PROCESSES FOR PRODUCING POLYUNSATURATED FATTY ACIDS IN TRANSGENIC ORGANISMS

Abstract

The present invention relates to polynucleotides from Ostreococcus lucimarinus which code for desaturases and elongases and which can be employed for the recombinant production of polyunsaturated fatty acids. The invention furthermore relates to vectors, host cells and transgenic nonhuman organisms which comprise the polynucleotides, and to the polypeptides encoded by the polynucleotides. Finally, the invention also relates to production processes for the polyunsaturated fatty acids and for oil, lipid and fatty acid compositions.

Claims

1. A process for the production of a substance which has the structure shown in the general formula I hereinbelow: ##STR00005## where the variables and substituents are as follows: R.sup.1=hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol, sphingo base or a radical of the formula II ##STR00006## R.sup.2=hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol or saturated or unsaturated C.sub.2-C.sub.24-alkylcarbonyl, R.sup.3=hydrogen, saturated or unsaturated C.sub.2-C.sub.24-alkylcarbonyl, or R.sup.2 and R.sup.3 independently of one another are a radical of the formula Ia: ##STR00007## in which n=2, 3, 4, 5, 6, 7 or 9, m=2, 3, 4, 5 or 6 and p=0 or 3; and wherein the process comprises the cultivation of: (i) a host cell comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with Ostreococcus lucimarinus-CoA-dependent delta-6 desaturase activity, which nucleic acid sequence comprises a. a nucleic acid sequence as shown in SEQ ID NO: 13, b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleic acid sequences of (a) or (b), and wherein the polynucleotide is operatively linked to an expression control sequence; or (ii) a transgenic, nonhuman organism comprising: I) an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with Ostreococcus lucimannus CoA-dependent delta-6 desaturase activity, which nucleic acid sequence comprises a. a nucleic acid sequence as shown in SEQ ID NO: 13, b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and wherein the polynucleotide is operatively linked to an expression control sequence; II) a vector comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with Ostreococcus lucimarinus CoA-dependent delta-6 desaturase activity, which nucleic acid sequence comprises a. a nucleic acid sequence as shown in SEQ ID NO: 13, b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and wherein the polynucleotide is operatively linked to an expression control sequence, or III) a host cell comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with Ostreococcus lucimarinus CoA-dependent delta-6 desaturase activity, which nucleic acid sequence comprises a. a nucleic acid sequence as shown in SEQ ID NO: 13, b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and wherein the polynucleotide is operatively linked to an expression control sequence, under conditions which permit the biosynthesis of the substance.

2. A process for the production of a substance which has the structure shown in the general formula I hereinbelow: ##STR00008## where the variables and substitutents are as follows: R.sup.1=hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol, sphingo base or a radical of the formula II ##STR00009## R.sup.2=hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol or saturated or unsaturated C.sub.2-C.sub.24-alkylcarbonyl, R.sup.3=hydrogen, saturated or unsaturated C.sub.2-C.sub.24-alkylcarbonyl, or R.sup.2 and R.sup.3 independently of one another are a radical of the formula Ia: ##STR00010## n=2, 3, 4, 5, 6, 7 or 9, m=2, 3, 4, 5 or 6 and p=0 or 3; and wherein the process comprises the cultivation of: (i) a host cell comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with the same substrate specificity and conversion rate as Ostreococcus lucimarinus CoA-dependent delta 6 desaturase, which nucleic acid sequence comprises a. a nucleic acid sequence as shown in SEQ ID NO: 13, b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleic acid sequences of (a) or (b) and wherein the polynucleotide is operatively linked to an expression control sequence; or (ii) a transgenic, nonhuman organism comprising: I) an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with the same substrate specificity and conversion rate as Ostreococcus lucimarinus CoA-dependent delta-6 desaturase, which nucleic acid sequence comprises a. a nucleic acid sequence as shown in SEQ ID NO: 13, b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and wherein the polynucleotide is operatively linked to an expression control sequence; II) a vector comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with the same substrate specificity and conversion rate as Ostreococcus lucimarinus CoA-dependent delta-6 desaturase, which nucleic acid sequence comprises a. a nucleic acid sequence as shown in SEQ ID NO: 13, b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b), and wherein the polynucleotide is operatively linked to an expression control sequence, or III) a host cell comprising an isolated polynucleotide comprising a nucleic acid sequence coding for a polypeptide with the same substrate specificity and conversion rate as Ostreococcus lucimarinus CoA-dependent delta-6 desaturase, which nucleic acid sequence comprises a. a nucleic acid sequence as shown in SEQ ID NO: 13, b. a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, or c. a nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b) and wherein the polynucleotide is operatively linked to an expression control sequence, under conditions which permit the biosynthesis of the substance.

3. The process according to claim 1, wherein the substituents R.sup.2 or R.sup.3 in the general formulae I and II are, independently of one another, saturated or unsaturated C.sub.15-C.sub.22-alkylcarbonyl.

4. The process according to claim 1, wherein the substituents R.sub.2 or R.sub.3 in the general formulae I and II are, independently of one another, unsaturated C.sub.18-, C.sub.20-, or C.sub.22-alkylcarbonyl with at least two double bonds.

5. The process according to claim 1, wherein the substance of formula I is stearidonic acid, eicosapentaenoic acid, ω3-docosapentaenoic acid, docosahexaenoic acid or mixtures of these.

6. The process according to claim 1, wherein the substance of formula I is stearidonic acid.

7. The process according to claim 1, wherein the transgenic, nonhuman organism is an animal, a plant or a multicellular microorganism.

8. The process according to claim 7, wherein the transgenic plant is an oil crop.

9. The process according to claim 8, wherein the transgenic plant is a Brassica species.

10. The process according to claim 9, wherein the Brassica species is canola.

11. A process for the production of an oil, lipid or fatty acid composition, according to claim 1 comprising the further step of formulating the substance as an oil, lipid or fatty acid composition.

12. The process according to claim 10, wherein the oil, lipid or fatty acid composition is formulated further to give a pharmaceutical, a cosmetic product, a foodstuff, a feeding stuff, or a food supplement.

13. The process according to claim 1, wherein the polypeptide encoded by the nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b) has the same delta 6 desaturase activity as Ostreococcus lucimarinus delta 6 desaturase.

14. The process according to claim 1, wherein the sequence identity to one of the nucleotide sequences of (a) or (b) is determined over the entire length of the sequences to be compared.

15. The process of claim 14 which implements the algorithm of Needleman and Wunsch to compare the sequences.

16. The process according to claim 2, wherein the substituents R.sup.2 or R.sup.3 in the general formulae I and II are, independently of one another, saturated or unsaturated C.sub.15-C.sub.22-alkylcarbonyl.

17. The process according to claim 2, wherein the substituents R.sub.2 or R.sub.3 in the general formulae I and II are, independently of one another, unsaturated C.sub.18-, C.sub.20-, or C.sub.22-alkylcarbonyl with at least two double bonds.

18. The process according to claim 2, wherein the substance of formula I is stearidonic acid, eicosapentaenoic acid, ω3-docosapentaenoic acid, docosahexaenoic acid or mixtures of these.

19. The process according to claim 2, wherein the substance of formula I is stearidonic acid.

20. The process according to claim 2, wherein the transgenic, nonhuman organism is an animal, a plant or a multicellular microorganism.

21. The process according to claim 20, wherein the transgenic plant is an oil crop.

22. The process according to claim 21, wherein the transgenic plant is a Brassica species.

23. The process according to claim 22, wherein the Brassica species is canola.

24. A process for the production of an oil, lipid or fatty acid composition, according to claim 2 comprising the further step of formulating the substance as an oil, lipid or fatty acid composition.

25. The process according to claim 23, wherein the oil, lipid or fatty acid composition is formulated further to give a pharmaceutical, a cosmetic product, a foodstuff, a feeding stuff, or a food supplement.

26. The process according to claim 2, wherein the polypeptide encoded by the nucleic acid sequence with at least 75% sequence identity to one of the nucleotide sequences of (a) or (b) has the same delta 6 desaturase activity as Ostreococcus lucimarinus delta 6 desaturase.

27. The process according to claim 2, wherein the sequence identity to one of the nucleotide sequences of (a) or (b is determined over the entire length of the sequences to be compared.

28. The process of claim 27 which implements the algorithm of Needleman and Wunsch to compare the sequences.

Description

FIGURES

[0172] FIG. 1 shows a sequence alignment of the Δ5- and Δ6-elongase amino acid sequences from O. lucimarinus (d5-elo-Olu (SEQ ID NO: 12); d6-elo-Olu (SEQ ID NO: 16)), O. tauri (d5-elo-Ota (SEQ ID NO: 18); d6-elo-Ota (SEQ ID NO: 20)), and T. pseudonana (d5-elo-Tps (SEQ ID NO: 34); d6-elo-Tps (SEQ ID NO: 36)) in the ClustalW comparison.

[0173] FIG. 2 shows a sequence alignment of the Δ4-desaturase amino acid sequences from O. lucimarinus (d4-des-Olu (SEQ ID NO: 6)), O. tauri (d4-des-Ota (SEQ ID NO: 24)), and T. pseudonana (d4-des-Tps (SEQ ID NO: 42)) in the ClustalW comparison.

[0174] FIG. 3 shows a sequence alignment of the Δ5-desaturase amino acid sequences from O. lucimarinus (first d5-des-Olu (SEQ ID NO: 8); second d5-des-Olu (SEQ ID NO: 10)), O. tauri (d5-des-Ota (SEQ ID NO: 28)), and T. pseudonana (d5-des-Tps (SEQ ID NO: 40)) in the ClustalW comparison.

[0175] FIG. 4 shows a sequence alignment of the Δ6-desaturase amino acid sequences from O. lucimarinus (d6-des-Olu (SEQ ID NO: 14)), O. tauri (d6-des-Ota (SEQ ID NO: 30)), and T. pseudonana (d6-des-Tps (SEQ ID NO: 38)) in the ClustalW comparison.

[0176] FIG. 5 shows a sequence alignment of the Δ12-desaturase amino acid sequences from O. lucimarinus (first d12-des-OI (SEQ ID NO: 4); second d12-des-OI (SEQ ID NO: 2)), O. tauri (d12-des-Ot (SEQ ID NO: 32)), and T. pseudonana (d12-des-Tp (SEQ ID NO: 44)) in the ClustalW comparison.

[0177] FIG. 6 shows the gas-chromatographic determination of the fatty acids from yeasts which have been transformed with the plasmid pYES (A, B) or pYES-D5Elo(OI) (C). The fatty acid 20:4Δ5,8,11,14 was fed (B, C).

[0178] FIG. 7 shows the gas-chromatographic determination of the fatty acids from yeasts which have been transformed with the plasmid pYES (A, B, C) or pYES-D6Elo(OI) (D,E). The fatty acids 18:3Δ6,9,12 or 18:4Δ6,9,12,15 were fed (B, D) and (C, E), respectively.

[0179] FIG. 8 shows the gas-chromatographic determination of the fatty acids from yeasts which have been transformed with the plasmid pYES (A, B) or pYES-D5Des(OI_2) (C). The fatty acid 20:3Δ5,8,11,14 was fed (B) and (C).

[0180] FIG. 9 shows the gas-chromatographic determination of the fatty acids from yeasts which have been transformed with the plasmid pYES (A) or pYES-D12Des(OI) (B).

[0181] FIG. 10 shows the gas-chromatographic determination of the fatty acids from yeast. pYes-d5Des(OI_1) in yeast strain InvSc without addition of fatty acids (A); pYes-d5Des(OI_1) in yeast strain InvSc after addition of the fatty acid 20:3n-6 (B), pYes-d5Des(OI_1) in yeast strain InvSc after addition of the fatty acid 20:4n-3 (C).

[0182] The present invention is illustrated in greater detail by the examples which follow, which are not to be construed as limiting.

EXAMPLES

Example 1

General Cloning Methods

[0183] 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, linkage of DNA fragments, transformation of Escherichia coli cells, bacterial cultures 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).

Example 2

Sequence Analysis of Recombinant DNA

[0184] Recombinant DNA molecules were sequenced with an ABI laser fluorescence DNA sequencer by the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA74, 5463-5467). Fragments obtained by polymerase chain reaction were sequenced and verified to avoid polymerase errors in constructs to be expressed.

Example 3

Lipid Extraction from Yeasts

[0185] The effect of the genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound (such as a fatty acid) can be determined by growing the modified microorganisms or the modified plant under suitable conditions (such as those described above) and analyzing the medium and/or the cellular components for the elevated production of the desired product (i.e. of the lipids or a fatty acid). These analytical techniques are known to the skilled worker and comprise spectroscopy, thin-layer chromatography, various types of staining methods, enzymatic and microbiological methods and analytical chromatography such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applications of HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, Chapter III: “Product recovery and purification”, p. 469-714, VCH: Weinheim; Belter, P. A., et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992) Recovery processes for biological Materials, John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

[0186] In addition to the abovementioned processes, plant lipids are extracted from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22):12935-12940 and Browse et al. (1986) Analytic Biochemistry 152:141-145. The qualitative and quantitative analysis of lipids or fatty acids is described by Christie, William W., Advances in Lipid Methodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1); “Progress in Lipid Research”, Oxford: Pergamon Press, 1 (1952)-16 (1977) under the title: Progress in the Chemistry of Fats and Other Lipids CODEN.

[0187] In addition to measuring the end product of the fermentation, it is also possible to analyze other components of the metabolic pathways which are used for the production of the desired compound, such as intermediates and by-products, in order to determine the overall production efficiency of the compound. The analytical methods comprise measuring the amount of nutrients in the medium (for example sugars, hydrocarbons, nitrogen sources, phosphate and other ions), measuring the biomass composition and the growth, analyzing the production of conventional metabolites of biosynthetic pathways and measuring gases which are generated during the fermentation. Standard methods for these measurements are described in Applied Microbial Physiology; A Practical Approach, P. M. Rhodes and P. F. Stanbury, Ed., IRL Press, p. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and references cited therein.

[0188] One example is the analysis of fatty acids (abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid chromatography/mass spectrometry; TAG, triacylglycerol; TLC, thin-layer chromatography).

[0189] The unambiguous proof for the presence of fatty acid products can be obtained by analyzing recombinant organisms using standard analytical methods: GC, GC-MS or TLC, as described on several occasions by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren [Gas chromatography/mass spectrometry methods], Lipide 33:343-353).

[0190] The material to be analyzed can be disrupted by sonication, grinding in a glass mill, liquid nitrogen and grinding or via other applicable methods. After disruption, the material must be centrifuged. The sediment is resuspended in distilled water, heated for 10 minutes at 100° C., cooled on ice and recentrifuged, followed by extraction for one hour at 90° C. in 0.5 M sulfuric acid in methanol with 2% dimethoxypropane, which leads to hydrolyzed oil and lipid compounds, which give transmethylated lipids. These fatty acid methyl esters are extracted in petroleum ether and finally subjected to a GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 μm, 0.32 mm) at a temperature gradient of between 170° C. and 240° C. for 20 minutes and 5 minutes at 240° C. The identity of the resulting fatty acid methyl esters must be defined using standards which are available from commercial sources (i.e. Sigma).

Example 4

Cloning and Characterization of Elongase Genes from Ostreococcus lucimarinus

[0191] By searching for conserved regions in the protein sequences in elongase genes, it was possible to identify two sequences with corresponding motifs in an Ostreococcus lucimarinus sequence database. In a further step, the genes were characterized by means of sequence alignment, gene prediction and the search for coding regions. The following coding regions were found:

TABLE-US-00001 TABLE 1 Coding regions Name of gene SEQ ID Amino acids D5Elo(Ol) 12 298 D6Elo(Ol) 16 287

[0192] FIG. 1 shows the sequence similarities with other algae (Ostreococcus tauri, Thalassiosira pseudonana) for the various elongase amino acid sequences in the ClustalW sequence alignment. Surprisingly, the O. lucimarinus sequences differ markedly from the other algae in their amino acid sequence.

TABLE-US-00002 TABLE 2 Sequence identities of individual elongases Name of gene SEQ ID Organism Identity in % D5Elo(Ol) 28 O. lucimarinus 100 O. tauri  77 T. pseudonana  21 D6Elo(Ol) 32 O. lucimarinus 100 O. tauri  71 T. pseudonana  25

[0193] The cloning procedure was carried out as follows:

[0194] 40 ml of an Ostreococcus lucimarinus culture in the stationary phase were spun down and resuspended in 100 μl of double-distilled water and stored at −20° C. The relevant genomic DNAs were amplified based on the PCR method. The corresponding primer pairs were selected in such a way that they contained the yeast consensus sequence for highly efficient translation (Kozak, Cell 1986, 44:283-292) next to the start codon. The amplification of the DNAs was carried out using in each case 1 μl of defrosted cells, 200 μM dNTPs, 2.5 U Taq polymerase and 100 pmol of each primer in a total volume of 50 μl. The conditions for the PCR were as follows: first denaturation at 95° C. for 5 minutes, followed by 30 cycles at 94° C. for 30 seconds, 55° C. for 1 minute and 72° C. for 2 minutes, and a final elongation step at 72° C. for 10 minutes.

[0195] To characterize the function of the Ostreococcus lucimarinus elongases, the open reading frames of the DNAs in question are cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1/V5-His-TOPO (Invitrogen), giving rise to pOLE1 and pOLE2.

[0196] The Saccharomyces cerevisiae strain 334 is transformed with the vector pOLE1 or pOLE2, respectively, by electroporation (1500 V). A yeast which is transformed with the blank vector pYES2 is used as control. The transformed yeasts are selected on complete minimal dropout uracil medium (CMdum) agar plates supplemented with 2% glucose. After the selection, in each case three transformants are selected for the further functional expression.

[0197] To express the OI elongases, precultures consisting of in each case 5 ml of CMdum dropout uracil liquid medium supplemented with 2% (w/v) raffinose are initially inoculated with the selected transformants and incubated for 2 days at 30° C. and 200 rpm. Then, 5 ml of CMdum (dropout uracil) liquid medium supplemented with 2% raffinose and 300 μM various fatty acids are inoculated with the precultures to an OD.sub.600 of 0.05. Expression is induced by the addition of 2% (w/v) galactose. The cultures were incubated for a further 96 hours at 20° C.

[0198] Yeasts which have been transformed with the plasmids pYES2, pOLE1 and pOLE2 are analyzed as follows:

[0199] The yeast cells from the main cultures are harvested by centrifugation (100×g, 5 min, 20° C.) and washed with 100 mM NaHCO.sub.3, pH 8.0 to remove residual medium and fatty acids. Starting with the yeast cell sediments, fatty acid methyl esters (FAMEs) are prepared by acid methanolysis. To this end, the cell sediments are incubated for one hour at 80° C. together with 2 ml of 1 N methanolic sulfuric acid and 2% (v/v) of dimethoxypropane. The FAMEs are extracted twice with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phases are washed in each case once with 2 ml of 100 mM NaHCO.sub.3, pH 8.0 and 2 ml of distilled water. Thereafter, the PE phases are dried with Na.sub.2SO.sub.4, evaporated under argon and taken up in 100 μl of PE. The samples are separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 μm, Agilent) in a Hewlett-Packard 6850 gas chromatograph equipped with flame ionization detector. The conditions for the GLC analysis are as follows: the oven temperature was programmed from 50° C. to 250° C. with an increment of 5° C./min and finally 10 min at 250° C. (holding).

[0200] The signals are identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Napier and Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.

[0201] Activity and Substrate Determination of D5Elo(OI):

[0202] To determine the activity and substrate specificity of d5Elo(OI), various fatty acids were fed (table 3). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts reveal the synthesis of novel fatty acids, the products of the d5Elo(OI) reaction. This means that the gene d5Elo(OI) was expressed functionally.

TABLE-US-00003 TABLE 3 Feeding of yeasts with the plasmids pYES and pYES-D5Elo(Ol) Sample name/ Expected fatty acid fed conversion Substrate Product pYES Control — — pYES-D5Elo(Ol_GA) 20:4->22:4 98.0 48.5 20:4 pYES-D5Elo(Ol_GA) 20:4->22:4 62.6 32.2 20:4

[0203] FIG. 6 shows the chromatograms of the individual experiments. In FIG. 6a, yeasts transformed with pYES were analyzed without the addition of fatty acids by way of control. In FIG. 1b, the pYES-transformed yeasts were fed the fatty acid 20:4Δ5,8,11,14. Here, the fed fatty acid can be detected in large amounts. The same experiment is carried out in FIG. 6C for yeasts transformed with the plasmid pYES-D5Elo(OI). As opposed to FIG. 6B, it is possible to detect, in the yeasts with pYES-D5Elo(OI), an additional fatty acid, which must be attributed to the D5Elo(OI) activity. With reference to the activity, it is possible to characterize D5Elo(OI) as a Δ5-elongase.

[0204] Summary of the D5Elo(OU) Results:

[0205] It was possible to demonstrate in the yeast feeding experiments that the cloned gene D5Elo(OI) SEQ ID12 was expressed functionally and that it has elongase activity. By reference of the fed fatty acid, it is possible to characterize D5Elo(OI) as a Δ5-elongase, i.e. C20-fatty acids with a Δ5-double bond are elongated specifically.

[0206] Activity and Substrate Determination of D6Elo(OI):

[0207] To determine the activity and substrate specificity of D6Elo(OI), various fatty acids were fed (table 4). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts reveal the synthesis of novel fatty acids, the products of the D6Elo(OI) reaction. This means that the gene D6Elo(OI) was expressed functionally.

TABLE-US-00004 TABLE 4 Feeding/conversion of various fatty acids with D6Elo(Ol) Sample name/ Expected fatty acid fed conversion Substrate Product pYES Control — — pYES-D6Elo(Ol_GA) γ18:3->20:3 236.5 232.1 γ18:3 pYES-D6Elo(Ol_GA) γ18:3->20:3 111.2 126.5 γ18:3 pYES-D6Elo(Ol_GA) 18:4->20:4 94.3 82.9 18:4 pYES-D6Elo(Ol_GA) 18:4->20:4 73.2 68.3 18:4

[0208] FIG. 7 shows the chromatograms of the individual experiments. In FIG. 7a, yeasts transformed with pYES were analyzed without the addition of fatty acids by way of control. In FIGS. 7b and 7c, the pYES-transformed yeasts were fed the fatty acid 18:3Δ6,9,12(b) and 18:4Δ6,9,12,15(c), respectively. Here, the fed fatty acids can be detected in large amounts. The same experiment is carried out in FIGS. 7C and 7D for yeasts transformed with the plasmid pYES-D6Elo(OI). As opposed to FIGS. 7B and 7C, it is possible to detect, in the yeasts with pYES-D6Elo(OI), an additional fatty acid, which must be attributed to the D6Elo(OI) activity. With reference to the activity, it is possible to characterize D6Elo(OI) as a Δ6-elongase.

[0209] Summary of the D6Elo(OI) Results:

[0210] It was possible to demonstrate in the yeast feeding experiments that the cloned gene D6Elo(OI) SEQ ID NO: 16 was expressed functionally and that it has elongase activity. By reference to the fed fatty acid, it is possible to characterize D6Elo(OI) as a Δ6-elongase, i.e. C18-fatty acids with a Δ6-double bond are elongated specifically.

Example 5

Cloning and Characterization of Ostreococcus lucimarinus Desaturase Genes

[0211] By searching for conserved regions in the protein sequences with the aid of conserved motifs (His boxes, Domergue et al. 2002, Eur. J. Biochem. 269, 4105-4113), it was possible to identify five sequences with corresponding motifs in an Ostreococcus lucimarinus sequence database (genomic sequences). In a further step, the genes were characterized by means of sequence alignment, gene prediction and the search for coding regions. The following coding regions were found:

TABLE-US-00005 TABLE 5 Coding regions Name of gene SEQ ID Amino acids D4Des(Ol)  6 466 D5Des(Ol)  8 459 D5Des_2(Ol) 10 491 D6Des(Ol) 14 482 D12Des(Ol)  4 442 D12Des_2(Ol)  2 362

[0212] To characterize the function of the Ostreococcus lucimarinus desaturase d6Des(OI) (=Δ6-desaturase), the open reading frame of the DNA is cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1/V5-His-TOPO (Invitrogen), giving rise to the corresponding pYES2.1-d6Elo(OI) clone. Further desaturase genes from Ostreococcus can be cloned accordingly.

[0213] The Saccharomyces cerevisiae strain 334 is transformed with the vector pYES2.1-d6Elo(OI), by electroporation (1500 V). A yeast which is transformed with the blank vector pYES2 was used as control. The transformed yeasts were selected on complete minimal dropout uracil medium (CMdum) agar plates supplemented with 2% glucose. After the selection, in each case three transformants were selected for the further functional expression.

[0214] To express the d6Elo(OI) desaturase, precultures consisting of in each case 5 ml of CMdum dropout uracil liquid medium supplemented with 2% (w/v) raffinose are initially inoculated with the selected transformants and incubated for 2 days at 30° C. and 200 rpm. Then, 5 ml of CMdum (dropout uracil) liquid medium supplemented with 2% raffinose and 300 μM various fatty acids are inoculated with the precultures to an OD.sub.600 of 0.05. Expression is induced by the addition of 2% (w/v) galactose. The cultures are incubated for a further 96 hours at 20° C.

[0215] In the ClustalW sequence alignment, FIGS. 2 to 5 show sequence similarities with other algae (Ostreococcus tauri, Thalassiosira pseudonana) for the various desaturase amino acid sequences. Surprisingly, the O. lucimarinus sequences differ markedly in their amino acid sequence from the other algae.

TABLE-US-00006 TABLE 6 Sequence identities of individual desaturases Name of gene SEQ ID Organism Identity in % D4Des(Ol)  6 O. lucimarinus 100 O. tauri  69 T. pseudonana  20 D5Des(Ol)  8 D5Des_2(Ol)  23 O. tauri_2  47 T. pseudonana  22 D5Des_2(Ol) 10 D5Des(Ol)  23 O. tauri_2  14 T. pseudonana  19 D6Des(Ol) 14 O. lucimarinus 100 O. tauri  62 T. pseudonana  15 D12Des(Ol)  4 D12Des_2(Ol)  51 O. tauri  82 T. pseudonana  34 D12Des_2(Ol)  2 D12Des(Ol)  51 O. tauri  47 T. pseudonana  32

[0216] The Genes are Characterized as Follows:

[0217] To express the desaturases in yeast cells are harvested from the main cultures by centrifugation (100×g, 5 min, 20° C.) and washed with 100 mM NaHCO.sub.3, pH 8.0 to remove residual medium and fatty acids. The yeast cell sediments are extracted for 4 hours using chloroform/methanol (1:1). The resulting organic phase is extracted with 0.45% NaCl, dried with Na.sub.2SO.sub.4 and evaporated in vacuo. Applying thin-layer chromatography (horizontal tank, chloroform:methanol:acetic acid 65:35:8), the lipid extract is separated further into the lipid classes phosphatidylcholine (PC), phosphatidylinosotol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE) and neutral lipids (NL). The various separated spots on the thin-layer plate are scraped off. For the gas-chromatographic analysis, fatty acid methyl esters (FAMEs) were prepared by acid methanolysis. To this end, the cell sediments are incubated for one hour at 80° C. together with 2 ml of 1 N methanolic sulfuric acid and 2% (v/v) dimethoxypropane. The FAMEs were extracted twice with petroleum ether (PE). To remove nonderivatized fatty acids, the organic phase is washed in each case once with 2 ml of 100 mM NaHCO.sub.3, pH 8.0 and 2 ml of distilled water. Thereafter, the PE phases are dried with Na.sub.2SO.sub.4, evaporated under argon and taken up in 100 μl of PE. The samples are separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 μm, Agilent) in a Hewlett-Packard 6850 gas chromatograph equipped with flame ionization detector. The conditions for the GLC analysis are as follows: the oven temperature is programmed from 50° C. to 250° C. with a 5° C./min increment and finally 10 min at 250° C. (holding).

[0218] The signals are identified by comparing the retention times with corresponding fatty acid standards (Sigma). The methodology is described for example in Napier and Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany. 52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al., 1998, FEBS Letters. 439(3):215-218.

[0219] Activity and Substrate Determination of D5Des_2 (OI):

[0220] To determine the activity and substrate specificity of D5Des_2 (OI) SEQ ID NO: 10, various fatty acids were fed (table 7). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts reveal the synthesis of novel fatty acids, the products of the D5Des_2 (OI) reaction. This means that the gene D5Des_2 (OI) was expressed functionally.

TABLE-US-00007 TABLE 7 Feeding/conversion of different fatty acids by D5Des(Ol_2). Sample name/ Expected fatty acid fed conversion Substrate Product pYES Control — — pYES-d5Des(Ol_GA) — w/o FS pYES-d5Des(Ol_GA) 20:3->20:4ara 11.1 0.9 20:3

[0221] FIG. 8 shows the chromatograms of the individual experiments. In FIG. 8a, yeasts transformed with pYES were analyzed without the addition of fatty acids by way of control. In FIG. 8b, the pYES-transformed yeasts were fed the fatty acid 20:3Δ8,11,14. Here, the fed fatty acid can be detected in large amounts. The same experiment is carried out in FIG. 8C for yeasts transformed with the plasmid pYES-D5Des(OI_2). As opposed to FIG. 8B, it is possible to detect, in the yeasts with pYES-D5Des(OI_2), an additional fatty acid, which must be attributed to the D5Des(OI_2) activity. With reference to the activity, it is possible to characterize D5Des(OI_2) as a Δ5-desaturase.

[0222] Summary of the D5Des_2 (OI) Results:

[0223] It was possible to demonstrate in the yeast feeding experiments that the cloned gene D5Des_2 (OI) SEQ ID NO: 10 was expressed functionally and that it has desaturase activity. By reference to the fed fatty acid, it is possible to characterize D5Des_2 (OI) as a Δ5-desaturase, i.e. C20-fatty acids with a Δ8-double bond are dehydrogenated specifically in the Δ5 position.

[0224] Activity and Substrate Determination of D12Des(OI):

[0225] To determine the activity and substrate specificity of D12Des(OI) SEQ ID NO: 4, various fatty acids were fed (table 8). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts reveal the synthesis of novel fatty acids, the products of the D12Des(OI) reaction. This means that the gene D12Des(OI) was expressed functionally.

TABLE-US-00008 TABLE 8 Feeding/conversion of different fatty acids by D12Des(Ol). Sample name/ Expected fatty acid fed conversion Substrate Product pYES Control — — pYES-D12Des(Ol) 18:1->18:2 24.9 1.1 pYES-D12Des(Ol) 18:1->18:2 24.1 1.0

[0226] FIG. 9 shows the chromatograms of the individual experiments. In FIG. 9a, yeasts transformed with pYES were analyzed without the addition of fatty acids by way of control. In FIG. 9b, the yeasts transformed with pYES-D12Des(OI) were analyzed. As opposed to FIG. 9a, it is possible to detect, in the yeasts with pYES-D12Des(OI), an additional fatty acid, which must be attributed to the D12Des(OI) activity. With reference to the activity, it is possible to characterize D12Des(OI) as a Δ12-desaturase.

[0227] Summary of the D12Des(OI) Results:

[0228] It was possible to demonstrate in the yeast feeding experiments that the cloned gene D12Des(OI) SEQ ID NO: 4 was expressed functionally and that it has desaturase activity. By reference to the fatty acid spectrum, it is possible to characterize D12Des(OI) as a Δ12-desaturase, i.e. C18-fatty acids with a Δ9-double bond are dehydrogenated specifically in the Δ12 position.

[0229] Activity and Substrate Determination of D5Des(OI):

[0230] To determine the activity and substrate specificity of D5Des(OI) SEQ ID NO: 8, various fatty acids were fed (table 9). The substrates fed can be detected in large amounts in all of the transgenic yeasts. The transgenic yeasts reveal the synthesis of novel fatty acids, the products of the D5Des(OI) reaction. This means that the gene D5Des(OI) was expressed functionally.

TABLE-US-00009 TABLE 9 Conversion of various fatty acids by D5Des(Ol) Expected Conversion Sample conversion Substrate Product rate [%] d5Des(Ol_febit) 20:3n-6->20:4ara 29.4 12.4 29.6 d5Des(Ol_febit) 20:3n-6->20:4ara 19.8 10.3 34.3 d5Des(Ol_febit) 20:4n-3->20:5 nd  1.2 >50%

[0231] FIG. 10 shows the gas-chromatographic analysis of yeast feeding experiments. After expression of pYes-d5Des(OI_1) in yeast strain InvSc without the addition of fatty acids (FIG. 10A), no conversion of the existing fatty acids was detected. pYes-d5Des(OI_1) expression in yeast strain InvSc after addition of the fatty acid 20:3n-6 (B) leads to the specific conversion of 20:3n-6 into 20:4n-6 (arachidonic acid), and expression of pYes-d5Des(OI_1) in yeast strain InvSc after addition of the fatty acid 20:4n-3 (C) leads to the specific conversion of 20:4n-3 into 20:5n-3 (eicosapentaenoic acid). The specific incorporation of d5 double bonds into the fed fatty acids shows the d5-desaturase activity of d5Des(OI).

[0232] Summary of the D5Des(OI) Results:

[0233] It was possible to demonstrate in the yeast feeding experiments that the cloned gene D5Des(OI) SEQ ID NO: 8 was expressed functionally and that it has desaturase activity. By reference to the fatty acid spectrum, it is possible to characterize D5Des(OI) as a Δ5-desaturase, i.e. C20-fatty acids with a 48-double bond are dehydrogenated specifically in the Δ5 position.