AU776417B2 - Plants expressing delta6-desaturase genes and oils from these plants containing pufas and method for producing unsaturated fatty acids - Google Patents
Plants expressing delta6-desaturase genes and oils from these plants containing pufas and method for producing unsaturated fatty acids Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0083—Miscellaneous (1.14.99)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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- C—CHEMISTRY; METALLURGY
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- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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Description
Plants expressing A6-desaturase genes, PUFAs-containing oils from these plants, and a process for the preparation of unsaturated fatty acids The present invention relates to an improved process for the preparation of unsaturated fatty acids and to a process for the preparation of triglycerides with an increased content of unsaturated fatty acids. The invention relates to the generation of a transgenic organism, namely of a transgenic plant or algae, with an increased content of fatty acids, oils or lipids with A6 double bonds owing to the expression of a moss A6-desaturase.
The invention furthermore relates to transgenic plants or algae containing a A6-desaturase gene, and to the use of the unsaturated fatty acids or triglycerides with an increased content of unsaturated fatty acids which have been prepared by the process.
Fatty acids and triglycerides have a multiplicity of uses in the S 20 food industry, in livestock nutrition, in cosmetics and in the pharmaceutical sector. They are suitable for a wide variety of uses depending on whether they are free saturated or unsaturated oooo fatty acids or triglycerides with an increased content of saturated or unsaturated fatty acids; thus, for example, polyunsaturated fatty acids are added to baby food 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 soybean, oilseed rape, sunflower and others, usually resulting in the form of their 30 triacyl glycerides. However, they can also be obtained from oeeo animal species such as fish. The free fatty acids are advantageously prepared by saponification.
Depending on the intended use, oils with saturated or unsaturated 35 fatty acids are preferred; thus, for example, lipids with S unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred in human nutrition because they have a beneficial effect on the blood cholesterol level and thus on the possibility of heart disease. A positive action on carcinogenesis is also attributed to the unsaturated fatty acids. Moreover, they are important starting materials for the synthesis of compounds which govern important biological processes within the organism.
They are therefore used in various dietetic foodstuffs or medicaments.
0093/00032 2 Owing to their beneficial properties, there has been no lack of attempts in the past to make available genes which are involved in the synthesis of fatty acids or triglycerides for the production of oils in various organisms with a modified content of unsaturated fatty acids. Thus, a A9-desaturase is described in WO 91/13972 and its US equivalent. WO 93/11245 claims a while WO 94/11516 claims a A12-desaturase.
A6-desaturases are described in Girke et al. (The Plant Journal, 1998: 39-48), Napier et al. (Biochem. 330, 1998: 611-614), Murata et al. (Biosynthesis of y-linolenic acid in cyanobacterium Spirulina patensis, pp. 22-32, In: y-linolenic acid, metabolism and its roles in nutrition and medicine, Huang, Y. and Milles, D.E. AOC Press, Champaign, Illinois), Sayanova et al. (Proc. Natl. Acad. Sci. USA, 94, 1997: 4211-4216), WO 98/46764, Cho et al. Biol. Chem., 274, 1999: 471-477), Aki et al. (Biochem. Biophys. Res. Commun., 255, 1999: 575-579), and Reddy et al. (Plant Mol. Biol., 27, 1993: 293-300).
Further 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. Further A6-desaturase are described in WO 93/06712, US 5,614,393, US5,614,393, WO 96/21022, WO 00/21557 and WO 99/27111. The biochemical characterization of the various desaturases is, however, inadequate as yet because the enzymes, being membrane-bound proteins, can be isolated and characterized only with great difficulty (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792). As a rule, membrane-bound desaturases are characterized by introducing them into a suitable organism which is subsequently tested for enzyme activity by analyzing the starting material and the product. The use for production in transgenic organisms described as in WO 98/46763 WO 98/46764, WO 98/46765 [sic]. The expression of various desaturases as in WO 99/64616 or WO 98/46776 and the formation of polyunsaturated fatty acids is also described and claimed here. As regards the expression efficacy of desaturases and their effect on the formation of polyunsaturated fatty acids, it must be noted that expression of an individual desaturase as described in the above prior art only led to, and leads to, low contents of unsaturated fatty acids, for example A-6-unsaturated [sic] fatty acids/lipids such as, for example, y-linoleic acid, being achieved.
There is thus still a great need for novel genes which are better suited and which encode enzymes which are involved in the biosynthesis of dnsaturated fatty acids and which allow Lhem- to be produced on an industrial scale. Furthermore, there is still a need for improved methods of obtaining the highest possible contents of polyunsaturated fatty acids.
There is disclosed herein a process for the preparation of unsaturated fatty acids using genes which encode, for example, desaturase enzymes and which are involved in the synthesis of polyunsaturated fatty acids in the seeds of an oil crop, thus increasing the content of polyunsaturated fatty acids.
In a first embodiment there is provided a process of preparing unsaturated fatty acids, which process comprises introducing, into a transgenic plant or algae, at least one isolated nucleic acid sequence encoding a polypeptide having A6-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having 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 SEQ ID oooo S: NO: i, oo• o. 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 have at least oo o homology at the amino acid level without substantially reducing the enzymatic action of the polypeptides, oooo ooo• S 30 and culturing the transgenic plant or algae, where the cultured •go• transgenic plant or algae contains at least 1 mol% of unsaturated go fatty acids based on the total fatty acid content in the *organism.
35 The organisms obtained by the processes according to the S invention contain, as a rule, unsaturated fatty acids in the form of bound fatty acids, i.e. the unsaturated fatty acids exist predominantly in the form of their mono-, di- or triglycerides, glycolipids, lipoproteins or phospholipids such as oils or lipids or else as fatty acids bound as esters or amides. Free fatty acids are also present in the organisms in the form of the free fatty acids or in the form of their salts. Advantageously, the free or bound unsaturated fatty acids have an increased content of fatty acids with A6 double bonds, such as, advantageously, ylinoleic acid, which is increased over that of the starting organisms. The organisms obtained by culturing in the process according to the invention, and the unsaturated fatty acids which they contain, can be used directly, for example for the production of pharmaceutical products, of agrochemicals, feeds or foodstuffs or else after isolation from the organisms. All steps of the purification of the unsaturated fatty acids can be used, that is to say that anything from crude extracts of the fatty acids up to fully purified fatty acids are suitable for preparing the abovementioned products. In an advantageous embodiment, the bound fatty acids can be liberated from the, for example, oils or lipids for example by hydrolysis with bases, such as, for example, with NaOH or KOH. These free fatty acids can be used directly in the mixture obtained or after further purification for producing pharmaceutical products, agrochemicals, feeds of foodstuffs. Also, the bound or free fatty acids can be used for transesterification or esterification, for example with other mono, di- or triglycerides or glycerol in order to increase the content of unsaturated fatty acids in these compounds, for example in the triglycerides.
20 The invention furthermore relates to a process for the preparation of triglycerides with an increased content of unsaturated fatty acids by incubating triglycerides with saturated or unsaturated or saturated and unsaturated fatty acids with at least one of the proteins encoded by the sequence 25 SEQ ID NO: 2. The processes are advantageously carried out in the presence of compounds which are capable of accepting or donating reduction equivalents. The fatty acids can subsequently be released from the triglycerides.
30 The abovementioned methods advantageously allow fatty acids of bound fatty acids such as triglycerides with an increased content of fatty acids with A6 double bonds to be synthesized.
Organisms which may be mentioned for the abovementioned processes 35 are, for example, plants such as Arabidopsis, barley, wheat, rye, oats, maize, soybean, rice, cotton, sugarbeet, tea, carrot, capsicum, canola, sunflower, flax, hemp, potato, triticale, tobacco, tomato, oilseed rape, coffee, tapioca, carcaba, arrowroot, tagetes, alfalfa, peanut, castor, coconut, oilpalm, safflower (Carthamus tinctorius), lettuce and the various tree, nut and grapevine species, or cacao bean, or algae. Preferred organisms are those which are naturally capable of synthesizing substantial amounts of oils, such as plants such as soybean, oilseed rape, coconut, oil palm, canola, safflower (Carthamus tinctorius), castor, calendula, linseed, borage, peanut, cacao bean or sunflower, with soybean, oilseed rape or sunflower being especially preferred.
Depending on the host organism, the organisms used in the processes are cultured or grown in the manner known to the skilled worker. They are usually cultured in a liquid medium which contains a carbon source, in most cases in the form of sugars, a nitrogen source, in most cases in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron salts, manganese salts and magnesium salts and, if appropriate, vitamins, at tenmperatures between 0°C and 100'C, preferably between 10 0 C and 60'C, either with passing in of oxygen or in the absence of oxygen, depending on the organism. It is possible in this context to maintain the pH of the medium at a fixed value, that is to say the pH is regulated during culturing or else the pH is not regulated and changes during culture. Culturing can be carried out batchwise, semi-batchwise or continuously. Nutrients can be introduced at the beginning of the fermentation or subsequently fed semicontinuously or continuously. A culture on solid media is S 20 also possible.
•eo After the transformation, plants are, as a rule, first :o :regenerated and then cultured or grown as customary. This can be .done in the greenhouse or in the open.
After cultivation, the lipids are obtained from the organisms in the customary manner. To this end, the organisms can first be disrupted after harvesting or else used directly. The lipids are o o advantageously extracted with suitable solvents such as apolar 30 solvents such as hexane or ethanol, isopropanol or mixtures such ooeo as hexane/isopropanol, phenol/chloroform/isoamyl alcohol at oo temperatures between 0°C to 80 0 C, preferably between 20 0 C to oo ~As a rule, the biomass is extracted with an excess of solvent, for example an excess of solvent to biomass of 1:4. The solvent 35 is subsequently removed, for example via distillation. Extraction S can also be effected by using supercritical CO 2 After extraction, the residual biomass can be removed for example by filtration.
The crude oil thus obtained can subsequently be purified further, for example by removing cloudiness by treating the oil with polar solvents such as acetone or chloroform, followed by filtration or centrifugation. A further purification by chromatographic methods, distillation or crystallization is also possible.
To obtain the free fatty acids from the triglycerides, they are saponified in the customary manner as described above.
The invention furthermore relates to unsaturated fatty acids and to triglycerides with an increased content of unsaturated fatty acids which have been prepared by the abovementioned methods, and to their use for the production of foodstuffs, feeds, cosmetics or pharmaceuticals. To this end, they are added in customary quantities to the foodstuffs, the feeds, the cosmetics or the pharmaceuticals.
In the process according to the expression, higher contents of unsaturated fatty acids such as y-linolenic acid were obtained by expressing a moss A6-desaturase in plants and algae, very especially preferably in oil crops such as oilseed rape, canola, linseed, soybean, sunflower, borage, castor, oilpalm, safflower (Carthamus tinctorius), coconut, peanut or cacao bean. Expression in field crops such as maize, wheat, rye, oats, triticale, rice, barley, alfalfa or bush plants (coffee, cacao, tea) is also 20 advantageous. Expression in the abovementioned organisms of a gene which encodes a moss A-6-desaturase and allows contents of unsaturated fatty acids of at least 1 mol%, preferably at least 3 mol%, especially preferably at least 4 mol%, very especially 2 preferably at least 5 mol%, to be achieved in the organisms.
SDerivative(s) are to be understood as meaning, for example, functional homologues of the enzymes encoded by SEQ ID NO: 1 or their enzymatic activity, that is to say enzymes which catalyze the same enzymatic reactions as those of SEQ ID NO: 1. These genes also make it possible advantageously to prepare unsaturated o. fatty acids with double bonds in position A6. Unsaturated fatty acids are to be understood hereinbelow as meaning doubly or polyunsaturated fatty acids which have double bonds. The double bonds can be conjugated or unconjugated. The sequence stated 35 in SEQ ID NO: 1 encodes an enzyme which has a A6-desaturase activity.
The enzyme A6-desaturase according to the invention advantageously introduces a cis double bond in position C 6 into fatty acid residues of glycerolipids (see SEQ ID NO: 1).
Moreover, the enzyme has a A6-desaturase activity which advantageously introduces exclusively a cis double bond in position C 6 into fatty acid residues of glycerolipids. The enzyme with the sequence stated in SEQ ID NO: 1 also has this activity, which is that of a monofunctional A6-desaturase.
The nucleic acids sequence(s) (the singular is intended to encompass the plural, and vice versa, for the application) or fragments thereof used in the process according to the invention can be used advantageously for isolating further genomic sequences via homology screening.
The derivatives mentioned can be isolated, for example, from other organisms namely eukaryotic organisms such as plants, such as, especially, mosses.
Derivatives or functional derivatives of the sequence stated in SEQ ID NO: 1 are furthermore to be understood as meaning, for example, allelic variants which have at least 50% homology at the deduced amino acid level, advantageously at least 70% homology, preferably at least 80% homology, especially preferably at least homology, and very especially preferably 90% homology. The homology was calculated over the entire amino acid region. The program PileUp, BESTFIT, GAP, TRANSLATE or BACKTRANSLATE constituent of the program package UWGCG, Wisconsin Package, 20 Version 10.0-UNIX, January 1999, Genetics Computer Group, Inc., Deverux et al., Nucleic. Acid Res., 12, 1984: 387-395) was used 0000 o(J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 1989: 151-153). The amino acid sequences deduced from the specified nucleic acids can be found in sequence SEQ ID NO: 2.
Homology is to be understood as meaning identity, i.e. the amino acid sequences are at least 50% identical. The sequences according to the invention have at least 65%, preferably at least 70%, especially preferably 75%, very especially preferably at o least 80%, homology at the nucleic acid level.
9000 S Allelic variants comprise, in particular, functional variants o. which can be obtained from the sequence shown in SEQ ID NO: 1 by deletion, insertion or substitution of nucleotides, while retaining the enzymatic activity of the deduced synthesized 35 proteins.
S Such DNA sequences can be isolated starting from the DNA sequence described in SEQ ID NO: 1 or parts of these sequences from other eukaryotes, such as, for example, those mentioned above, for example using customary hybridization methods or the PCR technique. These DNA sequences hybridize with the abovementioned sequences under standard conditions. For hybridization, it is advantageous to use short oligonucleotides, for example of conserved regions, which can be determined in a manner known to the skilled worker by comparisons with other desaturase genes. It is advantageous to use the histidine box sequences. However, it is also possible to use longer fragments of the nucleic acids according to the invention or the complete sequences for the hybridization. These standard conditions vary depending on the nucleic acid used: oligonucleotide, longer fragment c.r complete sequence, or depending on which type of nucleic acid, DNA or RNA, is used for the hybridization. Thus, for example, the melting temperatures for DNA:DNA hybrids are about 10°C lower than those for DNA:RNA hybrids of the same length.
Standard conditions mean, for example, depending on the nucleic acid, temperatures between 42 and 58°C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X 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°C in 5 x SSC, 50% formamide. The hybridization conditions for DNA:DNA hybrids are advantageously 0.1 x SSC and temperatures between about to 45°C, preferably between about 30°C to 45°C. The hybridization conditions for DNA:RNA hybrids are advantageously 0.1 x SSC and temperatures between about 30°C to 55°C, preferably between about 20 45°C to 55°C. These temperatures stated for the hybridization are 6 melting temperatures calculated by way of example for a nucleic acid with a length of about 100 nucleotides and a G C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant textbooks of 25 genetics such as, for example, Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated by the formulae known to the skilled worker, for example depending on the length of the nucleic acids, the nature of the hybrids or the G C content. Further information on 30 hybridization can be found by the skilled worker 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, SIRL Press at Oxford University Press, Oxford; Brown 1991, 35 Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.
Derivatives are also to be understood as meaning homologues of the sequence SEQ ID No: 1, for example eukaryotic homologues, truncated sequences, single-stranded DNA of the coding and noncoding DNA sequence or RNA of the coding and noncoding DNA 0093/00032 9 sequence.
Homologues of the sequence SEQ ID NO: 1 are furthermore to be understood as meaning derivatives such as, for example, promoter variants. These variants can be modified by one or more nucleotide exchanges, by insertion(s) and/or deletion(s), but without adversely affecting the functionality or efficacy of the promoters. Moreover, the promoters may have their efficacy increased by modification of their sequence, or be completely replaced by more effective promoters, even from heterologous organisms.
Derivatives are also advantageously understood as meaning variants whose nucleotide sequence in the region from -1 to -2000 in front of the start codon has been modified so that gene expression and/or protein expression is altered, preferably increased. Moreover, derivatives are also understood as meaning variants which have been modified at the 3' end.
The nucleic acid sequences encoding a A6-desaturase can be synthesized or obtained from nature or contain a mixture of synthetic or natural DNA constituents, or else be composed of various heterologous A6-desaturase gene sections from various organisms. In general, synthetic nucleotide sequences are produced using codons which are preferred by the host organisms in question, for example plants. As a rule, this leads to optimal expression of the heterologous genes. These codons which are preferred by plants may be determined from codons with the greatest protein frequency which are expressed in most plant species of interest. An example for Corynebacterium glutamicum is given in: Wada et al. (1992) Nucleic Acids Res. 20:2111-2118).
Experiments of this type can be carried out by standard methods and are known to those skilled in the art.
Functionally equivalent sequences encoding the A6-desaturase gene are those derivatives of the sequence according to the invention which still have the desired functions, i.e. the enzymatic activity of the proteins, despite deviating nucleotide sequence.
Functional equivalents thus encompass naturally occurring variants of the sequences described herein and artificial nucleotide sequences, for example obtained by chemical synthesis and adapted to the codon usage of a plant.
In addition, artificial DNA sequences are suitable as long as they confer, as described above, the desired property, for example the increase in the content of A6-double bonds in fatty acids, oils or lipids in the plant by overexpression of the-, 0093/00032 A6-desaturase gene in crop plants. Such artificial DNA sequences can be established for example, by backtranslation of proteins constructed by means of molecular modeling and having A6-desaturase activity, or by in-vitro selection. Techniques which are possible for the in-vitro evolution of DNA for modifying or improving the DNA sequences are described in Patten, P.A. et al., Current Opinion in Biotechnology 8, 724-733( 1997) or in Moore, J.C. et al., Journal of Molecular Biology 272, 336-347 (1997). Coding DNA sequences which have been obtained by backtranslating of a polypeptide sequence in accordance with the codon usage specific for the host plant are particularly suitable. This specific codon usage can be determined easily by a skilled worker familiar with methods of plant genetics by computer analysis of other, known genes of the plant to be transformed.
Further suitable equivalent nucleic acid sequences which must be mentioned are sequences which encode fusion proteins, where a A6-desaturase polypeptide or a functionally equivalent portion thereof is part of the fusion protein. The second portion of the fusion protein can be, for example, another polypeptide with enzymatic activity or an antigenic polypeptide sequence with the aid of which it is possible to detect A6-desaturase expression (for example myc-tag or his-tag). However, this preferably takes the form of a regulatory protein sequence such as, for example, an ER signal sequence, which guides the A6-desaturase protein to the desired site of action.
It is advantageously possible to combine the A6-desaturase gene in the process according to the invention with further genes of fatty acid biosynthesis. Examples of such genes are the acetyl transferases, further desaturases or elongases of unsaturated or saturated fatty acids as described in WO 00/12720. Advantageous for the in-vivo and, specifically, in-vitro synthesis is the combination with, for example, NADH cytochrome B5 reductases, which are able to accept or dominate reduction equivalents.
The proteins used in the process according to the invention are to be understood as meaning proteins which comprise an amino acid sequence shown in SEQ ID NO: 2 or a sequence which can be obtained therefrom by substitution, inversion, insertion or deletion of one or more amino acid residues, with the enzymatic activity of the protein shown in SEQ ID NO: 2 being retained or not substantially reduced. Not substantially reduced 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. It is moreover possible, for "0093Y00032 11 example, to replace particular amino acids by those with similar physico-chemical properties (bulk, basicity, hydrophobicity and the like). For example, arginine residues are replaced by lysine residues, valine residues by isoleucine residues or aspartic acid residues by glutamic acid residues. However, it is also possible for one or more amino acids to be transposed in their sequence, added or deleted, or several of these measures can be combined with each other.
Derivatives are also to be understood as functional equivalents which comprise, in particular, also natural or artificial mutations of an originally isolated sequence encoding a A6-desaturase and which additionally show the required function, that is to say the enzymatic activity is not substantially reduced. Mutations encompass substitutions, additions, deletions, transpositions or insertions of one or more nucleotide residues.
Thus, for example, the present invention also extends to those nucleotide sequences which are obtained by modification of the A6-desaturase nucleotide sequence. The aim of such a modification may be, for example, to localize further the coding sequence contained therein or, for example, also to insert further restriction enzyme cleavage sites.
Functional equivalents are also those variants whose function is, compared with the initial gene or gene fragment, attenuated not substantially reduced) or enhanced enzyme activity is greater than the activity of the initial enzyme, that is to say the activity is over 100%, preferably over 110%, particularly preferably over 130%).
The nucleic acid sequences mentioned above which can be used in the process according to the invention are advantageously inserted into an expression cassette in order to introduce them into a host organism. However, the nucleic acid sequences can also be introduced directly into the host organism. The nucleic acid sequence may advantageously be, for example, a DNA or cDNA sequence.
Coding sequences which are suitable for insertion into an expression cassette are, for example, those which encode a A6-desaturase with the above-described sequences and which impart, to the host, the ability of overproducing fatty acids, oils or lipids with double bonds in position A6. These sequences can be of homologous or heterologous origin.
An expression cassette nucleic acid construct or fragment) is to be-understood as meaning the sequence stated in, 0093'/00032 12 SEQ ID NO: 1 which is the result of the genetic code and/or its functional or nonfunctional derivatives which have advantageously been linked functionally to one or more regulatory signals to increase gene expression and which control expression of the coding sequence in the host cell. These regulatory sequences are intended to make specific expression of the genes and protein expression possible. This may mean, for example, depending on the host organism, that the gene is expressed and/or overexpressed only after induction, or that it is expressed and/or overexpressed immediately. For example, these regulatory sequences are sequences to which inducers or repressors bind and thus regulate expression of the nucleic acid. In addition to these novel regulatory sequences or in place of these sequences, it is possible for the natural regulation of these sequences still to be present in front of the actual structural genes and, where appropriate, to have been genetically modified so that natural regulation has been switched off and expression of the genes has been increased. However, the gene construct may also have a simple structure, that is to say no additional regulatory signals have been inserted in front of the nucleic acid sequence or its derivatives and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence has been mutated so that regulation no longer takes place and/or gene expression is increased. These modified promoters may also be placed alone in the form of subsequences promoter with parts of the nucleic acid sequences according to the invention) in front of the natural gene to increase the activity. In addition, the gene construct may advantageously comprise one or more enhancer sequences functionally linked to the promoter, which makes increased expression of the nucleic acid sequence possible. It is also possible to insert additional advantageous sequences at the 3' end of the DNA sequences, such as further regulatory elements or terminators. The A6-desaturase gene may be present in one or more copies in the expression cassette gene construct). Any genes which are coexpressed and which are advantageously involved in fatty acid biosynthesis may also be present in the expression cassette in one or more copies.
The regulatory sequences or factors may, as described above, preferably have a beneficial effect on the gene expression of the genes introduced, thus increasing it. Thus, enhancement of the regulatory elements can advantageously take place at the transcriptional level by using strong transcription signals such as promoters and/or enhancers. However, it is also possible to enhance translation by, for example, improving the stability of mRNA.
0093/00032 13 Suitable promoters in the expression cassette are, in principle, all promoters which are capable of controlling the expression of foreign genes in organisms, advantageously in plants of fungi. It is preferable to use in particular a plant promoter or promoters derived from, for example, a plant virus. Examples of advantageous regulatory sequences for the process according to the invention are present, for example, in promoters such as the cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, X-PR or in the X-PL promoter which are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are present, 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], RUBISCO SSU, OCS, B33, nos Nopaline Synthase Promoter) or in ubiquitin promoter. The expression cassette can also comprise a chemically inducible promoter by which expression of the exogenous A6-desaturase gene in the organisms, advantageously in the plants, can be controlled at a particular time. Examples of such advantageous plant promoters are the PRP1 promoter [Ward et al., Plant. Mol. Biol. 22 (1993), 361-366], a benzenesulfonamide-inducible promoter (EP 388186), a tetracycline-inducible promoter (Gatz et al., (1992) Plant J. 2,397-404), a salicylic acid-inducible promoter (WO 95/19443), an abscisic acid-inducible promoter (EP335528) and an ethanol- or cyclohexanone-inducible promoter (WO 93/21334). Further 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 amidotransferase promoter (see also Genbank Accession Number U87999) or a node-specific promoter in EP 249676 as can be advantageously used [sic]. Particularly advantageous plant promoters are those which ensure expression in tissues or plant parts/organs in which fatty acid biosynthesis or its precursors take place, such as, for example, in the endosperm or the developing embryo. Particular mention should be made of advantageous promoters which ensure seed-specific expression, such as, for example, the USP promoter or derivatives thereof, the LEB4 promoter, the phaseolin promoter or the napin promoter.
The USP promoter which has been stated in accordance with the invention and which is particularly advantageous, or its derivatives, mediate very early gene expression during seed development (Baeumlein et al., Mol Gen Genet, 1991, 225 459-67). Other advantageous seed-specific promoters which can be used for monocotyledonous and dicotyledonous plants are the promoters suitable for dicots such as, for example, the oilseed rape napin gene piomoter (US5,608,152), the Arabidopsib oleosin 009/00032 14 promoter (W098/45461), the Phaseolus vulgaris phaseolin promoter (US5,504,200), the Brassica Bce4 promoter (W091/13980) or the legume B4 promoter (LeB4, Baeumlein et al., Plant 2, 2, 1992: 233 239) or promoters which are suitable for monocots, such as the barley lpt2- or Iptl-gene promoters (W095/15389 and W095/23230) or the promoters of the barley hordein gene, of the rice glutelin gene, of the rice oryzin gene, of the rice prolamin gene, of the wheat gliadin gene, of the wheat glutelin gene, of the maize zein gene, of the oat glutelin gene, of the sorghum kasirin gene or of the rye secalin gene, which are described in W099/16890.
Further particularly preferred promoters are those which ensure expression in tissues or plant parts in which, for example, the biosynthesis of fatty acids, oils and lipids and their precursors takes place. Particular mention should be made of promoters which ensure seed-specific expression. Mention should be made of the oilseed rape napin gene promoter (US 5,608,152), the Vicia faba USP promoter (USP=unknown seed protein, Baeumlein et al., Mol Gen Genet, 1991, 225 459-67), of the Arabidopsis oleosin gene (W098/45461), of the phaseolin promoter (US 5,504,200) or of the legumin B4 gene promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 233-9). Mention should furthermore be made of promoters such as that of the barley lpt2 or Iptl gene (W095/15389 and W095/23230), which ensure seed-specific expression in monocots.
The expression cassette gene construct, nucleic acid construct) may, as described above, comprise other genes which are to be introduced into the organisms. These genes may be regulated separately or be in the same regulatory region as the A6-desaturase gene. These genes are advantageously further biosynthesis genes, advantageously of fatty acid biosynthesis, which allow increased synthesis. Examples which may be mentioned are the genes for A15-, A12-, A9-, A5- and A4-desaturase, the various hydroxylases, the acyl ACP thioesterases, P-ketoacyl synthases or f-ketoacyl reductases. It is advantageous to use the desaturase genes in the nucleic acid construct.
In principle, it is possible for all natural promoters with their regulatory sequences like those mentioned above to be used for the expression cassette according to the invention and the process according to the invention, as described below. It is also possible and advantageous to use synthetic promoters.
It is possible to manipulate various DNA fragments in order to -obtain a nucleotide sequence which is expediently'read in the 0093/00032 correct direction and which is equipped with a correct reading frame. To link the DNA fragments nucleic acids according to the invention) to each other adapters or linkers may be attached to the fragments.
Expediently, the promoter and terminator regions may be provided, in the direction of transcription, with a linker or polylinker comprising one or more restriction sites for 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. The size of the linker within the regulatory region is generally less than 100 bp, frequently less than 60 bp, but at least 5 bp. The promoter can be either native, or homologous, or else foreign, or heterologous, in relation to the host organism, for example the host plant. The expression cassette comprises in the 5'-3'-direction of transcription the promoter, a DNA sequence encoding a A6-desaturase gene used in the process according to the invention, and a region for transcriptional termination.
Various termination regions can be exchanged for each other as desired.
It is furthermore possible to employ manipulations which provide suitable restriction cleavage sites or which eliminate excess DNA or restriction cleavage sites. Where insertions, deletions or substitutions such as, for example, transitions and transversions, are suitable, in vitro mutagenesis, primer repair, restriction or ligation may be used. In suitable manipulations such as, for example, restriction, chewing back or filling in overhangs for blunt ends, complementary ends of the fragments may be provided for ligation.
Attachment of the specific ER retention signals SEKDEL (Schouten, A. et al., Plant Mol. Biol. 30 (1996), 781-792), may, inter alia, be of importance for advantageous high-level expression, thus tripling to quadrupling the average level of expression. It is also possible to employ other retention signals which occur naturally with plant and animal proteins localized in the ER for constructing the cassette.
Preferred polyadenylation signals are plant polyadenylation signals, preferably those which correspond essentially to T-DNA-polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of the T-DNA (octopin synthase) of the Ti-plasmids pTiACH5 (Gielen et al., EMBO J.3 (1984), 835 et seq.) or corresponding functional equivalents.
0093/00032 16 An expression cassette is generated by fusing a suitable promoter to a suitable A6-desaturase DNA sequence and to a polyadenylation signal by conventional recombination and cloning techniques as described, for example, in T. Maniatis, E.F. Fritsch and J.
Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
The DNA sequence encoding a Phsycomitrella [sic] patens A6-desaturase comprises all sequence characteristics which are necessary to achieve correct localization for the site of fatty acid, lipid or oil biosynthesis. No further targeting sequences are therefore necessary per se. However, such localization may be desirable and advantageous and can therefore be modified or enhanced artificially, so that such fusion constructs are also a preferred advantageous embodiment of the invention.
Particularly preferred sequences are those which ensure targeting into plastids.. Under certain circumstances, targeting into other compartments (see review in: Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423) for example into the vacuole, into the mitochondrion, into the endoplasmic reticulum peroxisomes, lipid bodies or, owing to the absence of suitable operative sequences, remaining in the compartment of formation, namely the cytosol, may also be desirable.
The nucleic acid sequences encoding A6-desaturase genes are advantageously cloned together with at least one reporter gene into an expression cassette which is introduced into the organism via a vector or directly into the genome. This reporter gene should make easy detection possible by a growth, fluorescence, chemo- or bioluminescence or resistance assay or by a photometric measurement. Examples of reporter genes are genes for resistance to antibiotics or herbicides, hydrolase genes, fluorescence protein genes, bioluminescence genes, sugar 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-desoxyglucose-6-phosphate phosphatase gene, the P-glucuronidase gene, P-lactamase gene, the neomycin phosphotransferase gene, the hygromycin phosphotransferase gene or the BASTA gluphosinate [sic] resistance) gene. These genes make it possible easily to measure and quantify the transcriptional activity and thus gene expression. It is thus possible to identify sites in the genome which show differences 009/00032 17 in productivity.
In a preferred embodiment, an expression cassette comprises upstream, i.e. at the 5' end of the coding sequence, a promoter and downstream i.e. at the 3' end, a polyadenylation signal, if appropriate, further regulatory elements which are operatively linked to the interposed coding sequence for the A6-desaturase DNA sequence. Operative linkage is to be understood as meaning the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, other regulatory elements in such a manner that each of the regulatory elements can carry out its function as intended in the expression of the coding sequence.
The sequences preferred for operative linkage are targeting sequences for ensuring subcellular localization in plastids.
However, targeting sequences to ensure subcellular localization in the mitochondrion, in the endoplasmatic reticulum in the nucleus, in oleoplasts or other compartments may also be employed if required, as well as translation enhancers such as the tobacco mosaic virus 5' leader sequence (Gallie et al., Nucl. Acids Res.
15 (1987), 8693-8711).
An expression cassette can comprise, for example, a constitutive promoter (preferably the USP or napin promoter), the gene to be expressed and the ER retention signal. The ER retention signal which is preferably used is the amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine).
For expression in a prokaryotic or eukaryotic host organism, for example a microorganism such as a fungus or plant, the expression cassette is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA which allows optimal expression of the genes in the host organism. Examples of suitable plasmids are in E. coli pLG338, pACYC184, pBR series such as, for example, pBR322, pUC series such as pUC18 or pUC19, M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 1 3-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, other advantageous fungal vectors being described by Romanos, M.A. et al., [(1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488] and by van den Hondel, C.A.M.J.J. et al. [(1991) "Heterologous gene expression in filamentous fungi] and in More Gene Manipulations in Fungi Bennet L.L.
Lasure, eds., pp. 396-428: Academic Press: San Diego] and in "Gene transfer systems and vector development for filamentous fungi" [van den Hondel, C.A.M.J.J. Punt, P.J. (1991) in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds.,- 0093/00032 18 pp. 1-28, Cambridge University Press: Cambridge]. Advantageous yeast vectors are, for example 2M, pAG-1, YEp6, YEpl3 or pEMBLYe23. Examples of algal or plant promoters are pLGV23, pGHlac+, pBIN19, pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer, 1988). The abovementioned vectors or derivatives of the abovementioned vectors constitute a small selection of plasmids which are possible. Further 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), Chap. 6/7, pp.
71 119 Advantageous vectors are shuttle vectors or binary vectors, which replicate in E. coli and Agrobacterium.
Apart from plasmids, vectors also mean all other vectors 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 cyclic DNA. These vectors are capable of autonomous replication or chromosomal replication in the host organism; chromosomal replication is preferred.
In a further embodiment of the vector, the expression cassette according to the invention can also advantageously be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism by heterologous or homologous recombination. This linear DNA may consist of a linearized plasmid or else only of the expression cassette as vector or the nucleic acid sequences according to the invention.
In a further advantageous embodiment, the nucleic acid sequence according to the invention can also be introduced alone into an organism.
If, in addition to the nucleic acid sequence according to the invention, further genes are to be introduced into the organism, it is possible to introduce them all together with a reporter gene in a single vector or each individual gene with a reporter gene in one vector in each case, or several genes together in various vectors, into the organism, in which case the various vectors can be introduced simultaneously or successively.
The vector advantageously comprises at least one copy of the nucleic acid sequences encoding a A6-desaturase, and/or of the expression cassette.
o09J/032 19 By way of example, the plant expression cassette can be incorporated into the transformation vector pRT Toepfer et al., 1993, Methods Enzymol., 217: 66-78; Toepfer et al. 1987, Nucl. Acids. Res. 15: 5890 et seq.).
As an alternative, a recombinant vector expression vector) can also be transcribed and translated in vitro, for example by using the T7 promoter and T7 RNA polymerase.
Expression vectors used in prokaryotes frequently make use of inducible systems with and without fusion proteins or fusion oligopeptides, it being possible for these fusions to take place both at the N terminus and at the C terminus or other domains of a protein which can be used. As a rule, such fusion vectors are intended to: increase the RNA expression rate, ii.) increase the protein synthesis rate which can be achieved, iii.) increase the solubility of a protein, or iv.) simplify purification by a binding sequence which can be used for affinity chromatography.
Proteolytic cleavage sites are frequently also introduced by fusion proteins, enabling elimination of part of the fusion protein also of the purification [sic]. Such recognition sequences for proteases recognize are [sic], for example, factor Xa, thrombin and enterokinase.
Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and (Pharmacia, Piscataway, NJ), which comprises glutathione S transferase (GST), maltose binding protein, or protein A.
Further examples of E. coli expression vectors are pTrc [Amann et al., (1988) Gene 69:301-315] and pET vectors [Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Stratagene, Amsterdam, The Netherlands].
Further advantageous vectors for use in yeasts are pYepSecl (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES derivatives (Invitrogen Corporation, San Diego, CA). Vectors for use in filamentous fungi are described in: van den Hondel, C.A.M.J.J. Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., eds., pp. 1-28, Cambridge University Press: Cambridge.
oo9/00o32 As an alternative, insect cell expression vectors may also be used advantageously, for example for the expression in Sf 9 cells. Examples of these are the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and of the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
Moreover, plant cells or algal cells may advantageously be used for gene expression. Examples of plant expression vectors are found in Becker, et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197 or in Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid. Res.
12: 8711-8721.
Moreover, the nucleic acid sequences encoding A6-desaturase may also be expressed in mammallian cells. Examples of suitable expression vectors are pCDM8 and pMT2PC, mentioned in: Seed, B.
(1987) Nature 329:840 or Kaufman et al. (1987) EMBO J. 6: 187-195). Promoters preferably to be used in such cases are of viral origin, such as, for example, promoters of polyoma virus, adenovirus 2, cytomegalovirus or simian virus 40. Further prokaryotic and eukaryotic expression systems are mentioned in Chapters 16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
The introduction of the nucleic acids according to the invention, of the expression cassette or of the vector into organisms, for example into plants, can in principle take place by all methods known to the skilled worker.
The skilled worker can find suitable methods for microorganisms 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.1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Habor Laboratory Press or 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. Use is made here of the above-described methods for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene cannon, the particle bombardment method, electroporation, incubation of dry embryos in DNA-containing solution, microinjection and agrobacterium-mediated gene transfer. The methods mentioned are described, for example, by 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 Potrykus, Annu. Rev.
Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The construct to be expressed advantageously clones into a vector suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
Agrobacteria transformed with such a vector can then be used in the known manner for transforming plants, in particular crop plants such as, for example tobacco plants, for example by bathing scarified leaves or leaf sections in an agrobacterial solution and subsequently growing them in suitable media. The transformation of plants with Agrobacterium tumefaciens is described, for example, by Hbfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known, inter alia, from F.F. White, 20 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.
Agrobacteria transformed with an expression vector as described 25 above can also be used in a known manner for transforming plants such as test plants such as Arabidopsis or crop plants such as cereals, maize, oats, rye, barley, wheat, soybean, rice, cotton, sugarbeet, canola, triticale, sunflower, flax, hemp, potato, tobacco, tomato, coffee, cacao, tea, carrot, capsicum, oilseed 30 rape, tapioca, carcaba, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and grapevine species, in particular oilcontaining crop plants such as soybean, peanut, castor, borrage, linseed, sunflower, canola, cotton, flax, oilseed rape, coconut, oilpalm, safflower (Carthamus tinctorius) or cacao bean, for 35 example by bathing scarified leaves or leaf sections in an agrobacterial solution and subsequently growing them in suitable media.
The genetically modified 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 Hofgen and Willmitzer.
Organisms or host organisms for the nucleic acids used in processes according to the invention, the expression cassette used or the vector used are, in principle and advantageously, all organisms which are capable of synthesizing fatty acids, specifically unsaturated fatty acids, or which are suitable for the expression of recombinant genes. Examples which nay be mentioned are plants such as Arabidopsis, Asteraceae such as calendula, or crop plants such as soybean, peanut, castor, sunflower, maize, cotton, flax, oilseed rape, coconut, oilpalm, safflower (Carthamus tinctorius) or cacao bean, and algae.
Preferred organisms are those which are capable of naturally synthesizing oils in substantial amounts, such as plants such as soybean, oilseed rape, coconut, oilpalm, safflower, castor, calendula, peanut, cacao bean or sunflower, with soybean, oilseed rape, sunflower or castor.
Host cells which can be used are also mentioned in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Expression strands which can be used, for example those which have a lower protease activity, are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic 20 Press, San Diego, California (1990) 119-128.
SDepending on the choice of the promoter, expression of the A6desaturase gene may take place specifically in the leaves, in the seeds, the tubers or other parts of the plant. The present 25 invention furthermore relates to such transgenic plants which overproduce fatty acids, oils or lipids, and to their propagation material and their plant cells, tissue or plant parts. A preferred subject according to the invention is transgenic plants of, for example, crop plants such as maize, oats, rye, wheat, 30 barley, rice, soybean, sugarbeet, canola, triticale, sunflower, flax, hemp, tobacco, tomato, coffee, cacao, tea, carrot, capsicum, oilseed rape, tapioca, carcaba, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and grapevine species, potatoes, in particular oil-containing crop plants such as 35 soybean, peanut, castor, borrage, linseed, sunflower, canola, cotton, flax, oilseed rape, coconut, oilpalm, safflower (Carthamus tinctorius) or cacao bean, laboratory plants such as Arabidopsis, or other plants such as mosses or algae comprising a functional nucleic acid sequence according to the invention or a functional expression cassette. Functional in this context means that an enzymatically active enzyme is formed.
The expression cassette or the nucleic acid sequences according to the invention comprising a A6-desaturase gene sequence can additionally also be used for the transformation of the organisms which have been mentioned above by way of example, such as bacteria, cyanobacteria, filamentous fungi, ciliates, animals or algae, with the aim of increasing the content in fatty acids, oils or lipids of A6-double bonds. Preferred transgenic organisms are bacteria, cyanobacteria, filamentous fungi or algae.
Transgenic organisms are to be understood as meaning organisms which comprise a foreign nucleic acid derived from another organism which encodes a A6-desaturase used in the process according to the invention. Transgenic organisms are also to be understood as meaning organisms which comprise a nucleic acid which is derived from the same organism and encodes a A6desaturase, this nucleic acid being present as an additional gene copy or not being present in the natural nucleic acid environment of the A6-desaturase gene. Transgenic organisms are also organisms in which the natural and/or 5'-region of the A6desaturase gene has been modified over the initial organisms by targeted, recombinant modifications. Preferred transgenic organisms are those into which a foreign DNA has been introduced.
Especially preferred are transgenic plants into which a foreign DNA has been introduced. Transgenic plants are to be understood 20 as meaning individual plant cells and their cultures, such as, for example, callus cultures on solid media or in liquid culture, the plant parts and intact plants.
*9 The invention furthermore relates to transgenic organisms 25 selected from transgenic plants and algae, comprising at least one isolated nucleic acid sequence encoding a polypeptide with A6-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID 30 NO: 1, b) nucleic acid sequences which, as a result of the degeneracy of the genetic code, are derived from [lacuna] shown in SEQ ID NO: 1, 0093/00032 24 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 have at least 50% homology at the amino acid level without substantially reducing the enzymatic action of the polypeptides.
Increasing the content of fatty acids, oils or lipids with A6-double bonds means for the purposes of the present invention for example the artificially acquired ability of an increased biosynthesis performance by functionally overexpressing the A6-desaturase gene in the organisms according to the invention, advantageously in the transgenic plants according to the invention, in relation to the nonrecombinant initial plants, at least for the duration of at least one plant generation.
The biosynthesis site of fatty acids, oils or lipids, for example, is generally the seed or cell layers of the seed, so that seed-specific expression of the A6-desaturase gene is meaningful. However, it is obvious that the biosynthesis of fatty acids, oils or lipids need not be restricted to the seed tissue, but may also take place in a tissue-specific manner in all remaining parts of the plant, for example in epidermis cells or in the tubers.
In addition, constitutive expression of the exogenous A6-desaturase gene is advantageous. However, inducible expression may also be desirable.
The efficacy of expression of the A6-desaturase gene can be determined for example in vitro by shoot meristem propagation. In addition, an expression of the A6-desaturase gene whose type and level has been modified, and its effect on fatty acid, oil or lipid biosynthetic activity can be tested in glasshouse experiments on test plants.
The invention relates to transgenic plants as described above, transformed with a nucleic acid sequence encoding a A6-desaturase, a vector or an expression cassette comprising a A6-desaturase gene sequence or DNA sequences hydribizing herewith, and to transgenic cells, tissue, parts and propagation material of such plants. Especially preferred in this context are transgenic crop plants as described above.
Plants for the purposes of the invention are monocots and dicots or algae.
The invention furthermore relates to: the use of a A6-desaturase DNA gene sequence with the sequence stated in SEQ ID NO:1 or DNA sequences hybridizing herewith for the generation of fungi, bacteria, animals or plants, preferably plants, with an increased content of fatty acids, oils or lipids with A6-double bonds by expressing this A6desaturase DNA sequence in plants.
the use of the proteins with the sequences SEQ ID NO: 2 for the preparation of unsaturated fatty acids in plants, fungi, bateria or animals, preferably plants.
The invention is illustrated in greater detail by the examples which follow: Examples Example 1: General cloning and culture methods: The cloning methods such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA-fragments, transformation of Escherichia coli cells, cultivation of organisms, and the sequence analysis of recombinant DNA, were carried out as described by Sambrook et al.
(1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
The protonema of Physcomitrella patens P. patens) was cultured in liquid medium as described by Reski et al. (Mol. Gen. Genet., 30 244, 1994: 352-359).
eo. Example 2: Recombinant DNA sequence analysis: Recombinant DNA molecules were sequenced using an ABI laser 35 fluorescence DNA sequencer by the method of Sanger (Sanger et al.
(1977) Proc. Natl. Acad. Sci. USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
Example 3: Analysis of lipid from the P. patens Protonema and from yeast cells The lipids were extracted from the S. patens Protonema or from yeast cells using chloroform/methanol as described by Siebertz et al. (Eur. J. Biochem., 101, 1979: 429-438) and purified with diethyl ether by thin-layer chromatography TLC). The fatty acids obtained were transmethylated to give the 009J/00032 26 corresponding methyl esters and analyzed by gas chromatography GC). The various methyl esters were identified using corresponding standards. Corresponding fatty acid pyrrolidides were obtained, and identified by GC-MS, as described by Anderson et al. (Lipids, 9, 1974: 185-190).
Example 4: Functional expression of the P. patens A6-desaturase cDNA in yeasts The expression experiments in yeasts were carried out with PPDES6 cDNA. Knock-out experiments had shown (data and experimental procedure not shown or described) that the knock-out effect leads to a loss of 20:311,14, 17 20:45,8,11, 14 20:45,11,14,17- and 20:5 5 ,8, 11 ,14, 17 -fatty acids. The 18:29,12- and 18:3 9 12 15 -fatty acids increase simultaneously. For expression in yeast, the PPDES6 cDNA was subcloned into the yeast expression vector pYES2 (Invitrogen). The vector obtained was named pYESdelta6.
Yeast cultures transformed with pYES2 (control) and pYESdelta6 (A6-desaturase cDNA) were cultured on uracil drop-out medium supplemented with 2% raffinose and 1% Tergitol NP-40 (for stabilizing the fatty acids). For expression, the cells were cultured with galactose (final concentration up to an optical density OD) of 0.5 and 600 nm. In feeding experiments, fatty acids were solubilized in 5% Tergitol and added at a final concentration of 0.0003%. The results of expression can be found in Table I. The synthesis of fatty acids with a double bond at position 6 is only possible in the presence of the expression construct with the A6-desaturase cDNA. This A6-desaturase enzyme had a greater activity in relation to fatty acids which already contain a double bond at position 9 or 12 (relative to carbon atom in the chain). The fatty acid methyl esters of all of the yeast lipids were analyzed by GC. The individual fatty acids synthesized are shown in the table in mol% of the overall fatty acids.
0093/00032 Table I: 27 Fatty acid composition in transformed yeasts in relation to the control Overall fatty acids pYES2 pYESdelta6 Fatty acids 18:29,12 +18:39,12, 1 16:0 16.4 16.1 23.8 25.8 16:19 54.0 55.5 38.1 31.4 16:26,9 4.2 1.7 18:0 3.2 2.4 4.0 18:19 24.9 19.7 19.1 19.2 18:26,9 0.6 0.2 18:29,12 8.5 18:36,9,12 4.0 18:39,12,15 11.7 18:46,9,12,15 Example 5: Transformation of P. patens The polyethylene glycol-mediated direct DNA transformation of protoplasts was carried out as described by Schafer et al. (Mol.
Gen. Genet., 226, 1991: 418-424). The transformants were selected on G418-containing medium (Girke et al., The Plant Journal, 1998: 39-48).
Example 6: Isolation of A6-desaturase cDNA and genomic clones of P. patens Eventually fragments of a A6-desaturase gene were cloned with the aid of a PCR reaction with the following degenerate oligonucleotides as primers: A: TGGTGGAA(A/G)TGGA(C/A)ICA(T/C)AA and B: GG(A/G)AA(A/C/G/T)A(A/G)(G/A)TG(G/A)TG(C/T)TC] and the following temperature program: 94°C, 3 min; [94°C, 20 sec; 45 0 C, 30 sec; 72 0 C, 1 min], 30 cycles; 72 0 C, 5 min. For cloning, poly(A)RNA was isolated from 12-day-old P. patens Protonema culture [sic]. The above-described PCR was carried out with this poly(A)RNA. Fragments of the expected fragment length (500 to 600 bp) were cloned into pUC18 and sequenced. The deduced amino acid sequence of a PCR fragment showed similarities with known A6-desaturases. Since it was known that P. patens has a A6-desaturase, it was assumed that this clone 0093/00032 28 encodes part of a A6-desaturase.
A complete cDNA clone PPDES6 cDNA) was isolated from P. patens cDNA library of 12-day-old Protonemata with the aid of the PCR fragment specified above. The nucleotide sequence is shown in SEQ ID NO:1. The deduced amino acid sequence can be seen from SEQ ID NO:2. The corresponding genomic sequence PPDES6 gene) was isolated with the aid of the PCR and the following oligonucleotides as primers: C: CCGAGTCGCGGATCAGCC D: CAGTACATTCGGTCATTCACC: Table II shows the results of the comparison between the novel P.
patens A6-desaturase over the entire nucleic acid sequence with the following, known A6-desaturase: Borago officinalis (U79010), Synechocystis sp (L11421), Spirulina platensis (X87094), Caenorhabiditis elegans (AF031477), Mortierella alpina (WO 98/46764), Homo sapiens (Cho et al., J. Biol. Chem., 274, 1999: 471-477), Rattus norvegicus (AB021980) and Mus musculus (Cho et al., J. Biol. Chem., 274, 1999: 471-477). The analysis was carried out with the Gap Program (GCG Package, Version 9.1) and the following analysis parameters: scoring matrix, blosum62, gap creation penalty, 12; gap extension penalty, 4. The results show the particular identity or similarity in percent in relation with the P. patens sequence.
Table II: Sequence comparison between P. patens A6-desaturase and other A6-desaturases eqAmino acid sequence identity [similarity] Borago officinalis 31 [38] Synechocystis sp. 21 [29] Spirulina platensis 20 [29] Caenorhabditis elegans 35 [43] Mortierella alpina 39 [47] Homo sapiens 27 [38] Rattus norvegicus 28 [39] Mus musculus 29 [39] Example 7: Cloning the Physcomitrella patens A6-desaturase The genomic A6-acyllipid desaturase from Physcomitrella patens was modified, isolated and used in the process according to the 0093/00032 29 invention on the basis of the published sequence (Girke et al., Plant 15, 1998: 39-48) using a polymerase chain reaction and cloning. To this end, a desaturase fragment was first isolated by means of polymerase chain reaction using two gene-specific primers, and inserted into the desaturase gene described in Girke et al. (see above).
Primer TG5: ccgctcgagcgaggttgttgtggagcggc and Primer TG3: 5'-ctgaaatagtcttgctcc-3' were first used for amplifying a gene fragment by means of polymerase chain reaction (30 cycles, 30 sec. at 94 0 V [sic], sec. at 50 0 C, 60 sec. at 72 0 C, post-incubation for 10 minutes at 72 0 C, in a Perkin Elmer thermocycler).
a) Cloning an expression plasmid expressing A6-desaturase under the control of the 35S CaMV [sic] promoter: An XhoI cleavage site was introduced into the fragment by the primer TG5. An XhoI/Eco47III fragment was obtained by restriction and transposed into the PPDES6 gene sequence described.in Girke et al. following analogous restriction with XhoI/Eco47III. The construct was named pZK. The insert of pZK was cloned into the XhoI/SmaI cleavage site of pRT99/35S as XhoI/HindIII fragment after filling up the HindIII cleavage site with nucleotides by treatment with the Klenow fragment of DNA polymerase I. The resulting plasmid pSK contains the 35S promoter [cauliflower mosaic virus, Franck et al. (1980) Cell 21, 285], the moss A6-desaturase and the 35S terminator in the vector pRT.
b) Construction of an expression construct under the control of the napin promoter: The resulting promoter desaturase fragment with terminator was cloned into the vector pJH3 by cleaving the plasmid pSK with XhoI, treatment with T4 DNA polymerase and PstI restriction. To this end, the vector BamHI was cleaved, the overhangs were filled up with Klenow enzyme, and this was followed by cutting with PstI. Ligation of the desaturase terminator fragment into the vector gave rise to the plasmid pJH7, which contains a napin promoter (Scofield et al., 1987, J. Biol. Chem. 262, 12202-8). The expression cassette of pJH7 was cleaved with Bspl20I and NotI and cloned into the binary vector pRE. This gave rise to the plasmid pRE-Ppdes6.
00O9/032 In a PCR reaction, the P. patens A6-desaturase cDNA according to the invention was used as template. With the aid of the oligonucleotides stated hereinbelow, a BamHI restriction cleavage site was introduced before the start codon and three adenine nucleotides were introduced into the A6-desaturase cDNA as consensus translation sequence for eukaryotes. A 1512 base pair fragment of the A6-desaturase was amplified and sequenced.
Pp-d6Desl: CC GGTACC aaaatggtattcgcgggcggtg -3' Pp-d6Des2: CC GGTACC ttaactggtggtagcatgct -3' The reaction mixtures contained approximately 1 ng/micro 1 [sic] template DNA, 0.5 mn of the oligonucleotides and, 200 pm deoxy-nucleotides (Pharmacia), 50 mM KC1, 10 mM Tris-HCl (pH 8.3 at 25 0 C, 1.5 mM MgC1 2 and 0.02 U/pl Pwo polymerase (Boehringer Mannheim) and are incubated in a Perkin Elmer PCR machine with the following temperature program: Annealing temperature: 50 0 C, 30 sec Denaturation temperature: 95 0 C, 30 sec Elongation temperature: 72 0 C, 90 sec Number of cycles: c) Construction of an expression construct under the control of the USP promoter: The resulting fragment of approx. 1.5 kB base pairs was ligated into the vector pBluescript SK- (Stratagene) which had been cleaved with EcoRV and was available for further clonings as BamHI fragment.
For the transformation of plants, a further transformation vector based on pBin-USP was generated, and this transformation vector contains the A6-desaturase BaMHI fragment. pBin-USP is a derivative of plasmid pBinl9. pBinUSP originated from pBinl9, by inserting an USP promoter into pBinl9 [Bevan et al. (1980) Nucl. Acids Res. 12, 8711] as EcoRI-BaMHI [sic] fragment. The polyadenylation signal is that of gene 3 of the T-DNA of the Ti-plasmid pTiACH5 (Gielen et al., (1984) EMBO J. 3, 835), where the nucleotides 11749-11939 were isolated as PvuII-HindIII fragment and, after the addition of SphI-linkers, cloned at the PvuII cleavage site between the SpHI-HindIII [sic] cleavage site of the vector. The USP promoter corresponds to the nucleotides 1-684 (Genbank Accession X56240), where part of the noncoding region of the USP gene was obtained in the promoter. The promoter fragment which is 684 base pairs in size was amplified with the aid of commercially available T7 standard primer (Stratagene) and with the aid of a synthesized primer via a PCR reaction using standard methods (primer sequence: GGATCTGCTGGCTATGAA-3'). The PCR fragment was subsequently cut with EcoRI/SalI and inserted into the vector pBinl9 with OCS terminator. This gave rise to the plasmid named pBinUSP.
d) Construction of an expression construct under the control of the Beta vulgaris vATPase C1 promoter: A construct using the v-ATPase cl promoter was generated analogously to the expression plasmid with the USP promoter.
The promoter was cloned into the plasmid pBinl9 with OCS terminator as an EcoRI/KpnI fragment and the P. patens A6desaturase gene was inserted between promoter and terminator via BaMHI. The promoter corresponds to a beta vulgaris 1153 base pair fragment (Plant Mol Biol, 1999, 39:463-475).
**o The construct was employed for the transformation of Arabidopsis thaliana and oilseed rape plants.
Example 8: Generation of transgenic oilseed rape plants 25 (modified according to Moloney et al., 1992, Plant Cell Reports, 8:238-242) To generate transgenic oilseed rape plants, binary vectors were made use of in Agrobacterium tumefaciens C58C1:pGV2260 or 30 Escherichia coli (Deblaere et al, 1984, Nucl. Acids. Res. 13, 4777-4788). To transform oilseed rape plants (var. Drakkar, NPZ Norddeutsche Pflanzenzucht, Hohenlieth, Germany), a 1:50 dilution of an overnight culture of a positively transformed agrobacterial colony grown in Murashige-Skoog Medium (Murashige and Skoog 1962 35 Physiol. Plant. 15, 473) supplemented with 3% of suciose (3MS medium) was used. Petioles or hypocotyls of freshly germinated sterile oilseed rape plants (in each case approx. 1 cm 2 were incubated for 5-10 minutes in a Petri dish together with a 1:50 agrobacterial dilution. This was followed by 3 days' incubation in the dark at 25_C on 3MS medium with 0.8% Bacto agar. After 3 days, the culture was continued under 16 hours light/8 hours dark, and continued in a weekly rhythm on MS medium supplemented with 500 mg/l Claforan (cefotaxime sodium), 50 mg/l kanamycin, -M benzylaminopurine (BAP) and 1.6 g/l glucose. Growing shoots were transferred to MS medium supplemented with sucrose, 250 mg/1 Claforan and 0.8% Bacto agar. If no roots have formed after three weeks, 2-indolebutyric acid was added to the medium as growth hormone for rooting.
Regenerated shoots were obtained on 2MS medium supplemented with kanamycin and Claforan, then, after rooting, transferred into soil and, after cultivation for two weeks, grown in a controlledenvironment cabinet or in the greenhouse and allowed to flower, and mature seeds were harvested and analyzed for A6-desaturase expression by means of lipid analyses. Lines with increased contents of or double bonds at the A6 position were identified.
In the stably transformed transgenic lines which functionally expressed the transgene, an increased content of double bonds at position A6 was found in comparison with untransformed control plants.
Example 9: Lipid extraction from seeds The plant material was first homogenized mechanically by comminuting in a pestle and mortar to make it more accessible to extraction.
Then, it was boiled for 10 minutes at 100°C and sedimented after cooling on ice. The cell sediment was hydrolyzed for one hour at 90°C with 1 N of methanolic sulfuric acid and 2% dimethoxypropane and the lipids were transmethylated. The resulting fatty acid 25 methyl esters (FAMEs) were extracted in petroleum ether. The extracted FAMEs were analyzed by gas liquid chromatooraphy using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature gradient of 170°C to 240°C over 20 minutes and 5 minutes at 240 0 C. The identity of the fatty acid 30 methyl esters was confirmed by comparison with corresponding FAME standards (sigma). The identity and position of the double bond could be analyzed further by suitable chemical derivatization of the FAME mixtures, for example to give 4,4dimethoxyoxazoline derivatives (Christie, 1997, in: Advances in 35 Lipid Methodology, 4 h Edition: Christie, Oily Press, Dundee, 119- 169, and 1998, Gaschromatographie-Massenspektrometrie Verfahren [Gas chromatography/mass spectrometry methods], Lipide 33:343- 353) using GC-MS. The GC analysis of the fatty acid methyl esters from the transgenic rapeseed which expressed A6-desaturase in a seed-specific fashion are shown in Table III. The transgenic rapeseed shows at least 4.95% y-linolenic acid in the seed.
Table III shows the GC analyses of the fatty acid methyl esters from mature, transgenic rapeseed which expressed A6-desaturase in a seed-specific fashion. The fatty acid composition is shown in [mol%] of the overall fatty acids. It can be stated that individual plants of the T2 generation which have been obtained from positively transformed, selfed plants contain up 4.95% of y-linolenic acid.
to approx.
Table III: GC analysis of the oilseed rape fatty acid methyl esters Name 18:0 18:1 18:2 18:3(y) 18:3( a) 18:4 R2-T2-11/la 1.98 53.58 22.63 3.86 11.38 0 R2-T2-11/lb 1.86 52.04 25.45 2.31 11.39 0 R2-T2-11/1c 1.95 49.17 24.30 2.84 9.20 0 R2-T2-11/3 1.82 49.83 24.54 3.88 10.12 0 R2-T2-11/4 1.72 48.02 24.66 4.95 9.52 0 R2-T2-11/5a 1.73 51.98 25.27 4.27 9.61 0 R2-T2-11/5b 2.02 56.19 25.08 0 9.33 0 R2-T2-11/5c 2.01 46.95 27.38 0 10.37 0 R2-T2-11/5d 1.83 49.49 24.15 4.40 8.65 0 R2-T2-11/6 2.08 54.52 23.94 2.05 9.29 0 R2-T2-11/10 1.94 53.92 22.81 4.06 9.44 0 R2-T2-WT 1.90 47.75 30.91 0 10.51 0 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.
9 9 *9 e 00 9 9 9 9 9 EDITORIAL NOTE APPLICATION NUMBER 65607/00 The following Sequence Listing pages 1-6 are part of the description.
The claims pages follow on pages 34-35 0093/00032 SEQUENCE PROTOCOL <170> Pate] <210> 1 <211> 2012 <212> DNA <213> Phys <220> <221> CDS <222> (319: <400> 1 ccgagtcgcg gaggaggagg tacctccggg ggagactgtt tctgtgagtg ntln Vers. 3omitreiia patens ).(1896) gatcagccat cgcccgccca gggccgcctg cagatgcgcg ggcgttggtg gagtcgtcat ttttggagcg ggcaaactct gttgcggctc gattttatgt cgggggcatt gccattgtgg tgcgtgcagc gccccgactg ccgcagagcg cattgtgtgg ccgaggatct ggaaggctat agagcggggg tctgtgtatg gga ctt cac Giy Leu Gir gacggtgttg actgcggcaa aggttcggca agactcagga acgaggttgt cag ggc Gin Giy tct ctc Ser Leu tgtggagcgg cttttgaa atg gta ttc gcg ggc ggt tct ctc gaa Ser Leu Giu ttc agc gac Phe Ser Asp gta cac agt Val His Ser gaa Glu ttc Phe Met 1 aac atc Asn Ile ttc agt Phe Ser Val Phe Ala gac gtc gag Asp Vai Giu 20 tat gtg tct Tyr Val Ser Giy 5 c ac His att gcc agt Ile Ala Ser atg Met Giy tca act gtt Ser Thr Val 35 aag Lys ggt Gly aag Lys tcg tgg agc Ser Trp Ser aag cgt gtt Lys Arg Vai ata caa cct Ile Gin Pro ttg Leu 50 cgc ctg acg Arg Leu Thr tcg gaa Ser Glu agt Ser gaa Glu 120 180 240 300 351 399 447 495 543 591 639 agc gct. gcc Ser Ala Ala tcg Ser gtg Val 65 act Thr caa tgt ata tca Gin Cys Ile Ser gct Ala 70 gtt cag aga Val Gin Arg aat Asn agt. acc cag Ser Thr Gin gga Gly agg gcg gag gca Ala Giu Ala ctc Leu 85 aag gca gaa tca gtc Ala Giu Ser Vai aag tcg aca cac gtg aag Val Lys ccc cta ccc acg aga cga tca tct cag tgg Pro Thr Arg Arg Arg Ser Ser Gin Trp 100 Lys Lys Ser Thr His Pro Leu 105 0093/00032 tca gaa gta Ser Glu Val 110 gca gta cac aac Ala Val His Asn aag Lys 115 cca agc gat tgc Pro Ser Asp Cys tgg Trp 120 att gtt gta Ile Val Val 687 aaa aac Lys Asn 125 aag gtg tat gat Lys Val Tyr Asp gtt Vai 130 tat Tyr tcc aat ttt gcg Ser Asn Phe Ala gac Asp 135 gag cat ccc gga Giu His Pro Gly gga Gly 140 tca gtt att agt Ser Val Ile Ser act Thr 145 ttt gga cga Phe Gly Arg gac Asp 150 ggc aca gat gtt Gly Thr Asp Val ttc Phe 155 735 783 831 879 tct agt ttt cat Ser Ser Phe His att ggt gac gtg Ile Gly Asp Vai 175 gca Ala 160 gct tct aca tgg Ala Ser Thr Trp aaa Ly s 165 att ctt caa gac Ile Leu Gin Asp ttt tac Phe Tyr 170 aaa gat Lys Asp gag agg gtg gag Giu Arg Vai Giu ccg Pro 180 act cca gag ctg Thr Pro Giu Leu ctg Leu 185 ttc cga gaa Phe Arg Giu 190 atg aga gct ctt ttc Met Arg Ala Leu Phe 195 ctg agg gag caa Leu Arg Giu Gin ctt Leu 200 ttc aaa agt Phe Lys Ser tcg aaa Ser Lys 205 ttg tac tat gtt Leu Tyr Tyr Vai atg Met 210 aag ctg ctc acg Lys Leu Leu Thr aat Asn 215 gtt gct att ttt Val Ala Ile Phe gct Ala 220 gcg agc att gca Ala Ser Ile Ala ata Ile 225 ata tgt tgg agc Ile Cys Trp Ser aag Lys 230 act att tca gcg Thr Ile Ser Ala gtt Val 235 975 1023 1071 1119 ttg gct tca gct Leu Ala Ser Ala cta tcc cat gat Leu Ser His Asp 255 tgt Cys 240 atg atg gct ctg Met Met Ala Leu tgt Cys 245 ttc caa cag tgc Phe Gin Gin Cys gga tgg Gly Trp 250 tgg ctt Trp Leu ttt ctc cac aat Phe Leu His Asn c ag Gin 260 gtg ttt gag aca Val Phe Giu Thr c gc Arg 265 aat gaa gtt Asn Giu Val 270 gtc ggg tat gtg Val Gly Tyr Val atc Ile 275 ggc aac gcc gtt Gly Asn Ala Val ctg Leu 280 ggg ttt agt Gly Phe Ser 1167 aca ggg Thr Gly 285 tgg tgg aag gag Trp Trp Lys Giu aag Ly s 290 cat aac ctt cat His Asn Leu His cat His 295 gct gct cca aat Ala Ala Pro Asn 1215 gaa tgc gat cag act tac caa cca att gat gaa gat att gat act ctc Giu Cys Asp Gin Thr Tyr Gin Pro Ile Asp Glu Asp Ile Asp Thr Leu 1263 0093/00032 300 315 ccc ctc att gcc Pro Leu Ile Ala tgg Trp 320 agc aag gac ata Ser Lys Asp Ile ctg Leu 325 gcc aca gtt gag Ala Thr Val Giu aat aag Asn Lys 330 1311 aca ttc ttg Thr Phe Leu tta ttt ttc Leu Phe Phe 350 cga Arg 335 atc ctc caa tac Ile Leu Gin Tyr cag Gin 340 cat ctg ttc ttc His Leu Phe Phe atg ggt ctg Met Gly Leu 345 aga tat acc Arg Tyr Thr 1359 1407 gcc cgt ggt agt Ala Arg Gly Ser tgg Trp 355 ctc ttt tgg agc Leu Phe Trp Ser tgg Trp 360 tct aca Ser Thr 365 gca gtg ctc tca Ala Val Leu Ser cct Pro 370 gtc gac agg ttg Val Asp Arg Leu ttg Leu 375 gag aag gga act Glu Lys Giy Thr gtt Val 380 ctg ttt cac tac Leu Phe His Tyr ttt Phe 385 tgg ttc gtc ggg Trp Phe Val Giy ac a Thr 390 gcg tgc tat ctt Ala Cys Tyr Leu ctc Leu 395 1455 1503 1551 cct ggt tgg aag Pro Gly Trp Lys cca Pro 400 tta gta tgg atg Leu Val Trp Met gc g Ala 405 gtg act gag ctc Val Thr Giu Leu atg tcc Met Ser 410 ggc atg ctg Gly Met Leu gtt tat aat Val Tyr Asn 430 ctg Leu 415 ggc ttt gta ttt Giy Phe Vai Phe gta Val 420 ctt agc cac aat Leu Ser His Asn ggg atg gag Gly Met Glu 425 gta tcc aca Val Ser Thr 1599 1647 tcg tct aaa gaa Ser Ser Lys Glu ttc Phe 435 gtg agt gca cag Val Ser Ala Gin atc Ile 440 cgg gat Arg Asp 445 atc aaa gga aac Ile Lys Gly Asn at a Ile 450 ttc aac gac tgg Phe Asn Asp Trp ttc Phe 455 act ggt ggc ctt Thr Giy Gly Leu aac Asn 460 tta Leu agg caa ata gag Arg Gin Ile Giu aac aaa ata gca Asn Lys Ile Ala 480 cat His 465 cat ctt ttc cca His Leu Phe Pro aca Thr 470 atg ccc agg cat Met Pro Arg His aat Asn 475 1695 1743 1791 cct aga gtg gag Pro Arg Vai Giu gtg Vai 485 ttc tgt aag aaa Phe Cys Lys Lys cac ggt His Gly 490 ctg gtg tac Leu Val Tyr gaa Giu 495 gac gta tct att Asp Val Ser Ile gct Ala 500 acc ggc act tgc Thr Gly Thr Cys aag gtt ttg Lys Vai Leu 505 1839 aaa gca ttg aag gaa gtc gcg gag gct gcg gca gag cag cat gct acc 18 1887 0093/00032 4 Lys Ala Leu Lys Glu Val Ala Giu Ala Ala Ala Giu Gin His Ala Thr 510 515 520 acc agt taa cagtctttgg aaagcttggc aattgatctt tattctccac 1936 Thr Ser 525 ggcagttgct tgtttgtttt ggggtgaatg accgaatgta ctggcatcca ttcttctgta 1996 gccatcaatt ttgaac 2012 <210> 2 <211> 525 <212> PRT <213> Physcomitrella patens <400> 2 Met Val Phe Ala Gly Gly Gly Leu Gin Gin Gly Ser Leu Glu Giu Asn 1 5 10 Ile Asp Val Giu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe 25 Ser Tyr Val Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gin 40 Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala 55 Val Gin Cys Ile Ser Ala .Giu Val Gin Arg Asn Ser Ser Thr Gin Gly 70 75 Thr Aia Glu Ala Leu Ala Giu Ser Val Val Lys Pro Thr Arg Arg Arg 90 Ser Ser Gin Trp Lys Lys Ser Thr His Pro Leu Ser Giu Val Ala Vai 100 105 110 His Asn Lys Pro Ser Asp Cys Trp Ile Vai Vai Lys Asn Lys Val Tyr 115 120 125 Asp Val Ser Asn Phe Ala Asp Giu His Pro Giy Gly Ser Vai Ile Ser 130 135 140 Thr Tyr Phe Gly Arg Asp Giy Thr Asp Val Phe Ser Ser Phe His Ala 145 150 155 160 Ala Ser Thr Trp Lys 1ie Leu Gin Asp Phe Tyr Ile Gly Asp Val Giu 165 170 175 0093/00032 Arc Ala Val Ile 225 Met Leu Tyr Giu Tyr 305 Ser Leu Gly Ser Phe 385 Leu Val Glu Pro 180 Leu Phe Leu 195 *Met Lys Leu 210 Sle Cys Trp Met Ala Leu His Asn Gin 260 Val Ile Gly 275 Lys His Asn 290 Gin Pro Ile Lys Asp Ile Gin Tyr Gin 340 Ser Trp Leu 355 Pro Val Asp 370 Trp Phe Vai Vai Trp Met Thr Pro Giu Leu Leu Lys Asp Phe Arg Giu Met Arg Arg Leu Ser Cys 245 Vai Asn Leu Asp Leu 325 His Phe Arg Giy Alia 405 ELeu Giu Gin Thr Asn 215 Lys Thr 230 Phe Gin Phe Giu Aia Vai His His 295 Giu Asp 310 Ala Thr Leu Phe Trp Ser Leu Leu 375 Thr Ala 390 Val Thr Ser His Leu 200 Vai Ile Gin Thr Leu 280 Ala Ile Val Phe Trp 360 Giu Cys Giu Asn Phe Aia Ser Cys Arg 265 Giy Aia Asp Giu Met 345 Arg Lys Tyr Leu Giy 425 Lys IlE Ala Giy 250 Trp Phe Pro Thr Asn 330 Giy Tyr Giy Leu Met 410 Met Ser Phe Vai 235 Trp Leu Ser Asn Leu 315 Lys5 Leu Thr Thr Leu 395 Ser Glu Ser Ala 220 Leu Leu Asn Thr Giu 300 Pro Thr Leu Ser Vai 380 Pro Giy V1al *Lys Leu 205 Aia Ser Ala Ser Ser His Giu Vai 270 Gly Trp 285 Cys Asp Leu Ile Phe Leu Phe Phe 350 Thr Aia 365 Leu Phe Gly Trp Met Leu Tyr Asn 430 Tyr Ile Al a Asp 255 Val Trp Gin Ala Arg 335 Al a Vai His Ly's Leu 415 Ser Tyr Ala Cys 240 Phe Gly Lys Thr Trp 320 Ile Arg Leu Tyr Pro 400 Gly Ser Phe Val Phe Val 420 Lys Glu Phe Val Ser Ala Gin Ile Val Ser Thr Arg Asp Ile Lys Gly 435 440 445 A O0§3/06032 Asn His 465 Pro Val Val Ile 450 His Arg Ser Ala Phe Leu Val Ile Giu 515 Asn Phe Giu Ala 500 Ala Asp Pro Val 485 Thr Trp Thr 470 Phe Gly Phe 455 Met Cys Thr 6 Thr Gly Pro Arg Lys Lys Cys Lys 505 Gin His 520 Gly His His 490 Val1 Ala Leu Asn 475 Gly Leu Thr Asn 460 Leu Leu Lys Thr Arg Asn Val Ala Ser 525 Gin Ile Giu Lys Ile Ala 480 Tyr Giu Asp 495 Leu Lys Giu 510 Ala Ala Giu
Claims (11)
1. A process of preparing unsaturated fatty acids, which process comprises introducing, into a transgenic plant or algae, at least one isolated nucleic acid sequence encoding a polypeptide having A6-desaturase activity, selected from the group consisting of: a) a nucleic acid sequence having the sequence shown in SEQ ID NO: 1i, b) nucleic acid sequences which, as a result of the degeneracy of the genetic code, are derived from 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 have at least homology at the amino acid level without substantially 20 reducing the enzymatic action of the polypeptides, and culturing the transgenic plant or algae, where the cultured transgenic plant or algae contains at least 1 mol% of unsaturated fatty acids based on the total fatty acid 25 content in the organism.
2. The process as claimed in claim 1, wherein the nucleic acid sequence is derived from a plant or algae. 30
3. The process as claimed in claim 1 or 2, wherein the nucleic acid sequence is derived from Physcomitrella patens.
4. The process as claimed in any one of claims 1 to 3, wherein the at least one isolated nucleic acid sequence is introduced into an oil crop.
The process as claimed in any one of claims 1 to 4, wherein the transgenic plant or algae contains at least 5% by weight of unsaturated fatty acids based on the total fatty acid content in the organism.
6. The process as claimed in any of claims 1 to 5, wherein the unsaturated fatty acids are isolated from the transgenic plant or algae.
7. A transgenic plant or algae comprising at least one isolated nucleic acid sequence encoding a polypeptide with A6- desaturase activity, selected from the group cons.isting of: a) a nucleic acid sequence having 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 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 have at least homology at the amino acid level without substantially reducing the enzymatic action of the polypeptides.
8. An oil, lipid or fatty acid or a fraction thereof, prepared by the process as claimed in any one of claims 1 to 6. oo0
9. A process of preparing unsaturated fatty acids, which process is substantially as hereinbefore described with reference to Example 8 or Example 9.
10. An oil, lipid or fatty acid or a fraction thereof, prepared by the process substantially as hereinbefore described with reference to Example 8 or Example 9. o.
11. The use of the oil, lipid or fatty acid composition as S" 30 claimed in claim 8 or claim 10 of a transgenic plant or algae as claimed in claim 7 in feed, foodstuffs, cosmetics or pharmaceuticals. DATED this 19th day of July 2004 BASF PLANT SCIENCE GMBH WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA P20716AU00
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DE10030976A DE10030976A1 (en) | 2000-06-30 | 2000-06-30 | Production of unsaturated fatty acids, useful e.g. in nutrition, cosmetics or pharmaceuticals, in organisms transformed with Physcomitrella patens delta-6-desaturase nucleic acid |
PCT/EP2000/006223 WO2001002591A1 (en) | 1999-07-06 | 2000-07-04 | Plants expressing δ6-desaturase genes and oils from these plants containing pufas and method for producing unsaturated fatty acids |
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US8816106B2 (en) | 2006-08-29 | 2014-08-26 | Commonwealth Scientific And Industrial Research Organisation | Synthesis of fatty acids |
US8816111B2 (en) | 2012-06-15 | 2014-08-26 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising polyunsaturated fatty acids |
US10005713B2 (en) | 2014-06-27 | 2018-06-26 | Commonwealth Scientific And Industrial Research Organisation | Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the sn-2 position |
US10125084B2 (en) | 2013-12-18 | 2018-11-13 | Commonwealth Scientific And Industrial Research Organisation | Lipid comprising docosapentaenoic acid |
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Also Published As
Publication number | Publication date |
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AU6560700A (en) | 2001-01-22 |
EP1190080A1 (en) | 2002-03-27 |
WO2001002591A1 (en) | 2001-01-11 |
CA2378423A1 (en) | 2001-01-11 |
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