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism by means of heterologous or homologous recombination. This linear DNA may consist of a linearized plasmid or only of the nucleic acid fragment as vector or of the nucleic acid sequence according to the invention.

The nucleic acid sequence according to the invention is advantageously cloned into a nucleic acid construct together with at least one reporter gene, and the nucleic acid construct is introduced into the genome. This reporter gene should allow easy detectability via a growth assay, a fluorescence assay, a chemo assay, a bioluminescence assay or a resistance assay, or via a photometric measurement. Examples of reporter genes which may be mentioned are genes for resistance to antibiotics or herbicides, hydrolase genes, fluorescence protein genes, bioluminescence genes, sugar metabolism genes or nucleotide metabolism genes, or biosynthesis genes such as the Ura3 gene, the Ilv2 gene, the luciferase gene, the P-galactosidase gene, the gfp gene, the 2-deoxyglucose-6-phosphate phosphatase gene, the P-glucuronidase gene, the p-lactamase gene, the neomycin phosphotransferase gene, the hygromycin phosphotransferase gene or the BASTA gluphosinate) resistance gene. These genes allow the 0050/50669 transcriptional activity, and thus gene expression, to be measured and quantified easily. In this way, genome sites which show different productivity can be identified.

In a further advantageous embodiment, the nucleic acid sequence according to the invention may also be introduced into an organism on its own.

If it is intended to introduce, into the organism, other genes in addition to the nucleic acid sequence according to the invention, all can be introduced into the organism in a single vector with a reporter gene, or each individual gene with a reporter gene per vector, it being possible for the various vectors to be introduced simultaneously or in succession.

The host organism advantageously contains at least one copy of the nucleic acid according to the invention and/or of the nucleic acid construct according to the invention.

In principle, the nucleic acid according to the invention, the nucleic acid construct or the vector can be introduced into organisms, for example plants, by all methods known to the skilled worker.

In the case of microorganisms, the skilled worker can find suitable methods in the textbooks by Sambrook, J. et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, by F.M. Ausubel et al. (1994) Current protocols in molecular biology, John Wiley and Sons, by D.M. Glover et al., DNA Cloning Vol.l, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press or by Guthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press.

The transfer of foreign genes into the genome of a plant is termed transformation. The described methods for the transformation and regeneration of plants from plant tissues or plant cells are used for transient or stable transformation.

Suitable methods are protoplast transformation by polyethylene-glycol-induced DNA uptake, the use of a gene gun, electroporation, the incubation of dry embryos in DNA-containing solution, microinjection and the agrobacterium-mediated gene transfer. The methods mentioned 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 by Potrykus, Annu.

0050/50669 11 Rev. Plant Physiol.Plant Molec.Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).

The transformation of plants with Agrobacterium tumefaciens is described, for example, by H6fgen and Willmitzer in Nucl. Acid Res. (1988) 16 [sic] 9877.

Agrobacteria which have been transformed with an expression vector according to the invention can also be used in the known manner to transform plants such as test plants like Arabidopsis or crop plants, in particular oil-containing crop plants such as soya, peanuts, castor, sunflowers, corn, cotton, flax, oilseed rape, coconut [lacuna], oil palms, safflower (Carthamus tinctorius) or cacao, for example by bathing wounded leaves or leaf sections in agrobacterial solution and subsequently culturing them in suitable media.

The genetically altered plant cells can be regenerated by all methods known to the skilled worker. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or H6fgen and Willmitzer.

Suitable organisms or host organisms for the nucleic acid according to the invention, the nucleic acid construct or the vector are, in principle, all organisms which are capable of synthesizing fatty acids, specifically unsaturated fatty acids, and which are suitable for the expression of recombinant genes.

Examples which may be mentioned are plants such as Arabidopsis, Asteraceae such as Calendula or crop plants such as soya, peanuts, castor, sunflowers, corn, cotton, flax, oilseed rape, coconut [lacuna], oil palms, safflower (Carthamus tinctorius) or cacao, microorganisms such as fungi, for example the genus Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, yeasts such as the genus Saccharomyces, algae or protozoans such as dinoflagellates such as Crypthecodinium.

Preferred organisms are those which are naturally capable of synthesizing oils in substantial amounts, like fungi such as Mortierella alpina, Pythium insidiosum or plants such as soya, oilseed rape, flax, coconut [lacuna], oil palms, safflower, castor, Calendula, peanuts, cacao or sunflowers, or yeasts such as Saccharomyces cerevisiae, with soya, oilseed rape, flax, sunflowers, Calendula or Saccharomyces cerevisiae being especially preferred. In principle, transgenic animals, for example Caenorhabditis elegans, are also [lacuna] as host organisms.

0050/50669 12 Another embodiment according to the invention are, as described above, transgenic plants which contain a functional or a nonfunctional nucleic acid or a functional or nonfunctional nucleic acid construct. The term nonfunctional is to be understood as meaning that an enzymatically active protein is no longer synthesized since the natural gene has been inactivated.

In addition, the term nonfunctional nucleic acids or nucleic acid constructs is also to be understood as meaning a so-called antisense DNA which leads to transgenic plants which show a reduction in, or lack, enzymatic activity. The antisense technology, specifically when combining, in the antisense DNA, the nucleic acid sequence according to the invention with other fatty acid synthesis genes, allows the synthesis of triglycerides with an elevated content of saturated fatty acids, or saturated fatty acids. Transgenic plants are to be understood as meaning single plant cells and their cultures on solid media or in liquid culture, parts of plants and entire plants.

The use of the nucleic acid sequence according to the invention or of the nucleic acid construct according to the invention for the generation of transgenic plants is therefore also subject matter of the invention.

The invention furthermore relates to an enzyme which converts a fatty acid of the structure I, 2 R -CH+ COOR (I) which has two double bonds separated from each other by a methylene group, to give a triunsaturated fatty acid of the structure II, 2 R J CH 2 -nCOOR' (II), the three double bonds of the fatty acid being conjugated and the substituents and variables in the compounds of the structures I and II having the following meanings: 0050/50669 13 R1= hydrogen, substituted or unsubstituted, unsaturated or saturated, branched or unbranched C 1 -Clo-alkyl-, -CH2 1

I

O 0 1 3 1 4 R R R2= substituted or unsubstituted, unsaturated or saturated

C

1 -C-Alkyl-

R

3 and R 4 independently of one another are hydrogen, substituted or unsubstituted, saturated or unsaturated, branched or unbranched C 1

-C

2 2 -alkylcarbonyl or phospho-, n 1 to 14, preferably 1 to 8, especially preferably 4 to 6, very especially preferably 6.

R

1 in the compounds of the formula I and II is hydrogen, substituted or unsubstituted, unsaturated or saturated, branched or unbranched

C

1 -Clo-alkyl-, or -CH2 0 0 13 14 R R Alkyl radicals which may be -mentioned are substituted or unsubstituted, branched or unbranched C 1 -Clo-alkyl chains such as, for example, methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl-, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, n-heptyl, n-octyl, n-nonyl or n-decyl.

Preferred radicals for R 1 are hydrogen and -CH 0 O 13 14 R R

R

2 in the compounds of the formula I and II denotes substituted or unsubstituted, unsaturated or saturated C 1

-C

9 -alkyl-.

Alkyl radicals which may be mentioned are substituted or unsubstituted, branched or unbranched C 1

-C

9 -alkyl chains such as, for example, methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl-, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 0050/50669 14 1-ethyipropyl, n-hexyl, 1,1-dimethyipropyl, 1,2-dimethyipropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethyipropyl, 1,2,2-trimethyipropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, n-heptyl, n-octyl or n-nonyl. Cl-C 5 -alkyl is preferred, C 5 -alkyl is especially preferred.

R

3 and R 4 independently of one another are hydrogen, substituted or unsubstituted, saturated or unsaturated, branched or unbranched C 1

-C

2 2 -alkylcarbonyl- or phospho-.

C

1

-C

2 2 -alkylcarbonyl such as methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, 1-methylethylcarbonyl, n-butylcarbonyl, 1-methylpropylcarbonyl, 2-methylpropylcarbonyl, 1,1-dimethylethylcarbonyl, n-pentylcarbonyl, 1-methylbutylcarbonyl, 2-methylbutylcarbonyl, 3-methylbutylcarbonyl, 1,1-dimethylpropylcarbonyl, 1,2-dimethylpropylcarbonyl, 2,2-dimethylpropylcarbonyl, 1-ethyipropylcarbonyl, n-hexylcarbonyl, 1-methylpentylcarbonyl, 2-methylpentylcarbonyl, 3-methylpentylcarbonyl, 4-methylpentylcarbonyl, 1,1-dimethylbutylcarbonyl, 1,2-dimethylbutylcarbonyl, 1,3-dimethylbutylcarbonyl, 2,2-dimethylbutylcarbonyl, 2,3-dimethylbutylcarbonyl, 3,3-dimethylbutylcarbonyl, 1-ethylbutylcarbonyl, 2-ethylbutylcarbonyl, 1,1,2-trimethylpropylcarbonyl, 1,2,2-trimethylpropylcarbonyl, 1-ethyl-1-methylpropylcarbonyl and 1-ethyl-2-methylpropylcarbonyl, heptylcarbonyl, nonylcarbonyl, decylcarbonyl, undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl, n-nonadecylcarbonyl or n-eicosylcarbonyl.

Preferred substituents for R 3 and R 4 are saturated or unsaturated

C

16

-C

2 2 -alkylcarbonyl.

Examples of substituents of the abovementioned radicals which may be mentioned are halogen such as fluorine or chlorine, alkyl or hydroxyl.

In the conversion with the enzyme according to the invention, one double bond is introduced into the fatty acid and one double bond is shifted so that the three double bonds which participate in the reaction are conjugated. Furthermore, one double bond is isomerized (from cis to trans).

0050/50669 The enzyme calendulic acid desaturase) advantageously catalyzes the conversion of linoleic acid (18:2, 9Z,12Z) to calendulic acid (18:3, 8E,10E,12Z). The enzyme introduces a trans double bond at position C8 and causes the specific shift of a cis double bond in position C9 to a trans double bond in position the isomerization being effected regiospecifically. A possible hypothetical reaction mechanism is shown in Fig. 1.

After deprotonation at C8 of the linoleic acid and a rearrangement of the free radical to C10, the elimination of water leads to a deprotonation at C11 and thus to the formation of calendulic acid. Simultaneously, bound FeIV is reduced to FeIII. Fig. 1 shows the hypothetical mechanism for (8,11)-linoleoyl desaturase (calendulic acid desaturase), modified after Svatos, A et al. (Insect Biochemistry and Molecular Biology 29,1999:225-232) based on the proposed catalytic mechanism for Ricinus A9 desaturase (Lindqvist, Y et al., EMBO Journal 15, 1996:4081-4092). Suitable substrates are still 6Z,9Z,12Z, 18:3-fatty acid and 9Z,12Z,15Z, 18:3-fatty acid, which, in turn, are then reacted to give 6Z,8E,10E,12Z- and 8E,10E,12Z,15Z-fatty acids, respectively.

The invention furthermore relates to a process for the preparation of unsaturated fatty acids, which comprises introducing at least one above-described nucleic acid sequence according to the invention or at least one nucleic acid construct according to the invention into a preferentially oil-producing organism, growing this organism, isolating that [sic] oil contained in the organism and liberating the fatty acids contained in the oil.

The invention also includes a process for the preparation of triglycerides with an increased content of unsaturated fatty acids, which comprises introducing at least one above-described nucleic acid sequence according to the invention or at least one nucleic acid construct according to the invention into a preferentially oil-producing organism, growing this organism and isolating the oil contained in the organism.

Both processes advantageously allow the synthesis of fatty acids of triglycerides with an increased content of unsaturated fatty acids such as calendulic acid.

The invention furthermore relates to a process for the preparation of saturated fatty acids, which comprises introducing at least one nonfunctional abovementioned nucleic acid sequence according to the invention or at least one nonfunctional nucleic acid construct according to the invention into an oil-producing 0050/50669 16 organism, growing this organism, isolating the oil contained in the organism and liberating the fatty acids contained in the oil, and to a process for the preparation of triglycerides with an increased content of saturated fatty acids, which comprises introducing at least one nonfunctional abovementioned nucleic acid sequence according to the invention or at least one nonfunctional nucleic acid construct according to the invention into an oil-producing organism, growing this organism and isolating that [sic] oil contained in the organism. Both processes involve the use of the so-called antisense technology (see above), or the inactivation of the lateral synthesis genes.

Examples of organisms for the abovementioned processes are plants such as Arabidopsis, soya, peanuts, castor, sunflowers, corn, cotton, flax, oilseed rape, coconut [lacuna], oil palms, safflower (Carthamus tinctorius) or cacao bean [sic], microorganisms such as [lacuna] fungi Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, yeasts such as the genus Saccharomyces, algae or protozoans such as dinoflagellates, for example Crypthecodinium. Preferred organisms are those which can naturally synthesize oils in substantial amounts, such as fungi, for example Mortierella alpina, Pythium insidiosum, or plants such as soya, oilseed rape, flax, coconut [lacuna], oil palms, safflower, castor, Calendula, peanuts, cacao bean [sic] or sunflowers, or yeasts such as Saccharomyces cerevisiae; soya, oilseed rape, flax, sunflowers, Calendula or Saccharomyces cerevisiae are especially preferred.

Depending on the host organism, the organisms used in the processes are grown or cultured in the manner known to those skilled in the art. As a rule, microorganisms are grown in a liquid medium which contains a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, a phosphate source such as potassium hydrogen phosphate, trace elements such as iron salts, manganese salts, magnesium salts and, if required, vitamins, at temperatures between 0 C and 100 0 C, preferably between 10 0 C to [sic] 60 0 C, while gassing in oxygen. The pH of the liquid medium can be maintained at a fixed value, i.e. the pH is regulated while culture takes place.

However, the microorganisms may also be cultured without pH regulation. Culturing can be effected by the batch method, the semi-batch method or continuously. Nutrients may be supplied at the beginning of the fermentation or fed in semicontinuously or continuously.

0050/50669 17 Post-transformation, plants are first regenerated as described above and then grown or planted as usual.

After the organisms have been grown, the lipids are obtained in the usual manner. To this end, the organisms can first be harvested and then disrupted, or they can be used directly. It is advantageous to extract the lipids with suitable solvents such as apolar solvents, for example hexane, or polar solvents, for example ethanol, isopropanol, or mixtures such as hexane/isopropanol, phenol/chloroform/isoamyl alcohol, at temperatures between OOC to [sic] 80°C, preferably between 20 0 C to (sic] 50 0 C. As a rule, the biomass is extracted with an excess of solvent, for example with an excess of solvent to biomass of 1:4.

The solvent is subsequently removed, for example by distillation.

The extraction may also be carried out with supercritical CO 2 After the extraction, the remainder of the biomass can be removed, for example, by filtration. Standard methods for the extraction of fatty acids from plants and microorganisms are described in Bligh et al. (Can. J. Biochem. Physiol. 37, 1959: 911-917) or Vick et al. (Plant Physiol. 69, 1982: 1103-1108).

The crude oil thus obtained can then be purified further, for example by removing cloudiness by adding polar solvents such as acetone or apolar solvents such as chloroform, followed by filtration or centrifugation. Further purification via columns or other techniques is also possible.

To obtain the free fatty acids from the triglycerides, the latter are hyrolyzed in the customary manner, for example using NaOH or

KOH.

The invention furthermore relates to unsaturated or saturated fatty acids and triglycerides with an increased content of saturated or unsaturated fatty acids which have been prepared by the abovementioned processes, and to their use for the preparation of foodstuffs, animal feed, cosmetics or pharmaceuticals. To this end, they are added to the foodstuffs, animal feed, cosmetics or pharmaceuticals in the customary quantities.

The invention is illustrated in greater detail in the examples which follow: 0050/50669 18 Examples A cDNA was cloned from Calendula officinalis mRNA using RT-PCR and RACE techniques. When expressing this cDNA in yeast, linoleic acid is converted into the octadecaconjutriene calendulic acid (8E, 10E, 12Z). As far as we know, this is the first time that a calendulic acid desaturase has been described. The enzyme causes a regiospecific shift of a cis double bond in position C9 to a trans double bond in position C10 and introduces a new trans double bond at position C8.

Transgenic yeasts and plants with an increased expression of calendulic acid desaturase cDNA show calendulic acid in their lipids.

Example 1: Isolation of RNA from Calendula officinalis seeds In order to be able to isolate cDNA clones for calendulic acid desaturase by means of PCR, RNA was isolated from Calendula officinalis seeds. Owing to the high fat content of the seeds, it was impossible to use standard protocols; the following method was used instead: Using a pestle and mortar, 20 g of plant material were ground in liquid nitrogen to give a powder. 100 ml of extraction buffer I [100 mM tris/HCl, pH 7.5, 20 mM EDTA, 2% lauryl sarcosyl, 4 M guanidinium thiocyanate, 5% PVP polyvinylpyrrolidone), 1% p-mercaptoethanol] were added, and the batch was mixed immediately and homogenized. The solution was transferred into 50-ml-vessels and shaken for approximately minutes. After centrifugation for 10-15 minutes at 4,000 g, the fatty layer or fat drops which had risen to the top were removed and the supernatant was transferred into fresh vessels.

This was followed by extraction with 1 volume of phenol/chloroform/isoamyl alcohol PCI, 25:24:1) and one extraction with chloroform; in each case, the mixture was shaken for 15 minutes and then centrifuged. The upper, aqueous phase was removed, placed on an 8-ml-CsCl cushion (5 M CsCl) and centrifuged for 18 hours at 18 0 C and 100,000 g. The supernatant was decanted off and the RNA precipitate was dried briefly. After a washing step with 70% ethanol, the RNA was dissolved in a mixture of 7.5 ml extraction buffer II (100 ml tris/HCl, pH 8.8, 100 mM NaC1, 5 mM EDTA, 2% SDS) and 10 ml of PCI, shaken for minutes and centrifuged. The upper, aqueous phase was extracted with chloroform and then an equal volume of 5 M LiCI was added. The RNA was precipitated overnight at 4 0 C. The mixture was then centrifuged for 60 minutes at 12,000 g and 4 C. The 0050/50669 19 precipitate was washed twice with 70% ethanol, dried and finally taken up in 500 il of H 2 0.

mRNA was isolated from the resulting Calendula total RNA using the Poly-Attract Kit (Promega, Mannheim) following the manufacturer's instructions. 1 Rg of this mRNA was translated into cDNA with the SuperscriptII reverse transcriptase by Gibco BRL (Eggenstein) using 200 pmol of oligo-dT primer following the manufacturer's instructions and employed as template in a polymerase chain reaction (PCR).

Example 2 Isolation and cloning of the Calendula officinalis calendulic acid desaturase In order to isolate, from Calendula officinalis, DNA sequences which encode a calendulic acid desaturase, various degenerate oligonucleotide primers were derived from amino acid sequences of the conserved histidine boxes of various A12 desaturases.

Primer A: 5' CCD TAY TTC TCI TGG AAR WWH AGY CAY CG 3' forward primer, derived from the amino acid sequence P Y F S W K Y/I S H R Primer B: 5' CCA RTY CCA YTC IGW BGA RTC RTA RTG 3' reverse primer, derived from the amino acid sequence H Y DS S/T E W D/N W The letters in primers A and B have the following meaning: R A/G Y C/T W A/T H A/C/T B C/G/T D A/G/T I inositol In a PCR with Calendula simplex cDNA (prepared as described in Example 1) as template, a DNA fragment with a length of 470 bp was amplified using primers A and B. The following PCR program was used: 1. 2 min 94 °C 2. 30 sec 94 °C 3. 45 sec 50 °C (annealing temperature) 4. 1 min 72 °C x 2. to 4.

0050/50669 0 sec 94 °C 6. 45 sec 50 °C 7. 1 min 72 oC, time increment 5 sec per cycle x 5. to 7.

8. 2 min 72 °C The TfI DNA polymerase from Biozym (Hess. Oldendorf) was used for the amplification. The 470 bp DNA fragment was cloned into the vector pCR 2.1-TOPO with the aid of the TOPO TA Cloning Kit (Invitrogen, Carlsbad, USA) and sequenced. The sequence of the 470 bp fragment corresponded to the sequence of nucleotide 466 to 893 of SEQ ID NO:1.

Example 3: Obtaining and sequencing complete cDNA clones In order to obtain a full-length clone, the fragment was extended by means of and 3'-RACE (rapid amplification of cDNA ends).

Starting from 1 ig of mRNA (isolated as described in Example 1), duplex cDNA was prepared using the "Marathon cDNA Amplification Kit" by CLONTECH (Heidelberg). After ligation of the adaptor, and 3'-RACE was carried out using the following primers: Specific primers for Primer C Primer D 5' GTG AGG GAG TGA GAG ATG GGT GTG GTG C 3' 5' AAC ACA CTT ACA CCT AGT ACT GGA ATT G 3' Specific primers for 3'-RACE: Primer E Primer F 5' TAT TCC AAA CTT CTT AAC AAT CCA CCC G 3' CAA TTC CAG TAC TAG GTG TAA GTG TGT T 3' First, a PCR was carried out with the adaptor-ligated duplex cDNA and primer C or E; then, a second PCR was carried out with primer D or F and a 1:50 dilution of the PCR product from the reaction with primer C or E as template.

The RACE-PCR was carried out using the following program: 1.

2.

3.

4.

5.

6.

1 min 30 sec 3 min x 2.

30 sec 30 sec 3 min x 4.

94°C 94°C 68 0

C

3.

94°C 65 0

C

68 0

C

6.

0050/50669 21 7. 5 min 68 0

C

The resulting DNA fragments were cloned into pCR 2.1-TOPO with the aid of the TOPO TA Cloning Kit (Invitrogen, Carlsbad, USA) and sequenced. The 5'-RACE product extended over the start codon into the 5'-untranslated region and the 3'-RACE over the stop codon into the 3'-UTR).

The composite sequence composed of the first PCR product and the RACE product is shown in SEQ ID NO: 1. The encoding region extends from nucleotide 42 (start codon) to 1175 (stop codon).

The and UTRs were only sequenced as simplexes, so that individual sequencing mistakes are possible here.

In order to obtain a general [sic] full-length clone, a PCR was carried out using the Expand High Fidelity System (Boehringer, Mannheim) and the primers G and H, with Calendula cDNA (see Example 1) as template.

Primer G 5' ATTAGAGCTCATGGGTGCTGGTGGTCGGATGTCG 3' forward primer (with SacI cleavage site) Primer H 5' ATTACTCGAGTGACATACACCTTTTTGATTACATCTTG 3' reverse primer (with Xhol cleavage site) The PCR was carried out using the following program: 1. 2 min 94°C 2. 30 sec 94°C 3. 35 sec 63 0

C

4. 2 min 72 0

C

x 2. 4.

30 sec 94°C 6. 35 sec 63 0

C

7. 2 min 72 0 C, time increment 5 seconds per cycle x 5. 7 8. 2 min 72 OC.

The 1.2 kb PCR product was cloned into the vector pGEM-T (Promega, Mannheim) and transformed into E. coli DH10B. The insert DNA was sequenced as duplex using a 373 DNA sequencer (Applied Biosystems). To this end, the following sequence-specific primers were used in addition to reverse primer and -21 primer: Primer I: 5' CGG TCT TCT CGC TGT ATT 3' 0050/50669 22 Primer J: 5' ATT ACC CAA GCT GCC C 3' The complete DNA sequence of calendulic acid desaturase (CalDes) is identical to the section from nucleotide 42 to 1193 of SEQ ID NO:1. The sequence encompasses the encoding region and a short section of the 3'-UTR.

A comparison of the derived amino acid sequence of Co-CalDes (SEQ ID NO:2) with annotated protein sequences of the SWISS-PROT and SP-TREMBL databases demonstrated the highest homology to a Crepis alpina A12-acetylenase (SP_PL: 081931, 74% identical amino acids), a Crepis palaestina A12-epoxygenase (SP_PL: 065771, 73% identical amino acids) and a Borago officinalis A12-desaturase (SP_PL: 082729, 62% identical amino acids) over the entire encoding region. The sequence comparisons are shown in Fig. 2.

Fig. 2 shows a comparisons of the amino acid sequences of Co-CalDes with Crepis alpina A12-acetylenase (Ca-Acetyl), Crepis palaestina A12-epoxygenase (Cp-Epoxy) and Borago officinalis A12-desaturase (Bo-Des).

Example 4: Expression of calendulic acid desaturase in yeast In a first approach, the encoding region of the cDNA was cloned in a yeast expression vector and expressed in S. cerevisiae, in order to demonstrate the functionality of CalDes. The calendulic acid desaturase produced in the yeast was meant to convert added linoleic acid into calendulic acid. The latter, in turn, was to be detected by HPLC in hydrolyzed lipid extracts.

In a second approach, the A. thaliana A12-desaturase FAD2 (Kajiwara et al., Appl. Environ. Microbiol., 62, 1996: 4309 4313) was expressed in yeast in addition to CalDes, so that the yeast cells endogenously produce linoleic acid which, in turn, can be converted into calendulic acid owing to the activity of CalDes. The calendulic acid, in turn, was to be detected by HPLC.

All solid and liquid media for yeast were prepared by protocols of Ausubel et al. (Current Protocols in Molecular Biology, John Wiley Sons, New York, 1995).

The CalDes cDNA was excised from the vector pGEM-T by restriction digest with SacI/XhoI and cloned into the SacI/XhoI-cut shuttle vector pYES2 (Invitrogen, Carlsbad, USA), and the resulting vector pYES2-CalDes was transformed into E. coli XL1 blue. After another plasmid preparation with the aid of the Plasmid Maxi Kit (QIAGEN), pYES2-CalDes was transformed into S. cerevisiae INCSv1 0050/50669 23 (Invitrogen, Carlsbad, USA) with the aid of the polyethylene glycol method (Von Pein PhD thesis, Heinrich Heine- Universitdt DUsseldorf, 1992), where the expression of the CalDes cDNA was under the control of the GAL1 promoter.

In order to be able to express, in the second approach, not only CalDes, but also FAD2, in yeast, the encoding region of the FAD2 gene was first amplified via PCR (protocol see Primers G and H) from A. thaliana cDNA with the aid of Tfl polymerase (Biozym).

The following primers were used for this purpose: Primer K: 5' AAACTCGAGATGGGTGCAGGTGGAAGAATGCCGG 3' forward primer (XhoI cleavage site) Primer L: 5' AAAAAGCTTTCATAACTTATTGTTGTACCAGTACACACC 3' reverse primer (HindIII cleavage site) The resulting PCR product was subjected to a restriction digest with XhoI/HindIII and then cloned into the XhoI/HindIII-cut yeast expression vector pESC-Leu (Stratagene), where the FAD2 DNA was under the control of the GAL1 promoter.

The expression of CalDes in S. cerevisiae INCSv1 was carried out using a modification of the procedure of Avery et al. (Appl.

Environ. Microbiol., 62, 1996: 3960 3966) and Girke et al. (The Plant Journal, 5, 1998: 39 48). To prepare a starter culture, ml of YPAD medium were inoculated with a single colony and the culture was incubated for 48 hours at 30 0 C at 200 rpm. Then, the cell culture was washed in 1 x YPA medium without sugar and centrifuged. The pelleted cells were resuspended in 2 ml of minimal medium without supplements and without sugar. 100 ml of minimal medium (dropout powder, 2% raffinose, 1% Tergitol in 500-ml-Erlenmeyer flasks were inoculated with 1 ml of this cell suspension and the culture was grown at 30 0 C and 200 rpm. At an OD 600 of 0.5, 2% of galactose were added and (in the case of the first batch) 0.003% of linoleic acid stock solution in Tergitol NP40). The cells were grown on until the stationary phase had been reached. They were then washed in minimal medium without supplements and stored at -20 0

C.

Example 5: Lipid extraction and HPLC analysis of the fatty acids from transgenic yeast The yeast cells were suspended in 30 ml of HIP solution (0.1 mM 2.6-di-tert-butyl-4-methylphenol in hexane: isopropanol (3:2 acidified with 150 [1 of concentrated HC1 and homogenized in an Ultra-Turrax (1 min, 24,000 rpm). The samples were then 0050/50669 24 shaken for 10 minutes at 4 0 C and centrifuged for 10 minutes at 5,000 g and 4 0 C. The supernatant was transferred into a fresh container and made up to 47.5 ml with 0.38 M K 2

SO

4 The samples, in turn, were shaken for 10 minutes at 4°C and centrifuged (see above). The hexane phase was withdrawn and evaporated to dryness under a stream of N 2 The residue was dissolved in 20 il of chloroform. For the alkaline hydrolysis of fatty acid esters, 400 il of methanol and 80 Rl of 40% strength KOH solution were added and the sample was incubated for 20 minutes at 60 0

C

under argon. The sample was subsequently cooled to room temperature, acidified to pH 3.0 with 35 il of concentrated HC1 and separated by HPLC.

The free fatty acids were separated using an ET 250/4 Nucleosil 120-5 C18-column (Macherey Nagel). The mobile phase used was methanol:H 2 0:glacial acetic acid (85:15:0.1 The separation was carried out at a flow rate of 1 ml/min and 25 0 C, and the absorption was measured at 268 nm to detect the conjutrienes.

Fig. 3 shows the elution profiles of the lipid extracts from transformed yeast cells following alkaline hydrolysis (Fig. 3B, elution profile of S. cerevisiae INCSv1 transformed with A. thaliana FAD2 DNA, and C, elution profile of S. cerevisiae INCSv1 transformed with Calendula officinalis pYES2-CalDes), and the elution profile of a calendulic acid standard (Fig. 3A).

Calendulic acid has a retention time of 12 minutes with a strong absorption at 268 nm, which is typical for conjutrienes. The hydrolyzed lipid extracts of yeast cells which were transformed with the blank vector pYES2 and grown with 0.003% of linoleic acid show no fatty acids with a retention time of calendulic acid (not shown). Equally, the hydrolyzed lipid extracts of yeast cells which express the FAD2 gene contain no calendulic acid (Fig. 3B).

In contrast, the HPLC analysis of the extracts of pYES2-CalDes-transformed yeast cells grown with 0.003% of linoleic acid showed a signal with the retention time of calendulic acid (Fig. 3C), which also showed the same absorption spectrum as the standard with a maximum of 268 nm and secondary maxima of 258 and 282 nm (Fig. 4A, standard, and C, elution profile of S. cerevisiae INCSv1 transformed with Calendula officinalis pYES2-CalDes). It was thus demonstrated that the expression of calendulic acid desaturase in yeast results in the biosynthesis of calendulic acid. Calendulic acid from transformed yeast cells was only successfully detected after hydrolysis of the lipids. No calendulic acid was detected in the free fatty acids of these cells, that is to say that, in yeast, calendulic 0050/50669 acid is incorporated into lipids. Since yeast contains no triacylglycerides, it must be assumed that the detected calendulic acid had been bound in the phospholipids of the yeast.

In addition, the lipid extracts of transgenic yeast cells which simultaneously express FAD2 and CalDes also contain calendulic acid (not shown).

Example 6: Expression of calendulic acid desaturase in Arabidopsis thaliana and Linum usitatissimum The expression of Calendula officinalis calendulic acid desaturase in transgenic plants is advantageous for increasing the calendulic acid content in these plants. To this end, the CalDes cDNA was cloned into binary vectors and transferred into A. thaliana and L. usitatissimum via Agrobacterium-mediated DNA transfer. The expression of the CalDes cDNA was under the control of the constitutive CaMV 35 S [sic] promoter or the seed-specific USP promoter.

The expression vectors used were the vector pBinAR (H6fgen and Willmitzer, Plant Science, 66, 1990: 221 230) and the pBinAR derivative pBinAR-USP, in which the CaMV 35 S [sic] promoter had been exchanged for the V. faba USP promoter. For recloning, the CalDes cDNA had to be excised from the vector pGEM-T. To this end, the latter was first cut with NcoI and filled up with Klenow to provide blunt ends; the insert was subsequently excised with SalI and cloned into the SmaI/SalI-cut vectors pBinAR and pBinAR-USP.

The resulting plasmids pBinAR-CalDes and pBinAR-USP-CalDes were transformed into Agrobacterium tumefaciens (H6fgen and Willmitzer, Nucl. Acids Res., 16, 1988: 9877). A. thaliana was transformed by "floral dip" (Clough and Bent, Plant Journal, 16, 1998: 735 743), and L. usitatissimum by coculturing linen [sic] hypocotyl sections with transformed A. tumefaciens cells.

The expression of the CalDes gene in transgenic Arabidopsis and Linum plants was studied by Northern Blot analysis. Selected plants were studied for their calendulic acid content in the seed oil.

To achieve seed-specific expression of CalDes, it is also possible to use the napin promoter analogously to the USP promoter.

0050/50669 26 SEQUENCE LISTING <110> Department of plant biochemistry <120> Fatty acid desaturase gene from plants <130> Sequence_desaturase <140> 50669 <141> 1999-08-31 <160> 2 <170> PatentIn Vers. <210> 1 <211> 1285 <212> DNA <213> Calendula officinalis <220> <221> CDS <222> (42)..(1175) <400> 1 aaaagctcac ttctctgtga gggtaattat atatcaacaa c atg ggt gct ggt ggt 56 Met Gly Ala Gly Gly cgg atg tcg gat cca tct gag gga aaa aac ate ctt gaa cgt Arg Met Ser Asp Pro Ser Glu Gly Lys Asn Ile Leu Glu Arg gtg cca Val Pro gtc gat cca Val Asp Pro ccg Pro ttc acg tta age Phe Thr Leu Ser gat Asp 30 ctg aag aaa gcg Leu Lys Lys Ala att cct acc Ile Pro Thr gtt gtt cat Val Val His cat tgc His Cys gat ctc Asp Leu ttt Phe gag cga tct gtc Glu Arg Ser Val ate Ile 45 cgg tca tca tac Arg Ser Ser Tyr tat Tyr att gtt gcc tat Ile Val Ala Tyr gtc Val 60 ttc tac tac ctt Phe Tyr Tyr Leu gca Ala aac acg tat atc Asn Thr Tyr Ile 200 248 296 cct Pro ctt att cct aca Leu Ile Pro Thr cct Pro 75 ctg gct tac cta Leu Ala Tyr Leu gca Ala 80 tgg ccc gtt tac Trp Pro Val Tyr tgg Trp ttt tgt caa get agc ate etc acc ggc ctc tgg gtc ate ggt cac gaa Phe Cys Gin Ala Ser Ile Leu Thr Gly Leu Trp Val Ile Gly His Glu 344 0050/50669 tgt ggt cac Cys Gly His gga ttc gtg Gly Phe Val 120 cat His 105 gca ttt agc gac Ala Phe Ser Asp tac Tyr 110 cag ttg att gat Gin Leu Ile Asp gac att gtt Asp Ile Val 115 tct tgg aaa Ser Trp Lys 392 440 ctc cat tcg gct Leu His Ser Ala ctc Leu 125 ctc acc ccg tat Leu Thr Pro Tyr ttc Phe 130 tat agc Tyr Ser 135 cac agg aat cac His Arg Asn His cac His 140 cgt Arg gcc aac aca aat Ala Asn Thr Asn tca Ser 145 ctc gat aac gat Leu Asp Asn Asp gaa Giu 150 ctt Leu gtt tac att cct Val Tyr Ile Pro ctt aac aat cca Leu Asn Asn Pro 170 aaa Lys 155 aag tcg aag Lys Ser Lys gtc Val 160 aag att tat tcc Lys Ile Tyr Ser aaa Lys 165 488 536 584 ccc ggg cga gtg Pro Gly Arg Val ttc Phe 175 act ttg gtg ttt Thr Leu Val Phe cgg ttg Arg Leu 180 act tta gga Thr Leu Gly ttt Phe 185 ccg tta tac ctc Pro Leu Tyr Leu tta Leu 190 act aat atc tcg Thr Asn Ile Ser ggc aag aaa Gly Lys Lys 195 632 tac ggg Tyr Gly gat cgt Asp Arg 215 agg Arg 200 ttt gcc aac cac Phe Ala Asn His ttt Phe 205 gat ccc atg Asp Pro Met gaa cgc gtt caa Giu Arg Val Gin gtt Val 220 ttg cta tcc gat LeU Leu Ser Asp agt cca att ttc Ser Pro Ile Phe 210 ttc ggt ctt ctc Phe Gly Leu Leu 225 aaa ggg gca gct Lys Gly Ala Ala aac Asn gct Ala tgg Trp 245 680 728 776 824 gta Val 230 ttt tat gca atc Phe Tyr Ala Ile aag Lys 235 ctt ctt gta gca Leu Leu Val Ala gca Ala 240 ggt Giy gta atc aac atg Val Ile Asn Met tac Tyr 250 gca att cca gta Ala Ile Pro Val cta Leu 255 gta agc gtg Val Ser Val ttc ttc Phe Phe 260 gtt ttg atc Val Leu Ile gat tca acc Asp Ser Thr 280 ac a Thr 265 tat ttg cac cac Tyr Leu His His acc Thr 270 cat ctc tca ctc His Leu Ser Leu cct cat tat Pro His Tyr 275 aca atc gat Thr Ile Asp 872 920 gaa tgg aac tgg Giu Trp Asn Trp atc Ile 285 aaa ggc gcc tta Lys Gly Ala Leu tca Ser 290 agg gat ttc ggg ttc ctg aat cgg gtt ttc cac gac gtt aca cac act 96 968 0050/50669 Arg Asp 295 Phe Gly Phe Leu Asn 300 Arg Val Phe His Asp 305 Val Thr His Thr cac His 310 gtc ttg cat cat Val Leu His His ttg Leu 315 atc tca tac att Ile Ser Tyr Ile cca Pro 320 cat tat cat gca His Tyr His Ala aag Lys 325 gaa gca agg gat Glu Ala Arg Asp gca Ala 330 atc aag cca gtg Ile Lys Pro Val ttg Leu 335 ggc gag tac tat Gly Glu Tyr Tyr aaa atc Lys Ile 340 gaa tgc Glu Cys 1016 1064 1112 gac agg act Asp Arg Thr atc tac atc Ile Tyr Ile 360 cca Pro 345 att ttc aaa gca Ile Phe Lys Ala atg Met 350 tat aga gag gct Tyr Arg Glu Ala aag Lys 355 gag ccc gat gag Giu Pro Asp Giu gat Asp 365 agc gag cac aaa Ser Giu His Lys ggt Gly 370 gtg ttc tgg Val Phe Trp 1160 tac cac aag atg Tyr His Lys Met 375 taa tcaaaaaggt gtatgtcaat gcaattgtat gcttaattaa 1215 gttgttaaac tttctattcc gtgtaataaa ttatcattaa gagaaaaaaa aaaaaaaaaa 1275 aaaaaaaaaa 1285 <210> 2 <211> 377 <212> PRT <213> Calendula off icinalis <400> 2 Met Gly 1 Ala Gly Gly Arg Met 5 Ser Asp Pro Ser Glu Gly Lys 10 Asn Ile Leu Giu Arg Val1Pro Val Asp Pro Pro 25 Phe Thr Leu Ser Asp Leu Lys Arg Ser Ser Lys Ala Ile Pro Thr His Cys Phe Giu Arg Ser Val Ile Tyr Tyr Val Val His Asp Leu 55 Ile Vai Ala Tyr Val Phe Tyr Tyr Leu Ala Trp Asn Thr Tyr Ile Pro Val Tyr Trp Pro 70 Leu Ile Pro Thr Pro 75 Leu Ala Tyr Leu Ala Leu Trp Phe Cys Gin Ala Ser 90 Ile Leu Thr Gly 0050/50669 29 Val Ile Gly His Glu Cys Giy His His Ala Phe Ser Asp Tyr Gin Leu 100 I lE Tyx Ser 145 Lys Leu Ile Ser Phe 225 Lys Val1 Ser Leu Asp 305 His Giu Asp Phe 130 Leu Ile Vai Ser Pro 210 Gly Gly Ser Leu Ser 290 Vai Tyr Tyr Asr 115 Ser Asp Tyr Phe Gly 195 Ile Leu Ala Vai Pro 275 Thr Thr Hlis Tyr Ile *Trp Asn *Ser Arg 180 Lys Phe Leu Ala Phe 260 His Ile His Ala Lys 340 Val Lys Asp Lys 165 Leu Lys Asn Ala Trp 245 Phe Tyr Asp rhr Lys 325 Ile *Gly *Tyr Giu 150 Leu Thr Tyr Asp Val 230 Val1 Val Asp Arg His1 310 Glu2 Asp2 Phe Ser 135 Val Leu Leu Gly Arg 215 Phe Ile Leu Ser Asp 295 Val1 kla krg 105 Vai Leu 120 His Arg Tyr Ile Asn Asn Gly Phe 185 Arg Phe 200 Glu Arg Tyr Ala Asn Met Ile Thr 265 Thr Glu 280 Phe Gly Leu His His Asn Pro Prc 170 Pro Ala Val Ile Tyr 250 Tyr Trp Phe His Ala 330 Ile Ser His Lys 155 Pro Leu Asn Gin Lys 235 Ala Leu Asn Leu Leu 315 Ile Phe Ala His 140 Arg Giy Tyr His Val 220 Leu Ile His Trp Asn 300 Ile Lys Lys Leu 125 Ala Lys Arg Leu Phe 205 Leu Leu Pro His Ile 285 Arg Ser Pro Ala 110 Leu Asn Thr Thr Ser Lys Val Phe 175 Leu Thr 190 Asp Pro Leu Ser Val Ala Val Leu 255 Thr His 270 Lys Gly Val Phe Tyr Ile Val Leu 335 Met Tyr 350 Pro Asn Val 160 Thr Asn Met Asp Ala 240 Gly Leu Ala His Pro 320 Gly Arg Arg Thr Asp Pro 345 Giu Ala Lys Glu Cys Ile Tyr Ile Giu Pro Asp Glu Asp Ser Giu His 360 365 Lys Gly Val Phe Trp Tyr His Lys Met 370 375 Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

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Claims (19)

1. 2. An amino acid sequence encoded by a nucleic acid sequence as claimed in claim i.
2. 3. An amino acid sequence as claimed in claim 2, encoded by the sequence shown in SEQ ID NO: i.
3. 4. A nucleic acid construct comprising a nucleic acid sequence as claimed in claim i, where the nucleic acid sequence is linked to one or more regulatory signals.
4. 5. A vector comprising a nucleic acid sequence as claimed in •claim 1 or a nucleic acid construct as claimed in claim 4. oooo
5. 6. An organism excluding a human being, comprising at least one nucleic acid sequence as claimed in claim 1 or at least one Se• nucleic acid construct as claimed in claim 4.
6. 7. An organism as claimed in claim 6, which is selected from a plant, a microorganism and an animal.
7. 8. A transgenic plant comprising a functional or nonfunctional nucleic acid sequence as claimed in claim 1 or a functional or nonfunctional nucleic acid construct as claimed in o. claim 4. o• S S
8. 9. A process for the preparation of unsaturated fatty acids, which process comprises introducing at least one nucleic acid sequence as claimed in claim 1 or at least one nucleic acid construct as claimed in claim 4 into an oil-producing organism, growing this organism, isolating the oil contained in the organism and liberating the fatty acids contained in the oil. A process for the preparation of triglycerides with an increased content of unsaturated fatty acids, which comprises introducing at least one nucleic acid sequence as claimed in claim 1 or at least one nucleic acid construct as claimed in claim 4 into an oil-producing organism, growing this organism and isolating the oil contained in the organism.
9. 11. A process for the preparation of saturated fatty acids, which comprises introducing at least one nonfunctional nucleic acid sequence as claimed in claim 1 or at least one nonfunctional nucleic acid construct as claimed in claim 4 into an oil- producing organism, growing this organism, isolating the oil contained in the organism and liberating the fatty acids contained in the oil.
10. 12. A process for the preparation of triglycerides with an increased content of saturated fatty acids, which process comprises introducing at least one nonfunctional nucleic acid sequence as claimed in claim 1 or at least one nonfunctional nucleic acid construct as claimed in claim 4 into an oil- producing organism, growing this organism and isolating the oil contained in the organism. o 13. A process as claimed in claim 9 or 10, wherein the unsaturated fatty acids have an increased calendulic acid content.
11. 14. A method as claimed in any one of claims 9 to 12, wherein the organism is a plant or a microorganism. An unsaturated fatty acid prepared by a process as claimed in :o:claim 9. off.
12. 16. A triglyceride with an increased content of unsaturated fatty acids prepared by a process as claimed in claim oO S o ooS o•
13. 17. A saturated fatty acid prepared by a process as claimed in claim 11.
14. 18. A triglyceride with an increased content of saturated fatty acids prepared by a process as claimed in claim 12.
15. 19. The use of a nucleic acid sequence as claimed in claim 1 or of a nucleic acid construct as claimed in claim 4 for the generation of transgenic plants. The use of a nucleic acid sequence as claimed in claim 1 or of a fragment of said nucleic acid sequence having substantially the same properties thereof, for isolating a genomic sequence via homology screening.
16. 21. The use of unsaturated or saturated fatty acids as claimed in claim 15 or 17 or triglycerides with an increased content of unsaturated or saturated fatty acids as claimed in claim 16 or 18 for the preparation of foodstuffs, animal feed, cosmetics or pharmaceuticals.
17. 22. An enzyme which is encoded by a nucleic acid sequence as claimed in claim 1 and which converts a fatty acid of the structure I, CH2 COOR (I) which has two double bonds separated from each other by a methylene group, to give a triunsaturated fatty acid of the 00 structure II, 2 ooo R C H- 2 (COOR oeoo *n n the three double bonds of the fatty acid being conjugated and the substituents and variables in the compounds of the e: structures I and II having the following meanings: ooo* •go*o* go* R hydrogen, substituted or unsubstituted, unsaturated or saturated, branched or unbranched C 1 -Co-alkyl-, -CH2I 93 94 R R R 2 substituted or unsubstituted, unsaturated or saturated C,-C,-Alkyl- R 3 and R 4 independently of one another are hydrogen, substituted or unsubstituted, saturated or unsaturated, branched or unbranched C 1 -C,-alkylcarbonyl or phospho-, n 1 to 14
18. 23. An unsaturated fatty acid prepared by a process as claimed in claim 9 or claim 11.
19. 24. A triglyceride with an increased content of unsaturated fatty acids prepared by a process as claimed in claim 10 or claim 12. DATED this 25th day of September 2004 BASF AKTIENGESELLSCHAFT WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD •HAWTHORN VICTORIA 3122 AUSTRALIA go CJH/JMN g* oo