EP2054509A2 - Processes for producing polyunsaturated fatty acids in transgenic organisms - Google Patents
Processes for producing polyunsaturated fatty acids in transgenic organismsInfo
- Publication number
- EP2054509A2 EP2054509A2 EP07820931A EP07820931A EP2054509A2 EP 2054509 A2 EP2054509 A2 EP 2054509A2 EP 07820931 A EP07820931 A EP 07820931A EP 07820931 A EP07820931 A EP 07820931A EP 2054509 A2 EP2054509 A2 EP 2054509A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- acid
- fatty acids
- fatty acid
- desaturase
- vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- 150000003573 thiols Chemical class 0.000 description 1
- 150000004764 thiosulfuric acid derivatives Chemical class 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 235000017468 valeriana Nutrition 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 150000004669 very long chain fatty acids Chemical class 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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- A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- 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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- 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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- 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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Definitions
- the present invention relates to polynucleotides from Ostreococcus lucimarinus which encode desaturases and elongases and can be used for the recombinant production of polyunsaturated fatty acids. Furthermore, the invention relates to vectors, host cells and transgenic non-human organisms containing the polynucleotides, as well as 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.
- Fatty acids and triacylglycerides have a variety of uses in the food, animal nutrition, cosmetics and pharmaceutical industries. Depending on whether they are free saturated and unsaturated fatty acids or triacylglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a wide variety of applications.
- Polyunsaturated fatty acids such as linoleic and linolenic acid are essential for mammals because they can not be produced by them. Therefore, polyunsaturated ⁇ -3 fatty acids and ⁇ -6 fatty acids are an important component of animal and human food.
- polyunsaturated ⁇ -3 fatty acids which are preferred in fish oils, is particularly important for food.
- the unsaturated fatty acid DHA is thereby attributed a positive effect on the development and maintenance of brain functions.
- polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA, polyunsaturated fatty acids, long chain poly unsaturated fatty acids, LCPUFA, long-chain polyunsaturated fatty acids).
- the free fatty acids are advantageously prepared by saponification.
- Common natural sources of these fatty acids are fish such as herring, salmon, sardine, perch, eel, carp, trout, halibut, mackerel, zander or tuna or algae.
- oils with saturated or unsaturated fatty acids are preferred.
- lipids with unsaturated fatty acids especially polyunsaturated fatty acids
- the polyunsaturated ⁇ -3 fatty acids thereby a positive effect on the cholesterol level in the blood and thus the possibility of preventing heart disease is attributed.
- ⁇ -3 fatty acids By adding these ⁇ -3 fatty acids to the diet, the risk of heart disease, stroke or hypertension can be significantly reduced.
- inflammatory especially chronic inflammatory processes in the context of immunological diseases such as rheumatoid arthritis can be positively influenced by ⁇ -3 fatty acids. They are therefore added to foods especially dietary foods or found in medicines application.
- ⁇ -6 fatty acids such as arachidonic acid tend to have a negative effect on these diseases in these rheumatic diseases due to our usual food composition.
- ⁇ -3- and ⁇ -6 fatty acids are precursors of tissue hormones, the so-called eicosanoids such as the prostaglandins derived from dihomo- ⁇ -linolenic acid, arachidonic acid and eicosapentaenoic acid, the thromoxanes and leukotrienes derived from arachidonic acid and Derive the eicosapentaenoic acid.
- Eicosanoids (so-called PG 2 series), which are formed from ⁇ -6 fatty acids promote in the Usually inflammatory reactions, while eicosanoids (so-called PG3 series) of ⁇ -3 fatty acids have little or no proinflammatory effect.
- ⁇ -6 desaturases are described in WO 93/06712, US Pat. No. 5,614,393, US Pat. No. 5,614,393, WO 96/21022, WO00 / 21557 and WO 99/2711 1 and also the application for production in transgenic organisms as described in WO98 / 46763 WO98 / 46764, WO9846765.
- the expression of various desaturases as described in WO99 / 64616 or WO98 / 46776 and formation of polyunsaturated fatty acids is also described and claimed. Concerning.
- microorganisms for the production of PUFAs are microorganisms such as microalgae such as Phaeodactylum tricornutum, Porphiridium species, Thraustochytria species, Schizochytria species or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor and / or mosses such as Physcomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen (1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22: 1060-1062; Akimoto, M.
- microalgae such as Phaeodactylum tricornutum, Porphiridium species, Thraustochytria species, Schizochytria species or Crypthecodinium species, ciliates such as Stylonychia or Colpidium
- EPA eicosapentaenoic acid
- DHA docosahexaenoic acid
- Chain length - instead of the ⁇ 4-desaturation, a further elongation step at 24 C, a further ⁇ 6-desaturation and finally beta-oxidation to the C 22 is carried out here.
- the so-called spokesman synthesis route is not suitable because the regulatory mechanisms are not known.
- the polyunsaturated fatty acids can be divided according to their desaturation pattern into two major classes, ⁇ -6 or ⁇ -3 fatty acids, which have metabolically and functionally different activities.
- the starting material for the ⁇ -6 pathway is the fatty acid linoleic acid (18: 2 ⁇ 9 12 ), while the ⁇ -3 pathway is via linolenic acid (18: 3 ⁇ 9 12 / I5 ).
- Linolenic acid is formed by the activity of an ⁇ -3-desaturase (Tocher et al., 1998, Prog. Lipid Res., 37, 73-117, Domergue et al., 2002, Eur. J. Biochem., 269, 4105-4113). Mammals, and therefore also humans, have no corresponding desaturase activity ( ⁇ -12 and ⁇ -3-desaturase) and must ingest these fatty acids (essential fatty acids) through the diet.
- the elongation of fatty acids by elongases by 2 or 4 C atoms is of crucial importance for the production of C 2 O or C 22 PUFAs.
- This process runs over 4 stages.
- the first step is the condensation of malonyl-CoA on the fatty acyl-CoA by ketoacyl-CoA synthase (KCS, hereinafter referred to as elongase).
- KCS ketoacyl-CoA synthase
- KCR ketoacyl-CoA reductase
- dehydratase dehydration step
- enoyl-CoA reductase enoyl-CoA reductase
- Higher plants contain polyunsaturated fatty acids such as linoleic acid (C18: 2) and linolenic acid (C18: 3).
- ARA, EPA and DHA are absent or only found in the seed oil of higher plants (E. Ucciani: Germany Dictionnaire des Huiles Vegetales, Technique & Documentation - Lavoisier, 1995. ISBN: 2-7430-0009-0).
- oilseeds such as oilseed rape, linseed, sunflower and soybeans, as this will enable large quantities of high quality LCPUFAs to be obtained inexpensively for the food, animal and pharmaceutical industries.
- gene coding for enzymes of biosynthesis must be advantageous via genetic engineering methods introduced and expressed by LCPUFAs in oilseeds.
- These genes can be advantageously isolated from microorganisms and lower plants that produce LCPUFAs and incorporate them into the membranes or triacylglycerides.
- ⁇ 6-desaturase genes from the moss Physcomitrella patens and ⁇ 6 elongase genes from P. patens and the nematode C. elegans have been isolated.
- the present invention thus relates to a polynucleotide comprising a nucleic acid sequence which is selected from the group consisting of:
- nucleic acid sequence encoding a polypeptide having an amino acid sequence as in any one of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16;
- nucleic acid sequence encoding a polypeptide that is at least 70% identical to a polypeptide encoded by the nucleic acid sequences of (a) or (b), the polypeptide having desaturase or elongase activity; and (d) a nucleic acid sequence for a fragment of a nucleic acid of (a), (b) or (c), wherein the fragment encodes a polypeptide having a desaturase or elongase activity.
- polynucleotide relates to polynucleotides comprising nucleic acid sequences encoding polypeptides having desaturase or elongase activity.
- the desaturase or elongase activities are preferably required for the biosynthesis of lipids or fatty acids. Particularly preferred are the following desaturase or elongase activities: ⁇ -4-desaturase, ⁇ -5-desaturase, ⁇ -5 elongase, ⁇ -6-desaturase, ⁇ -6 elongase or ⁇ -12-desaturase.
- the desaturases and / or elongases are preferably involved in the synthesis of polyunsaturated fatty acids (PUFAs) and more preferably in the synthesis of long-chain PUFAs (LCPUFAs).
- PUFAs polyunsaturated fatty acids
- LCPUFAs long-chain PUFAs
- Suitable detection systems for these desaturase or elongase activities are described in the examples or in WO2005 / 083053. With regard to substrate specificities and conversion rates, the abovementioned activities are particularly preferably those of the respective enzymes from Ostereococcus lucimarinus.
- the specific polynucleotides of the invention i. the polynucleotides having a nucleic acid sequence according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 were obtained from Ostereococcus lucimarinus.
- polynucleotides according to the invention are:
- Polynucleotides which encode a polypeptide having ⁇ -12-desaturase activity and (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 1 or 3, (ii) a nucleic acid sequence encoding a polypeptide as in SEQ ID NO: 2 or 4, (iii) comprise a nucleic acid sequence which is at least 83% identical to any of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acid of (i), (ii ) or (iii).
- Polynucleotides which encode a polypeptide having ⁇ -4-desaturase activity and (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 5, (ii) comprise a nucleic acid sequence coding for a polypeptide as shown in SEQ ID NO: 6, (iii) comprise a nucleic acid sequence which is at least 72% identical to one of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acid of (i), (ii) or (iii).
- Polynucleotides encoding a ⁇ 5-desaturase activity polypeptide (i) a
- Nucleic acid sequence comprise, as shown in SEQ ID NO: 7 or 9, (ii) a nucleic acid sequence encoding a polypeptide as in SEQ ID NO: 8 or 10 (iii) comprise a nucleic acid sequence which is at least 72% identical to any one of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acid of (i), (ii) or (iii) ,
- Polynucleotides which encode a ⁇ 5-elongase activity polypeptide and comprise a nucleic acid sequence as shown in SEQ ID NO: 11, (ii) comprise a nucleic acid sequence encoding a polypeptide as shown in SEQ ID NO: 12, (iii) comprise a nucleic acid sequence at least 78% identical to any one of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a nucleic acid fragment of (i), (ii) or (iii ).
- Polynucleotides which encode a polypeptide having ⁇ -6-desaturase activity and (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 13, (ii) comprise a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, (iii) comprise a nucleic acid sequence which is at least 72% identical to any one of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acid of (i), (ii) or (iii ).
- polynucleotide also encompasses variants of the aforementioned specific polynucleotides. These may be homologs, orthologues or paralogue sequences. Such varainates comprise nucleic acid sequences which have at least one base exchange, one base addition or one base deletion, wherein the variants should still encode a polypeptide having the aforementioned biological activity of the respective starting sequence.
- Varieties include polynucleotides that can hybridize with the aforementioned polynucleotides, preferably under stringent conditions. Particularly preferred stringent conditions are known in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6.
- a preferred example of stringent hybridization conditions are hybridizations in 6x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 50-65 ° C. It is known to those skilled in the art that these hybridization conditions differ with respect to the type of nucleic acid and, for example, when organic solvents are present, with respect to the temperature and the concentration of the buffer. The temperature differs, for example under "standard hybridization conditions" depending on the type of nucleic acid between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2).
- the temperature is about 42 ° C under standard conditions.
- the hybridization conditions for DNA: DNA hybrids are, for example, 0.1 x SSC and 20 0 C to 45 ° C, preferably between 30 0 C and 45 ° C.
- the hybridization conditions for DNA: RNA hybrids are, for example, 0.1 x SSC and 30 0 C to 55 ° C, preferably between 45 ° C and 55 ° C.
- conserveed sequences can be determined by sequence comparisons with polynucleotides encoding polypeptides of similar activity.
- DNA or cDNA from bacteria, fungi, plants or animals can be used as a template.
- DNA fragments obtained by the PCR can be used to screen appropriate genomic or cDNA libraries to isolate, if necessary, the complete open reading frame of the polynucleotide and to determine by sequencing.
- Other variants include polynucleotides comprising a nucleic acid sequence comprising at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%.
- At least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or at any other percentage referred to herein, is identical to any one of the aforementioned specific nucleic acid sequences and a polypeptide encodes the particular biological activity.
- polypeptide having an amino acid sequence comprising nucleic acid sequences encoding a polypeptide having an amino acid sequence, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or at any other percentage referred to herein is identical to one of aforementioned specific amino acid sequences and wherein the polypeptide has the respective biological activity of the starting sequence.
- the percentage of identical nucleotides or amino acids preferably refers to a sequence segment of at least 50% of the sequences to be compared and most preferably the full length of the sequences to be compared.
- a variety of programs that implement algorithms for such comparisons are known in the art and commercially available. In particular, reference is made to the algorithms of Needleman and Wunsch or Smith and Waterman, which provide particularly reliable results. These algorithms may preferably be implemented by the following programs: PiIeUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153), Gap and Bestfit (Needleman and Wunsch Biol. 48: 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math.
- the percentage (%) of sequence identity within the scope of the present invention is determined with the GAP program over the entire sequence with the following fixed sizes: Gap Weight: 50, Length Weight: 3, Average Match: 10,000, and Average Mismatch: 0.000.
- a polynucleotide comprising only a fragment of the aforementioned nucleic acid sequences is also a polynucleotide of the invention.
- the fragment is intended to encode a polypeptide which has the biological activity of the starting sequence or of the polypeptide encoded therefrom.
- Polypeptides encoded by such polynucleotides therefore comprise or consist of domains of the aforementioned specific polypeptides (parent polypeptides) which mediate biological activity.
- a fragment according to the invention preferably comprises at least 50, at least 100, at least 250 or at least 500 consecutive nucleotides of the abovementioned specific sequences or encodes an amino acid sequence comprising at least 20, at least 30, at least 50, at least 80, at least 100, or at least 150 consecutive amino acids of any one of the aforementioned specific amino acid sequences.
- polynucleotide variants of the invention preferably have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the respective biological activity of the polypeptide is encoded by the starting sequence.
- polypeptides encoded by the polynucleotides of the invention may be involved in the metabolism of compounds necessary for building fatty acids, fatty acid esters such as di-glycylglycerides and / or triacylglycerides in an organism, preferably in a plant or plant cell, or transporting molecules through membranes participate, wherein Ci ⁇ -, C 2 0 or C 22 carbon chains in the fatty acid molecule with double bonds at least two, preferably three, four, five or six positions are meant.
- the polynucleotides of the invention either comprise or consist of the aforementioned specific nucleic acid sequences. That is to say, the polynucleotides according to the invention may in principle also comprise further nucleotides. These may preferably be 3 ' or 5 ' untranslated regions of the genomic nucleic acid sequence. These preferably consist of at least 100, 200 or 500 nucleotides at the 5 'terminus and at least 20, 50 or 100 nucleotides at the 3' terminus of the coding region.
- Other polynucleotides comprising additional nucleic acid sequences are those encoding fusion proteins. Such fusion proteins may further encode polypeptide or polypeptide portions in addition to the aforementioned polypeptides.
- the additional polypeptide or polypeptide portion may be other enzymes of lipid or fatty acid biosynthesis.
- polypeptides which can serve as markers for expression green, yellow, red, blue-fluorescent proteins, alkaline phosphatase, etc.
- tags as markers or aid for purification (eg FLAG tags , 6-histidine tags, MYC tags, etc.).
- Polynucleotide variants can be isolated from a variety of natural or artificial sources. For example, they can be generated artificially by in vitro or in vivo mutagenesis. Homologs or orthologues of the specific sequences can be obtained from a wide variety of animals, plants or microorganisms. Preferably, they are obtained from algae.
- algae of the family Prasinophyceae as from the genera Heteromastix, Mammiella, Mantoniella, Micromonas, Nephroselmis, Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda, Pycnococcus, Pyramimonas, Scherffelia or Tetraselmis as the genera and species Heteromastix longifillis, Mamiella gilva, Mantoniella squama- ta, Micromona pusilla, Nephroselmis olivacea, Nephroselmis pyriformis, Nephroselmis rotunda, Ostreococcus tauri, Ostreococcus sp.
- the polynucleotides are derived from algae of the genera Mantonielle or Ostreococcus.
- algae such as Isochrysis or Crypthecodinium
- algae / diatoms such as Thalassiosira, Phaeodactylum or Thraustochytrium
- mosses such as Physcomitrella or Ceratodon
- algae of the genera Mantonielle or Ostreococcus or the diatoms of the genera Thalassiosira or Crypthecodinium most preferably the algae of the genera Mantonielle or Ostreococcus or the diatoms of the genera Thalassiosira or Crypthecodinium.
- the polynucleotides may also preferably be obtained from higher plants such as the primulaceae such as Aleuritia, Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides, microorganisms such as fungi such as Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella, bacteria such as Shewanella, yeasts or animals such as Nematodes eg Caenorhabditis, insects or fish.
- the polynucleotide variants are also preferably derived from an animal of the vertebrate order.
- the polynucleotides are of the vertebrate class; Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus and, most preferably, from the order of Salmoniformes such as the family Salmonidae such as the genus Salmo, for example, from the genera and species Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario.
- the polynucleotides according to the invention can in this case be isolated by means of standard molecular biological techniques and the sequence information provided here.
- a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level can be identified. These may be used as a hybridization probe as well as standard hybridization techniques (such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CoId Spring Harbor Laboratory, ColD Spring Harbor Laboratory Press, ColD Spring Harbor, NY, 1989) Isolation of other useful in the process of nucleic acid sequences can be used.
- polynucleotides or fragments thereof can be isolated by polymerase chain reaction (PCR) using oligonucleotide primers based on this sequence or portions thereof (eg, a nucleic acid molecule comprising the complete sequence or a portion thereof can be polymerase chain-reaction utilized isolated from oligonucleotide primers prepared on the basis of this same sequence).
- PCR polymerase chain reaction
- mRNA can be isolated from cells (eg, by the guanidinium thiocyanate extraction method of Chirgwin et al.
- oligonucleotide primers for amplification by polymerase chain reaction can be prepared on the basis of the amino acid sequences shown in the SEQ ID numbers.
- a nucleic acid of the invention may be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques.
- the thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis.
- Oligonucleotides corresponding to a desaturase nucleotide sequence can be prepared by standard synthetic methods, for example with an automated DNA synthesizer.
- the polynucleotides of the invention may be provided either as isolated polynucleotides (i.e., isolated from their natural origin, e.g., the genomic locus) or in genetically altered form (i.e., the polynucleotides may also be present at their natural genetic locus, but must then be genetically engineered).
- An isolated polynucleotide preferably comprises less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequence naturally present in its vicinity.
- the polynucleotide according to the invention can be in the form of a single-stranded or double-stranded nucleic acid molecule and can be genomic DNA, cDNA or RNA.
- the polynucleotides of the invention include all orientations of the sequences shown in the SEQ ID Nos., I. also complementary strands and reverse or reverse-complementary orientations. Further, the term also includes chemically modified nucleic acids, such as the naturally occurring methylated DNA molecules, or artificial nucleic acids, e.g. biotinylated nucleic acids.
- the invention also encompasses oligonucleotides of at least 15 bp, preferably at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, or at least 50 bp, which can specifically hybridize with one of the aforementioned polynucleotides under stringent conditions.
- the oliguncleotides may consist of DNA or RNA or both.
- Such oligonucleotides can be used as primers for PCR, as expression-inhibiting, antisense oligonucleotides, for RNA interference (RNAi) or for chimeric or genomic approaches.
- RNAi RNA interference
- RNAi methods are described, for example, in Fire et al., Nature (1998) 391: 806-811; Fire, Trends Genet. 15, 358-363 (1999); Sharp, RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond et al. Nature Rev. Genet. 2, 11 10-11 19 (2001); Tuschl, Chem. Biochem. 2, 239-245 (2001); Hamilton et al., Science 286, 950-952 (1999); Hammond et al., Nature 404, 293-296 (2000); Zamore et al., Cell 101, 25-33 (2000); Bernstein et al., Nature 409, 363-366 (2001); Elbashir et al., Genes Dev.
- the polynucleotides according to the invention can be used particularly effectively for the recombinant production of polyunsaturated fatty acids in host cells and transgenic organisms.
- the proteins encoded by the polynucleotides of the invention encode polypeptides with ⁇ - 12 desaturase, ⁇ -4-desaturase, ⁇ -5 desaturase, ⁇ -5-elongase, ⁇ -6-desaturase and ⁇ -6-elongase activity can Ci 8 -, C 2 O and C 22 fatty acids with one, two, three, four or five double bonds, and preferably polyunsaturated Ci 8 fatty acids with one, two or three double bonds, such as C18: 1 ⁇ 9, C18: 2 ⁇ 9 '12 or C18: 3 ⁇ 9 ' 12 ' 15 , polyunsaturated C 2 o fatty acids having three or four double bonds such as C20: 3 ⁇ 8 ' 11 ' 14 or C20: 4
- the fatty acids are desaturated in phospholipids or CoA fatty acid esters, advantageously in the CoA fatty acid esters.
- a simple, inexpensive production of these polyunsaturated fatty acids is possible especially in eukaryotic systems.
- the unsaturated fatty acids prepared by means of the polynucleotides of the invention can then be formulated as oil, lipid and fatty acid compositions and used accordingly.
- the present invention further relates to a vector comprising the polynucleotide of the invention.
- vector denotes a nucleic acid molecule which can transport another nucleic acid molecule, such as the polynucleotides according to the invention, to which it is bound.
- vector is a circular double-stranded plasmid
- DNA loop can be ligated into the additional DNA segments.
- Another vector type is a viral vector, with additional DNA segments inserted into the viral vector
- Genome can be ligated.
- Certain vectors can autonomously replicate in a host cell into which they have been introduced (eg bacterial vectors with bacterial learn replication origin). Other vectors are advantageously integrated into the genome of a host cell upon introduction into the host cell and thereby replicated together with the host genome.
- certain vectors may direct the expression of genes to which they are operably linked. These vectors are also referred to herein as expression vectors.
- expression vectors suitable for recombinant DNA techniques are in the form of plasmids.
- the terms plasmid and vector can be used interchangeably since the plasmid is the most commonly used vector form.
- the invention is intended to encompass these other forms of expression vectors, such as viral vectors that perform similar functions.
- vector is also intended to encompass other vectors known to those skilled in the art, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA, artificial chromosomes.
- phages viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA, artificial chromosomes.
- viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA, artificial chromosomes.
- constructs for targeted, ie homologous recombination, or heterologous insertion of polynucleotides are examples of polynucleotides.
- Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
- transformation and “transfection”, conjugation and transduction are intended to encompass a variety of methods known in the art for introducing foreign nucleic acid (eg DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE- Dextran-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment.
- Suitable methods for transforming or transfecting host cells, including plant cells can be found in Sambrook et al.
- Suitable cloning vectors are well known to those skilled in the art. These include, in particular, vectors which can be replicated in microbial systems, ie in particular vectors which ensure efficient cloning in yeasts or fungi, and which enable stable transformation of plants. Particular mention should be made of various binary and co-integrated vector systems suitable for T-DNA-mediated transformation. Such vector systems are usually characterized in that they contain at least the vir genes required for the Agrobacterium-mediated transformation as well as the T-DNA limiting sequences (T-DNA border). Preferably, these vector systems also comprise further cis-regulatory regions such as promoters and terminators and / or selection markers. ker, with which appropriately transformed organisms can be identified.
- binary systems are based on at least two vectors, one of them vir genes, but no T-DNA and a second T-DNA, but no carries vir gene.
- these binary vectors include vectors of the series pBI B-HYG, pPZP, pBecks, pGreen. Bin19, pB1101, pBinAR, pGPTV and pCAMBIA are preferably used according to the invention.
- the vectors with the inserted polynucleotides according to the invention can be stably propagated in microorganisms, in particular Escherichia coli and Agrobacterium tumefaciens, under selective conditions and enable a transfer of heterologous DNA into plants or microorganisms.
- the polynucleotides according to the invention can be introduced into organisms such as microorganisms or plants and thus be used for plant transformation. Suitable vectors for this are published in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp.
- the vector is an expression vector.
- the polynucleotide is in operative (ie, functional) connection to an expression control sequence.
- the expression control sequence together with the polynucleotide and optionally further sequence elements of the vector is also referred to as expression cassette.
- the expression co-rolling sequence ensures that the polynucleotide can be expressed in a host cell after transformation or transfection.
- the expression control sequence to be used preferably contains cis-regulatory elements such as promoter and / or enhancer nucleic acid sequences which are recognized by the transcription machinery of the host cells.
- the term also includes other expression control elements such as polyadenylation signals and RNA stabilizing sequences.
- the polynucleotides according to the invention may be present in one or more copies in the expression cassette or the expression vector according to the invention (for example in the form of several expression cassettes).
- the regulatory sequences or factors can, as described above, preferably positively influence the gene expression of the introduced genes and thereby increase them.
- enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
- an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
- Further expression control sequences for the purposes of the present invention are translation terminators at the 3 'end of the polynucleotides to be translated.
- the OCS1 terminator can be used here.
- a different terminator sequence should be used here for each polynucleotide to be expressed.
- Preferred expression control sequences or regulatory sequences are present in promoters, such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3 , gal, trc, ara, SP6, ⁇ -PR or ⁇ -PL promoter and are advantageously used in Gram-negative bacteria.
- 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 CaMV / 35S [ Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter.
- inducible promoters such as those in EP-AO 388 186 (benzylsulfonamide-inducible), Plant J.
- EP-AO 335 528 Abzisinic inducible
- WO 93/21334 ethanol or cyclohexenol inducible promoters.
- Further suitable plant promoters are the promoter of cytosolic FBPase or the ST LSI promoter of the potato (Stockhaus et al., EMBO J. 8, 1989, 2445), the glycine max phosphoribosylprophosphate amidotransferase promoter (Genbank Accession No. U87999) or the nodia-specific promoter described in EP-A-0 249 676.
- promoters which allow expression in tissues involved in fatty acid biosynthesis.
- seed-specific promoters such as the USP Promoter but also other promoters such as the LeB4, DC3, phaseolin or napin promoter.
- Further particularly advantageous promoters are seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and in US Pat. No. 5,608,152 (napin promoter from rapeseed), WO 98/45461 (oleosin promoter from Arobidopsis), US Pat. No.
- the polynucleotides of the present invention should preferably be seed-specifically expressed in oilseeds.
- seed-specific promoters can be used, or such promoters that are active in the embryo and / or in the endosperm.
- seed-specific promoters can be isolated from both dicotolydone and monocotolydone plants.
- acyl Carrier protein [US 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO 98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [US 5,504,200], Bce4 [WO 91/13980] , Legumes B4 (LegB4 promoter) [Bäumlein et al., Plant J., 2,2, 1992], Lpt2 and lpt1 (barley) [WO 95/15389 u. WO95 / 23230], seed-specific promoters from rice, maize and the like.
- Plant gene expression can also be facilitated by a chemically inducible promoter (see review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108).
- Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
- each of the polynucleotides of the invention should be expressed under the control of their own preferred, a different promoter, since repeating sequence motifs can lead to instability of the T-DNA or to recombination events.
- the expression cassette is advantageously constructed in such a way that a promoter is followed by a suitable interface for insertion of the nucleic acid to be expressed, advantageously followed by a terminator behind the polylinker in a polylinker.
- This sequence is repeated several times, preferably three, four or five times, so that up to five genes can be brought together in one construct and thus introduced into the transgenic plant for expression.
- the sequence is repeated up to three times.
- each nucleic acid sequence has its own promoter and optionally its own terminator.
- Such advantageous constructs are disclosed for example in DE 10102337 or DE 10102338.
- the insertion site or the sequence of the inserted nucleic acids in the expression cassette is not of decisive importance, that is to say a nucleic acid sequence can be inserted at the first or last position in the cassette, without this significantly influencing the expression.
- the recombinant expression vectors used may be designed for expression in prokaryotic or eukaryotic cells. This is advantageous since intermediate steps of the vector construction are often carried out in microorganisms for the sake of simplicity.
- the ⁇ 12-desaturase, ⁇ 6-desaturase, ⁇ 6-elongase, ⁇ 5-desaturase, ⁇ 5-elongase and / or ⁇ -4-desaturase genes can be expressed in bacterial cells , Insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA, et al., 1992 "Foreign gene expression in yeast: a review", Yeast 8: 423-488, van den Hondel, CAMJJ, et al.
- Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- the recombinant expression vector may alternatively be transcribed and translated in vitro using, for example, T7 promoter regulatory sequences and T7 polymerase.
- fusion expression vectors include pGEX (Pharmacia Biotech Ine, Smith, DB, and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ). in which glutathione S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.
- GST glutathione S-transferase
- suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al.
- Target gene expression from the pTrc vector is based on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter.
- Target gene expression from the pET 11 d vector is based on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1).
- This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident ⁇ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
- Other suitable vectors in prokaryotic organisms are known to those skilled in the art, these vectors are for example in E.
- the pBR series such as pBR322
- the pUC Series such as pUC18 or pUC19
- the M1 13mp series pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24
- pLG200
- the expression vector is a yeast expression vector.
- yeast expression vectors for expression in the yeast S. cerevisiae include pYeDesaturased (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 pYES2 (Invitrogen Corporation, San Diego, CA).
- Vectors and methods for constructing vectors suitable for use in other fungi, such as filamentous fungi include those described in detail in: van den Hondel, C.A.M.J.J., & Punt, PJ.
- yeast vectors are, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.
- polynucleotides of the present invention may also be expressed in insect cells using baculovirus expression vectors.
- Baculovirus vectors that are available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and U.S. Pat pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
- Preferred plant expression vectors include those described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border Biol. 20: 1 195-1 197; and Bevan, MW (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 871 1-8721; Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, pp. 15-38.
- a plant expression cassette preferably contains expression control sequences which can direct gene expression in plant cells and are operably linked so that each sequence can fulfill its function, such as termination of transcription, for example polyadenylation signals.
- Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACH ⁇ known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835ff.) Or functional equivalents thereof, as well all other terminators functionally active in plants are suitable.
- a plant expression cassette preferably contains other operably linked sequences such as translation enhancers such as the overdrive sequence containing the tobacco mosaic virus 5 'untranslated leader sequence which increases the protein / RNA ratio (Gallie et al , 1987, Nucl. Acids Research 15: 8693-871 1).
- Plant gene expression, as described above, must be operably linked to a suitable promoter that performs gene expression in a timely, cell or tissue-specific manner.
- Useful promoters are constitutive promoters (Benfey et al., EMBO J.
- telomeres are preferred sequences necessary to direct the gene product into its corresponding cell compartment (see review in Kermode, Crit., Plant, 15, 4 (1996) 285) -423 and references cited therein), for example to the vacuole, the nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes, and other compartments of plant cells.
- plastids such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes, and other compartments of plant cells.
- Plant gene expression can also be facilitated by a chemically inducible promoter as described above (see review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108).
- Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
- Promoters which react to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp ⁇ O promoter Tomato (US 5,187,267), the potato inducible alpha-amylase promoter (WO 96/12814) or the wound inducible pinI I promoter (EP-A-0 375 091).
- the pathogen-induced PRP1 gene promoter Ward et al., Plant Mol. Biol. 22 (1993) 361-366
- the heat-inducible hsp ⁇ O promoter Tomato US 5,187,267
- the potato inducible alpha-amylase promoter WO 96/1281
- the wound inducible pinI I promoter EP-A-0 375 091
- promoters which induce gene expression in tissues and organs in which the fatty acid, lipid and oil biosynthesis takes place in sperm cells such as the cells of the endosperm and the developing embryo.
- Suitable promoters are the rapeseed napkin promoter (US 5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), the Arabidopsis oleosin promoter (US Pat. WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US Pat. No.
- Bce4 promoter Brassica WO 91/13980
- legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9)
- promoters expressing seed-specific expression in monocotyledonous plants such as maize To bring in barley, wheat, rye, rice, etc.
- Suitable noteworthy promoters are the lpt2 or lpt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene , the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene).
- promoters which induce plastid-specific expression since plastids are the compart- ment in which the precursors as well as some end products of lipid biosynthesis are synthesized.
- Suitable promoters such as the viral RNA polymerase promoter, are described in WO 95/16783 and WO 97/06250, and the cIpP promoter from Arabidopsis described in WO 99/46394.
- the expression vector may, as described above, also include other genes to be introduced into the organisms. It is possible and preferred to introduce into the host organisms regulatory genes, such as genes for inducers, repressors or enzymes, which intervene by their enzyme activity in the regulation of one or more genes of a biosynthetic pathway, and to express therein. These genes may be of heterologous or homologous origin. Heterologous genes or polynucleotides are derived from a parent organism that differs from the target organism into which the genes or polynucleotides are to be introduced. For homologous genes or polynucleotides, the target organism and the parent organism are the same.
- the vector comprises at least one further polynucleotide encoding a further enzyme involved in the biosynthesis of lipids or fatty acids.
- CoA lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (s), Fatty acid desaturase (s), fatty acid acetylenase (s), lipoxygenase (s), triacylglycerol lipase (s), allene oxide synthase (s), hydroperoxide lyase (s), fatty acid elongase (s), ⁇ 4-desaturase (n), ⁇ 5-desaturase (s), ⁇ 6-desaturase (s), ⁇ 8-desaturase (s), ⁇ 9-desaturase (s), ⁇ 12-desaturase (s), ⁇ 5-elongase (s), ⁇ 6-elongase ( n) and ⁇ 9 elongase (
- the invention also relates to a host cell comprising the polynucleotide or vector of the invention.
- Host cells may in principle be any eukaryotic or prokaryotic cells. They may be primary cells from animals, plants or multicellular microorganisms, e.g. from those mentioned elsewhere in the description. Furthermore, the term also includes cell lines that can be obtained from these organisms.
- host cells according to the invention may also be unicellular microorganisms, for example bacteria or fungi.
- Particularly preferred microorganisms are fungi selected from the family of Chaetomiaceae, Choanephoraceae, Cryptococceae, Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae or Tuberculariaceae.
- Choanephoraceae such as the genera Blakeslea, Choanephora eg the genera and species Blakeslea trispora, Choanephora cucurbiturum, Choanephora infundibulifera var.
- Mortierellaceae such as the genus Mortierella eg the genera and species Mortierella isabellina, Mortierella polycephala , Mortierella ramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae such as the genera Phytium, Phytophthora eg the genera and species Pythium debaryanum, Pythium intermedium, Irregular Pythium, Pythium megalacanthum, Pythium paro-candrum, Pythium sylvaticum, Pythium ultimum, Phytophthora cactorum, Phytophthora cinnamomi , Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, Phytophthora erythroseptica, Phytophthora lateralis, Phytophthora mega
- Minuta Pichia minuta var nonfermentans, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia philodendri, Pichia pini, Pichia polymorpha, Pichia quercuum , Pichia rhodanensis, Pichia sargentensis, Pichia stipitis, Pichia strasburgensis, Pichia subpelliculosa, Pichia toletana, Pichia trehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti, Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces bisporus, Saccharomyces capensis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae , Saccharomyces cerevisiae var.
- pombe Thraustochytriaceae as the genera Althornia, Aplanochytrium, Japonochytrium, Schizochytrium, Thraustochytrium eg the species Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium mangrovei, Schizochytrium minutum, Schizochytrium octosporum, Thraustochytrium aggregatum, Thraustochytrium amoeboideum, Thraustochytrium antacticum, arudimentale Thraustochytrium, Thraustochytrium aureum, benthicola Thraustochytrium, Thraustochytrium globosum, Thraustochytrium indicum, Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytrium multi Rudi mental, Thraustochytrium pachyder-
- bacteria selected from the group of the families Bacillaceae, Enterobacteriacae or Rhizobiaceae are particularly preferred. Particularly preferred are the following bacteria selected from the group: Bacillaceae such as the genus Bacillus eg the genera and species Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus sphaericus subsp.
- Bacillaceae such as the genus Bacillus eg the genera and species Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagul
- Enterobacteriacae such as the genera Citrobacter, Edwardsieila, Enterobacter, Erwinia, Escherichia, Klebsiella, Salmonella or Serratia eg the genera and species Citrobacter amalonaticus, Citrobacter diversus, Citrobacter freundii, Citrobacter genomospecies, Citrobacter gillenii, Citrobacter intermedium, Citrobacter koseri, Citrobacter murliniae, Citrobacter sp , Edwardsiella hoshinae, Edwardsiella ictaluri, Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwiniaananatis, Erwinia aphidicola, Erwinia billingiae, Erwinia cacticida, Erwinia carcinogena, Erwinia carnegieana, Erwinia caro
- Escherichia coli mutabile Escherichia fergusonii
- Escherichia hermannii Escherichia sp.
- Escherichia vulneris Escherichiella aerogenes
- Klebsiella edwardsii subsp. atlantae Klebsiella ornithinolytica
- Klebsiella oxytoca Klebsiella planticola
- Klebsiella pneumoniae Klebsiella pneumoniae subsp.
- Salmonella daressalaam Salmonella enterica subsp. houtenae, Salmonella enterica subsp. salamae, Salmonella enteritidis, Salmonella gallinarum, Salmonella heidelberg, Salmonella panama, Salmonella senftenberg, Salmonella typhimurium, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia marcescens, Serratia marcescens subsp.
- Rhizobiaceae such as the genera Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium eg the genera and species Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium gelatinovorum, Agrobacterium larrymoorei, Agrobacterium meteori, Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi, Agrobacterium stellulatum, Agrobacterium tumefaciens, Agrobacterium vitis, Carbophilus carboxidus, Chelatobacter heintzii
- Useful host cells are also mentioned in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- Useful expression strains e.g. those which have lower protease activity are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.
- These include plant cells and certain tissues, organs and parts of plants in all their forms such as anthers, fibers, root hairs, stems, embryos, cilia, kotelydones, petioles, crops, plant tissue, reproductive tissue and cell cultures transgenic plant is derived and / or can be used to produce the transgenic plant.
- Polynucleotides or vectors may be introduced into the host cell using transformation or transfection techniques known in the art, as previously described. Conditions and media for the cultivation of the host cells are also known to the person skilled in the art.
- the host cell according to the invention preferably additionally comprises at least one further enzyme, which is involved in the biosynthesis of lipids or fatty acids.
- Enzyme can be present endogenously in the host cell, ie the host cell already naturally expresses a gene which codes for a corresponding enzyme.
- a heterologous polynucleotide may be introduced into the host cell encoding the enzyme. Suitable methods and measures for the expression of a Heterologous polynucleotides are known in the art and described herein in connection with the polynucleotides, vectors and host cells of the present invention.
- the invention also relates to a method for producing a polypeptide having desaturase or elongase activity comprising the steps:
- the polypeptide can be obtained by all common methods for protein purification.
- the methods include, for example, affinity chromatography, molecular sieve chromatography, high-pressure liquid chromatography or protein precipitation optionally with specific antibodies.
- the method need not necessarily provide a pure preparation of the polypeptide.
- the invention thus also relates to a polypeptide which is encoded by the polynucleotide according to the invention or which is obtainable by the abovementioned method according to the invention.
- polypeptide refers to both a substantially pure polypeptide and a polypeptide preparation that contains other components or contaminants.
- the term is also used for fusion proteins or protein aggregates comprising the polypeptide of the invention and additionally further components.
- the term also refers to chemically modified polypeptides. Chemical modifications in this context include artificial modifications or naturally occurring modifications, eg, post-translational modifications such as phosphorylation, myristylation, glycosylation, etc.
- polypeptide, peptide, or protein are interchangeable and are used accordingly in the description and in the prior art.
- polypeptides according to the invention have the abovementioned biological activities, ie desaturase or elongase activities, and can influence the biosynthesis of polyunsaturated fatty acids (PUFAs), preferably the long-chain PUFAs (LCPUFAs), as described herein.
- PUFAs polyunsaturated fatty acids
- LCPUFAs long-chain PUFAs
- the invention also encompasses an antibody which specifically recognizes the polypeptide according to the invention.
- Antibodies to the polypeptide of the invention may be prepared by known methods using purified polypeptide or fragments thereof with appropriate epitopes as the antigen. Suitable epitopes can be determined by known antigenicity determination algorithms based on the amino acid sequences of the polypeptides of the invention provided herein. The corresponding polypeptides or fragments can then be synthesized or recombinantly recovered. After immunization of animals, preferably mammals e.g. Rabbits, rats or mice, the antibodies can then be recovered from the serum by known methods. Alternatively, monoclonal antibodies or antibody fragments may be provided by known methods; see, e.g. Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Colard Spring Harbor, 1988, or Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3.
- the antibodies are preferably monoclonal or polyclonal antibodies, single-chain antibodies or chimeric antibodies and fragments thereof such as Fab, Fv or scFv. Further antibodies within the meaning of the invention are bispecific antibodies, synthetic antibodies or their chemically modified derivatives.
- the antibodies of the invention are intended to specifically recognize the polypeptides of the invention, i. they should not crossreact with other proteins to any significant extent. This can be tested by methods known in the art.
- the antibodies can be used, for example, for immunoprecipitation, for immunohistochemistry or for protein purification (for example affinity chromatography).
- the invention further relates to a transgenic non-human organism comprising the polynucleotide, the vector or the host cell of the present invention.
- the transgenic, non-human organism is an animal, a plant or a multicellular microorganism.
- transgenic is to be understood as meaning that a heterologous polynucleotide, ie a polynucleotide which does not naturally occur in the particular organism, is introduced into the organism, which can be achieved either by random insertion of the polynucleotide or by homologous recombination it is also possible to introduce the vector according to the invention instead of the polynucleotide become.
- Methods for introducing polynucleotides or vectors for random insertion or homologous recombination are known in the art and also described in more detail below.
- Host cells containing the polynucleotide or vector may also be introduced into an organism to produce a transgenic organism. However, such an organism is then a chimeric organism in which only the cells which are derived from the introduced cells are transgenic, ie comprise the heterologous polynucleotide.
- transgenic non-human organisms oil-producing organisms that is, used for the production of oils, such as fungi such as Mortierella or Thraustochytrium, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylum or plants.
- oils such as fungi such as Mortierella or Thraustochytrium, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylum or plants.
- all plants can be used as transgenic plants, ie both dicotyledonous and monocotyledonous plants.
- they are oilseed crops containing high levels of lipid compounds such as peanut, rapeseed, canola, sunflower, safflower (Carthamus tinctoria), poppy, mustard, hemp, castor, olive, sesame, calendula, punica, evening primrose, mullein, thistle , Wild roses, hazelnut, almond, macadamia, avocado, bay leaf, pumpkin, flax, soy, pistachio, borage, trees (oil palm, coconut or walnut) or crops such as corn, wheat, rye, oats, triticale, rice, barley, cotton , Cassava, pepper, tagetes, solanaceae plants such as potato, tobacco, aubergine and tomato, Vicia species, pea, alfalfa or bush plants (coffee, cocoa, tea), Salix species and perennial grass
- Preferred plants according to the invention are oil crop plants, such as peanut, rapeseed, canola, sunflower, safflower, poppy, mustard, hemp, castor, olive, calendula, punica, evening primrose, pumpkin, flax, soy, borage, trees (oil palm, coconut).
- Particularly preferred are C18: 2 and / or C18: 3 fatty acid rich plants such as sunflower, safflower, tobacco, mullein, sesame, cotton, pumpkin, poppy, evening primrose, walnut, flax, hemp, thistle or safflower.
- plants such as safflower, sunflower, poppy, evening primrose, walnut, flax or hemp.
- plants which are able to synthesize fatty acids as all dicotyledonous or monocotyledonous plants, algae or mosses.
- Advantageous plants are selected from the group of the plant families Adelotheciaceae, Anacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae, Cannabaceae, Convolvulceae, Chenopodiaceae, Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagaceae, Ericaceae, Euphorbiaceae, Fabaceae , Geraniaceae, Gramineae, Juglandaceae, Lauraceae, Leguminosae, Linaceae, Prasinophyceae or vegetables or ornamental plants such as Tagetes.
- Adelotheciaceae such as the genera Physcomitrella eg the genus and species Physcomitrella patens
- Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium eg the genus and species Pistacia vera [pistachio], Mangifer indica [ Mango] or Anacardium occidentale [Cashew]
- Asteraceae such as the genera Calendula, Carthus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana eg the genus and species Calendula officinalis [Gardening Marigold], Carthamus tinctorius [ Safflower], Centaurea cyanus [cornflower], Cichorium intybus [chicory], Cynara scolymus [Artichoke], Helianthus annus [sunflower], Lactuca sativa, Lact
- [Cayenne pepper] such as the genera Hordeum, Seeale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea (maize), Triticum eg the genera and species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeum hexastichonum, Hordeum hexastichum, Hordeum irregular, Hordeum sativum, Hordeum secalinum [barley], Seeale cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.
- Multicellular microorganisms that can be used as transgenic non-human organisms are preferably protists or diatoms selected from the group of the family Dinophyceae, Turaniellidae or Oxytrichidae such as the genera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum, Stylonychia mytilus, Stylonychia pustulata, Stylonychia putrina , Stylonychia notophora, Stylonychia sp., Colpidium campylum or Colpidium sp.
- protists or diatoms selected from the group of the family Dinophyceae, Turaniellidae or Oxytrichidae such as the genera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum, Stylonychia mytilus, Stylonychia pustulata, Stylonychia putrina
- the invention relates to a process for producing a substance having the structure shown in the following general formula I.
- R 1 hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol,
- Lysophosphatidylserine lysophosphatidylinositol, sphingolase or a radical of formula II
- R 2 hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol or saturated or unsaturated C 2 -C 24 -alkylcarbonyl,
- R 3 hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl, or R 2 and R 3 are independently of one another a radical of the formula Ia:
- the method comprises culturing (i) a host cell of the invention or (ii) a transgenic non-human of the invention under conditions permitting biosynthesis of the substance.
- the aforementioned substance is provided in an amount of at least 1% by weight based on the total content of the lipids in the host cell or the transgenic organism.
- R 1 in general formula I denotes hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lyso-phosphatidylethanolamine, lyso-phosphatidylglycerol, lyso-diphosphatidylglycerol, lyso-phosphatidylserine, lyso-phosphatidylinositol, sphingobase, or a radical of the general formula II
- R 1 are always bonded in the form of their thioesters to the compounds of general formula I.
- R 2 in the general formula II denotes hydrogen, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, lyso-phosphatidylglycerol, lyso-diphosphatidylglycerol, lyso-phosphatidylserine, lyso-phosphatidylinositol or saturated or unsaturated C 2 -C 24 - alkylcarbonyl.
- Suitable alkyl radicals are substituted or unsubstituted, saturated or unsaturated C 2 -C 24 -alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl, n- Octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl, n- Octadecylcarbonyl, n-nonadecylcarbonyl,
- C 1 -C 22 -alkylcarbonyl radicals such as n-decylcarbonyl, n- Undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n- Docosanylcarbonyl or n-tetracosanylcarbonyl- containing one or more double bonds are preferred.
- saturated and / or unsaturated C 0 -C 22 - radicals such as Cio-alkylcarbonyl, Cn-alkylcarbonyl, Ci2-alkylcarbonyl, Ci 3 - alkylcarbonyl, Ci 4 -alkylcarbonyl, C 6 alkylcarbonyl, Ci ⁇ -alkylcarbonyl, C 20 - alkylcarbonyl or C 22 -alkylcarbonyl radicals which contain one or more double bonds.
- C 1 -C 22 -alkylcarbonyl radicals such as C 1 -C 6 -alkylcarbonyl, C 1-6 -alkylcarbonyl, C 20 -alkylcarbonyl or C 22 -alkylcarbonyl radicals which contain one or more double bonds.
- These advantageous radicals may contain two, three, four, five or six double bonds.
- the particularly advantageous radicals having 20 or 22 carbon atoms in the fatty acid chain contain up to six double bonds, advantageously three, four, five or six double bonds, more preferably five or six double bonds. All these radicals are derived from the corresponding fatty acids.
- R 3 in the general formula II is hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl.
- Suitable alkyl radicals are substituted or unsubstituted, saturated or unsaturated C 2 -C 24 -alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl, n- Octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-pentadecylcarbonyl, n-
- C 1 -C 22 -alkylcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl , n-octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl containing one or more double bonds are preferred.
- C 1 -C 22 -alkylcarbonyl radicals such as C 1 -C 6 -alkylcarbonyl, C 1-6 -alkylcarbonyl, C 20 -alkylcarbonyl or C 22 -alkylcarbonyl radicals which contain one or more double bonds.
- These Advantageous radicals may contain two, three, four, five or six double bonds.
- the particularly advantageous radicals having 20 or 22 carbon atoms in the fatty acid chain contain up to six double bonds, advantageously three, four, five or six double bonds, more preferably five or six double bonds. All these radicals are derived from the corresponding fatty acids.
- R 1 , R 2 and R 3 may be substituted by hydroxyl and / or epoxy groups and / or may contain triple bonds.
- the polyunsaturated fatty acids prepared in the process according to the invention contain at least two, advantageously three, four, five or six double bonds. Particularly advantageously, the fatty acids contain four five or six double bonds.
- Fatty acids produced in the process advantageously have 18, 20 or 22 carbon atoms in the fatty acid chain, preferably the fatty acids contain 20 or 22 carbon atoms in the fatty acid chain.
- saturated fatty acids are little or not reacted with the nucleic acids used in the process. Little is understood to mean that compared to polyunsaturated fatty acids, the saturated fatty acids have less than 5% of the activity, advantageously less than 3%, more preferably less than 2%, most preferably less than 1; 0.5; 0.25 or 0.125% are implemented. These produced fatty acids can be produced as the only product in the process or present in a fatty acid mixture.
- the substituents R 2 or R 3 in the general formulas I and II independently of one another denote saturated or unsaturated C 8 -C 22 -alkylcarbonyl, particularly advantageously independently they denote unsaturated C 8 -, C 2 o- or C 22 -alkylcarbonyl - with at least two double bonds.
- the polyunsaturated fatty acids produced in the process are advantageously bound in membrane lipids and / or triacylglycerides, but may also be present as free fatty acids or bound in the form of other fatty acid esters in the organisms. They may be present as "pure products" or advantageously in the form of mixtures of different fatty acids or mixtures of different glycerides.
- the different fatty acids bound in the triacylglycerides can thereby be derived from short-chain fatty acids having 4 to 6 C atoms, medium-chain fatty acids having 8 to 12 C atoms or long-chain fatty acids having 14 to 24 C atoms, preferably the long-chain fatty acids are particularly preferred the long-chain fatty acids LCPUFAs of Ci 8 , C 2 o and / or C 22 fatty acids.
- the fatty acid ester with polyunsaturated C 8 -, C 2 0 and / or C 22 -Fettklare- molecules can be isolated from the organisms which have been used for the production of the fatty acid ester, in the form of an oil or lipid, for example in the form of compounds such as sphingolipids, Phosphoglycerides, lipids, glycolipids such as glycosphingolipids, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl-coenzymeA esters which contain at least two polyunsaturated fatty acids Preferably, they are isolated in the form of their diacylglycer
- the polyunsaturated fatty acids are also included as free fatty acids or bound in other compounds in the organisms beneficial to the plants.
- the various abovementioned compounds (fatty acid esters and free fatty acids) in the organisms have an approximate distribution of 80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10% by weight of monoglycerides , 1 to 5 wt .-% of free fatty acids, 2 to 8 wt .-% phospholipids ago, wherein the sum of the various compounds to 100 wt .-% complements.
- the LCPUFAs produced are present in a content of at least 3% by weight, advantageously of at least 5% by weight, preferably of at least 8% by weight, more preferably of at least 10% by weight, very particularly preferably at least 15 wt .-% based on the total fatty acids in the transgenic organisms advantageously produced in a transgenic plant.
- advantageously Ci 8 - and / or C 2 o fatty acids in the host organisms are present, at least 10%, advantageously at least 20%, more preferably at least 30%, most preferably at least 40% in the corresponding products such as DPA or DHA, to name only two exemplified implemented.
- the fatty acids are prepared in bound form.
- these unsaturated fatty acids can be brought to the sn1, sn2 and / or sn3 position of the advantageously prepared triglycerides.
- the starting compounds linoleic acid (C18: 2) or linolenic acid (C18: 3) undergo several reaction steps in the process according to the invention, the end products of the process, such as, for example, arachidonic acid (ARA), eicosapentaenoic acid (EPA), ⁇ -6-docosapentaenoic acid or DHA, are precipitated not as absolute pure products, there are always also small traces of precursors in the final product included.
- ARA arachidonic acid
- EPA eicosapentaenoic acid
- DHA ⁇ -6-docosapentaenoic acid
- the end products such as ARA, EPA or DHA are present as mixtures.
- the precursors should advantageously not more than 20 wt .-%, preferably not more than 15 wt .-%, more preferably not more than 10 wt .-%, most preferably not more than 5 wt .-% based on the amount of the respective Final product amount.
- ARA, EPA or only DHA are bound in the process according to the invention or produced as free acids in a transgenic plant as end products.
- the compounds ARA, EPA and DHA are prepared simultaneously, they are advantageously used in a ratio of at least 1: 1: 2 (EPA: ARA: DHA), preferably of at least 1: 1: 3, preferably of 1: 1: 4 preferably prepared from 1: 1: 5.
- Fatty acid esters or fatty acid mixtures which have been prepared by the process according to the invention advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid; 7 - 85% oleic acid; 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachidic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids in each case based on 100% and on the total fatty acid content of the organisms.
- polyunsaturated fatty acid in the fatty acid esters or fatty acid mixtures are preferably at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1% based on the total fatty acid content of arachidonic acid.
- the fatty acid esters or fatty acid mixtures prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8,9 -Methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentenodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,1-dienanoic acid), vernonoic acid (9,10-epoxyoctadec-12-enoic acid) , Taric acid (6-octadecynoic acid), 6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (t10-h
- fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they are less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, more preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1% ago.
- the nucleic acid sequences according to the invention or the nucleic acid sequences used in the method according to the invention can increase the yield of polyunsaturated fatty acids by at least 50%, advantageously by at least 80%, particularly advantageously by at least 100%, very particularly advantageously by at least 150% compared to the non-transgenic starting organism
- a yeast, an alga, a fungus, or a plant such as Arabidopsis or flax can be obtained by comparison in GC analysis, see Examples.
- chemically pure polyunsaturated fatty acids or fatty acid compositions can be prepared by the methods described above.
- the fatty acids or fatty acid compositions from the organism such as the microorganisms or plants or the culture medium in which or on which the organisms were grown, or from the organism and the culture medium in a known manner, for example via extraction, distillation, crystallization, chromatography or Isolated combinations of these methods.
- These chemically pure fatty acids or fatty acid compositions are advantageous for applications in the food industry, the cosmetics industry and especially the pharmaceutical industry.
- acyl-CoA dehydrogenase s
- acyl-CoA dehydrogenase s
- genes selected from the group of ⁇ -4-desaturases, ⁇ -5-desaturases, ⁇ -6-desaturases, ⁇ -9-desaturases, ⁇ -12-desaturases, ⁇ -6-elongases or ⁇ - 5- elongases used in combination with the polynucleotides of the invention, wherein single genes or multiple genes can be used in combination.
- the desaturases used in the process of the invention convert their respective substrates in the form of the CoA fatty acid esters.
- the respective desaturation products are thereby synthesized in higher amounts, since the elongation step usually takes place on the CoA fatty acid esters, while the desaturation step takes place predominantly on the phospholipids or on the triglycerides.
- An exchange reaction that would require another possibly limiting enzyme reaction between the CoA fatty acid esters and the phospholipids or triglycerides is thus not required.
- the enzymatic activity of the polypeptides used in the process according to the invention makes it possible to prepare a wide variety of polyunsaturated fatty acids in the process according to the invention.
- the various polyunsaturated fatty acids or individual polyunsaturated fatty acids such as EPA or ARA in free or bound form.
- fatty acids derived from C18: 2 fatty acids such as GLA, DGLA or ARA or those derived from C18: 3 fatty acids derive, such as SDA, ETA or EPA.
- linoleic acid LA, C18: 2 A9 '12
- GLA, DGLA and ARA can arise as products of the process which may be present as free fatty acids or bound.
- ⁇ -linolenic acid ALA, C18: 3 A9 ' 12 ' 15
- SDA, ETA, EPA and / or DHA free fatty acids or bound as described above.
- Enzymes ⁇ -5-desaturase, ⁇ -6-desaturase, ⁇ -4-desaturase, ⁇ -12-desaturase, ⁇ -5 elongase and / or ⁇ -6 elongase can be targeted in the abovementioned organisms advantageously in the abovementioned plants produced only individual products.
- Due to the activity of ⁇ -6-desaturase and ⁇ -6 elongase for example, GLA and DGLA or SDA and ETA are formed, depending on the starting plant and unsaturated fatty acid. Preference is given to DGLA or ETA or mixtures thereof.
- ARA, EPA and / or DHA are additionally produced.
- ARA, EPA or DHA or mixtures thereof are synthesized, depending on the fatty acids present in the organism or in the plant, which serves as the starting substance for the synthesis. Since these are biosynthetic chains, the respective end products are not present as pure substances in the organisms. There are always small amounts of precursor compounds in the final product.
- small amounts are less than 20 wt .-%, advantageously less than 15 wt .-%, more preferably less than 10 wt .-%, most preferably less than 5, 4, 3, 2 or 1 wt .-% based to the end product DGLA, ETA or mixtures thereof or ARA, EPA, DHA or mixtures thereof advantageously EPA or DHA or mixtures thereof.
- the fatty acids can also be fed from the outside.
- Preferred substrates are linoleic acid (C18: 2 A9 '12 ), ⁇ -linolenic acid (C18: 3 ⁇ 6 ' 9 '12 ), eicosadienoic acid (C20: 2 ⁇ 11 ' 14 ), dihomo- ⁇ -linolenic acid (C20: 3 ⁇ 8 ' 11 ' 14 ), the arachidonic acid (C20: 4 ⁇ 5A11 '14 ), the docosatetraenoic acid (C22: 4 ⁇ 7 ' 10 ' 13 ' 16 ) and the docosapentaenoic acid (C22: 5 ⁇ 4 ' 7 ' 10 ' 13 ' 15 ).
- nucleic acid sequences or their derivative or homologs which code for polypeptides which still possess the enzymatic activity of the proteins encoded by nucleic acid sequences.
- sequences are cloned individually or in combination with the polynucleotides of the invention in expression constructs and used for introduction and expression in organisms. By their construction, these expression constructs enable a favorable optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.
- the method further comprises the step of obtaining a cell or whole organism containing the nucleic acid sequences used in the method, wherein the cell and / or organism with a polynucleotide of the invention is a gene construct or a vector as described below, alone or in combination with other nucleic acid sequences coding for proteins of the fatty acid or lipid metabolism.
- this method further comprises the step of recovering the oils, lipids or free fatty acids from the organism or from the culture.
- the culture may be, for example, a fermentation culture, for example, in the case of culturing microorganisms such as e.g.
- the cell or organism thus produced is advantageously a cell of an oil-producing organism such as an oil crop such as peanut, canola, canola, flax, hemp, peanut, soybean, safflower, hemp, sunflower or borage.
- Cultivation is, for example, culturing in the case of plant cells, tissue or organs on or in a nutrient medium or the whole plant on or in a substrate, for example in hydroponics, potting soil or on arable land.
- Suitable organisms or host cells for the process according to the invention are those which are able to synthesize fatty acids, especially unsaturated fatty acids, or are suitable for the expression of recombinant genes.
- Examples include plants such as Arabidopsis, Asteraceae such as Calendula or crops such as soybean, peanut, castor, sunflower, corn, cotton, flax, rape, coconut, oil palm, dyer safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example, the genus Mortierella, Thraustochytrium, Saprolegnia, Phytophthora or Pythium, bacteria such as the genus Escherichia or Shewanella, yeasts like the Genus Saccharomyces, cyanobacteria, ciliates, algae such as Mantoniella or Ostreococcus or protozoa such as dinoflagellates such as Thalassiosira or Crypthe
- transgenic animals are also advantageously suitable for non-human animals, for example C. elegans. Other suitable host cells and organisms have previously been described in detail.
- Transgenic plants which contain the polyunsaturated fatty acids synthesized in the process according to the invention can advantageously be marketed directly without the synthesized oils, lipids or fatty acids having to be isolated.
- Plants in the process according to the invention include whole plants and all plant parts, plant organs or plant parts such as leaves, stems, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, callosis, kotelydons, petioles, crop material, plant tissue, reproductive tissue, Cell cultures that can be derived from the transgenic plant and / or used to produce the transgenic plant.
- the seed includes all seed parts such as the seed shells, epidermis and sperm cells, endosperm or embryonic tissue.
- the compounds prepared in the process according to the invention can also be isolated from the organisms advantageously plants in the form of their oils, fat, lipids and / or free fatty acids.
- Polyunsaturated fatty acids produced by this process can be harvested by harvesting the organisms either from the culture in which they grow or from the field. This can be done by pressing or extraction of the plant parts, preferably the plant seeds.
- the oils, fats, lipids and / or free fatty acids can be obtained by so-called cold pressing or cold pressing without supplying heat by pressing.
- the plant parts, especially the seeds, to be easier to digest they are first crushed, steamed or roasted. The pretreated seeds can then be pressed or extracted with solvents such as warm hexane.
- the solvent is removed again.
- these are harvested after harvesting, for example, directly without further working steps, or else extracted after digestion by various methods known to the person skilled in the art. In this way, more than 96% of the compounds prepared in the process can be isolated.
- the products thus obtained are further processed, that is refined.
- the so-called degumming can be carried out enzymatically or, for example, chemically / physically by adding acid, such as phosphoric acid.
- the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution.
- the product obtained is thoroughly washed with water to remove the lye remaining in the product and dried.
- the products are subjected to bleaching with, for example, bleaching earth or activated carbon.
- the product is still deodorized, for example, with steam.
- the PUFAs or LCPUFAs produced by this process are C 8 -, C 2 O- or C 22 -fatty acid molecules and advantageously C 2 o- or C 22 -fatty acid molecules having at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds.
- These Ci 8 -, C 2 o- or C 22 -Fettkladklaküle can be isolated from the organism in the form of an oil, lipid or a free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.
- One embodiment of the invention is therefore oils, lipids or fatty acids or fractions thereof which have been prepared by the method described above, more preferably oil, lipid or fatty acid composition comprising PUFAs derived from transgenic plants.
- oils, lipids or fatty acids advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid as described above; 7 - 85% oleic acid; 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachidic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids in each case based on 100% and on the total fatty acid content of the organisms.
- polyunsaturated fatty acid in the fatty acid esters or fatty acid mixtures are preferably at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1% based on the total fatty acid content of arachidonic acid.
- the fatty acid esters or fatty acid mixtures prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8,9 -Methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentenodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,11-dienoic acid), vernonic acid (9,10-epoxyoctadec-12-enoic acid), tartric acid ( 6- octadecynoic acid), 6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen
- fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they are less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, more preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1% ago.
- the oils, lipids or fatty acids according to the invention preferably contain at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, 7%, 8%, 9% or 10%, particularly advantageously at least 11%, 12%, 13%, 14% or 15% ARA or at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, or 7%, more preferably at least 8 %, 9% or 10% EPA and / or DHA based on the total fatty acid content of the production organism advantageously a plant, particularly advantageous an oil crop such as soy, rapeseed, coconut, oil palm, safflower, flax, hemp, castor, calendula, peanut, cocoa bean , Sunflower or the above-mentioned other monocotyledonous or dicotyledonous oil crops.
- an oil crop such as soy, rapeseed, coconut, oil palm, safflower, flax, hemp, cast
- Another embodiment of the invention is the use of the oil, lipid, fatty acids and / or fatty acid composition in feed, food, cosmetics or pharmaceuticals.
- the oils, lipids, fatty acids or fatty acid mixtures according to the invention may be mixed with other oils, lipids, fatty acids or fatty acid mixtures of animal origin, such as those described in the art, for example. Fish oils are used. These oils, lipids, fatty acids or fatty acid mixtures, which consist of vegetable and animal components, can be used for the production of feed, food, cosmetics or pharmaceuticals.
- oil is understood as meaning a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s). It is preferred that the oil, lipid or fat contain a high proportion of polyunsaturated free or advantageously esterified fatty acid (s), in particular linoleic acid, ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, ⁇ -linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid.
- s polyunsaturated free or advantageously esterified fatty acid
- the proportion of unsaturated esterified fatty acids is about 30%, more preferred is a proportion of 50%, even more preferred is a proportion of 60%, 70%, 80% or more.
- the proportion of fatty acid after conversion of the fatty acids into the methyl esters can be determined by transesterification by gas chromatography.
- the oil, lipid or fat may contain various other saturated or unsaturated fatty acids, eg calendulic acid, palmitic, palmitoleic, stearic, oleic acid, etc. In particular, depending on the starting organism, the proportion of the various fatty acids in the oil or fat may vary.
- the polyunsaturated fatty acids having advantageously at least two double bonds which are produced in the process are, as described above, for example sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
- the polyunsaturated fatty acids containing, for example, an alkali treatment such as aqueous KOH or NaOH or acid hydrolysis advantageously in the presence of an alcohol such as methanol or ethanol or via an enzymatic cleavage liberate and isolate via, for example, phase separation and subsequent acidification via, for example, H 2 SO 4 .
- an alkali treatment such as aqueous KOH or NaOH or acid hydrolysis
- an alcohol such as methanol or ethanol or via an enzymatic cleavage
- the release of the fatty acids can also be carried out directly without the workup described above.
- the nucleic acids used in the method can advantageously be either a plant cell or plant, either on a separate plasmid or advantageously integrated into the genome of the host cell.
- integration may be at random or by such recombination as replacing the native gene with the incorporated copy, thereby modulating production of the desired compound by the cell, or by using a gene in "trans".
- the nucleic acids are introduced into the organisms via multi-expression cassettes or constructs for multiparallel expression advantageous for multiparallel seed-specific expression of genes brought into the plants.
- Moose and algae are the only known plant systems that produce significant amounts of polyunsaturated fatty acids, such as arachidonic acid (ARA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).
- nucleic acid molecules which are isolated from strains which also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the process according to the invention and thus for modification of the lipid and PUFA production system in a host, in particular plants, such as oilseed plants, for example Rapeseed, canola, flax, hemp, soy, sunflower, borage. They are therefore advantageous for use in the process according to the invention.
- acyl-CoA-dehydrogenase s
- acyl-ACP [acyl carrier protein] desaturase
- acyl-ACP thioesterase s
- Fatty acid acyltransferase s
- acyl-CoA lysophospholipid acyltransferase
- fatty acid synthase s
- fatty acid hydroxylase s
- acetyl coenzyme A carboxylase s
- acyl coenzyme A- Oxidase s
- fatty acid desaturase s
- fatty acid acetylenase s
- lipoxygenase s
- the polyunsaturated C 16 -fatty acids must first be desaturated by the enzymatic activity of a desaturase and then be extended by at least two carbon atoms via an elongase. After one round of elongation this enzyme activity leads to C 2 o-fatty acids, and after two rounds of elongation to C 22 -fatty acids.
- Desaturases and elongases used in the activity of the process according to the invention preferably leads to Ci ⁇ -, C 2 0 and / or C 22 fatty acids advantageously having at least two double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, especially preferably C 2 o- and / or C 22 -fatty acids having at least two double bonds in the fatty acid molecule, preferably having three, four, five or six double bonds, very particularly preferably having five or six double bonds in the molecule.
- further desaturation and elongation steps such as desaturation at ⁇ -5 and ⁇ -4 positions may occur.
- Particularly preferred products of the process according to the invention are dihomo- ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and / or docosaheic acid.
- the C 2 o fatty acids having at least two double bonds in the fatty acid can be extended by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
- the preferred biosynthesis site of fatty acids, oils, lipids or fats in the advantageously used plants is, for example, generally the seeds or cell layers of the seed, so that a seed-specific expression of the nucleic acids used in the method is useful.
- biosynthesis of fatty acids, oils or lipids need not be limited to the seed tissue, but may also be tissue-specific in all other parts of the plant - for example in epidermal cells or in the tubers.
- microorganisms such as yeasts such as Saccharomyces or Schizosaccharomyces
- fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium algae such as Isochrysis, Mantoniella, Ostreococcus, Phaeodactylum or Crypthecodiniu
- yeasts such as Saccharomyces or Schizosaccharomyces
- fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium algae such as Isochrysis, Mantoniella, Ostreococcus, Phaeodactylum or Crypthecodiniu
- these organisms are advantageously attracted to fermentation.
- the polyunsaturated fatty acids prepared in the process can be at least 5%, preferably at least 10%, more preferably at least 20%, very particularly preferably at least 50%. be increased compared to the wild type of organisms that do not contain the nucleic acids recombinantly.
- the polyunsaturated fatty acids produced in the organisms used in the process can in principle be increased in two ways.
- the pool of free polyunsaturated fatty acids and / or the proportion of esterified polyunsaturated fatty acids produced by the process can be increased.
- the process according to the invention increases the pool of esterified polyunsaturated fatty acids in the transgenic organisms.
- microorganisms are used as organisms in the process according to the invention, they are grown or grown, depending on the host organism, in a manner known to the person skilled in the art.
- Microorganisms are usually in a liquid medium containing a carbon source usually in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally contains vitamins, at temperatures between 0 0 C and 10O 0 C, preferably between 10 0 C to 60 ° C attracted under oxygen fumigation.
- the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation or not.
- the cultivation can be batchwise, semi-batch wise or continuous. Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously.
- the polyunsaturated fatty acids prepared can be isolated from the organisms by methods known to those skilled in the art as described above. For example, extraction, distillation, crystallization, optionally salt precipitation and / or chromatography. The organisms can be opened up for this purpose yet advantageous.
- the inventive method when it is in the host organisms are microorganisms, advantageously carried out at a temperature between 0 0 C to 95 °, preferably between 10 0 C to 85 ° C, more preferably between 15 ° C to 75 ° C, most preferably carried out between 15 ° C to 45 ° C.
- the pH is advantageously maintained between pH 4 and 12, preferably between pH 6 and 9, more preferably between pH 7 and 8.
- the process according to the invention can be operated batchwise, semi-batchwise or continuously.
- a summary of known cultivation methods is in the textbook by Chmiel (Bioreatechnik 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) Find.
- the culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington D.C, USA, 1981).
- these media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
- Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
- sugars such as mono-, di- or polysaccharides.
- very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
- Sugar can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It can also be advantageous to add mixtures of different carbon sources.
- oils and fats such as soybean oil, sunflower oil, peanut oil and / or coconut oil, fatty acids such as palmitic acid, stearic acid and / or linoleic acid, alcohols and / or polyalcohols such.
- fatty acids such as palmitic acid, stearic acid and / or linoleic acid
- alcohols and / or polyalcohols such as glycerol, methanol and / or ethanol and / or organic acids such as acetic acid and / or lactic acid.
- Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
- Exemplary nitrogen sources include ammonia in liquid or gas form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast extract, meat extract and others.
- the nitrogen sources can be used singly or as a mixture.
- Inorganic salt compounds which may be included in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
- sulfur-containing fine chemicals in particular methionine
- inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic sulfur compounds, such as mercaptans and thiols can be used.
- Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
- Chelating agents can be added to the medium to keep the metal ions in solution.
- Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
- the fermentation media used according to the invention for the cultivation of microorganisms usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
- Growth factors and salts are often derived from complex media components, such as yeast extract, molasses, corn steep liquor, and the like.
- suitable precursors can be added to the culture medium.
- the exact composition of the media compounds will depend heavily on the experiment and will be specific to each specific see case individually decided. Information about the media optimization is available from the textbook "Applied Microbiol Physiology, A Practical Approach” (Ed PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).
- Growth media may also be obtained from commercial suppliers such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
- All media components are sterilized either by heat (20 min at 1, 5 bar and 121 0 C) or by sterile filtration.
- the components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or added randomly or batchwise, as desired.
- the temperature of the culture is usually between 15 ° C and 45 ° C, preferably at 25 ° C to 40 0 C and can be kept constant or changed during the experiment.
- the pH of the medium should be in the range of 5 to 8.5, preferably around 7.0.
- the pH for cultivation can be controlled during cultivation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid.
- acidic compounds such as phosphoric acid or sulfuric acid.
- the foaming anti-foaming agents such.
- As fatty acid polyglycol are used.
- the medium can be selected selectively acting substances such. As antibiotics, are added.
- oxygen or oxygen-containing gas mixtures such as ambient air
- the temperature of the culture is nor- mally at 20 0 C to 45 ° C and preferably 25 ° C to 40 0 C.
- the culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.
- the fermentation broths thus obtained in particular containing polyunsaturated fatty acids, usually have a dry matter content of 7.5 to 25% by weight.
- the fermentation broth can then be further processed.
- the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation or a combination of these methods are removed from the fermentation broth or completely left in it.
- the biomass is worked up after separation.
- the fermentation broth can also without cell separation with known methods such. B. with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or be concentrated. This concentrated fermentation broth may eventually be worked up to recover the fatty acids contained therein.
- polynucleotides or polypeptides of the present invention which are involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds via membranes, are advantageously used in plants according to the invention for modulating the production of PUFAs in transgenic organisms
- Solanacaen - plants such as potato, tobacco, aubergine and tomato, Vicia species, pea, cassava, alfalfa, bush plants (coffee, cocoa, tea), Salix species, trees (oil palm, coconut) and perennial grasses and forage crops, either directly ( For example, if overexpression or optimization of a fatty acid biosynthesis protein has a direct impact on
- PUFAs polyunsaturated fatty acids
- Brasicaceae for example stearidonic acid, eicosapentaenoic acid and docosahexaenoic acid
- Brasicaceae for example stearidonic acid, eicosapentaenoic acid and docosahexaenoic acid
- boraginaceous plants for example stearidonic acid, eicosapentaenoic acid and docosahexaenoic acid
- linaceae particularly advantageous is Lein (Linum usitatissimum) for the production of PUFAS with the nucleic acid sequences of the invention advantageously, as described, in combination with other desaturases and elongases.
- the lipid synthesis can be divided into two sections: the synthesis of fatty acids and their attachment to sn-glycerol-3-phosphate and the addition or modification of a polar head group.
- Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and phosphoglycerides. The OO
- Fatty acid synthesis begins with the conversion of acetyl-CoA into malonyl-CoA by the acetyl-CoA carboxylase or into acetyl-ACP by the acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions, so that a saturated fatty acid molecule with the desired chain length is obtained.
- the production of unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically by molecular oxygen or anaerobically (for fatty acid synthesis in microorganisms see FC Neidhardt et al., (1996) E.
- fatty acids thus bound to phospholipids must then be converted again for the further elongations from the phospholipids into the fatty acid CoA ester pool. This is facilitated by acyl-CoA3sophospholipid acyltransferases. Furthermore, these enzymes can transfer the elongated fatty acids again from the CoA esters to the phospholipids. This reaction sequence can optionally be run through several times.
- Precursors for the PUFA biosynthesis are, for example, oleic acid, linoleic acid and linolenic acid. These Ci ⁇ -carbon fatty acids must be extended to C 2 o and C 22 in order to obtain fatty acids of the eicosa- and docosa-chain type.
- the desaturases used in the process such as the ⁇ -12, ⁇ -4, ⁇ -5 and ⁇ -6 desaturases and / or the ⁇ -5, ⁇ -6 elongases, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or Docosahexaen Anlagenre advantageously eicosapentaenoic acid and / or docosahexaenoic acid are prepared and then used for various purposes in food, feed, cosmetic or pharmaceutical applications.
- C 2 - and / or C 22 -fatty acids having at least two, preferably at least three, four, five or six double bonds in the fatty acid molecule, preferably C 2 0 or C 22 -fatty acids with advantageously four, five or six double bonds, can be used with the abovementioned enzymes be prepared in the fatty acid molecule.
- the desaturation can take place before or after elongation of the corresponding fatty acid.
- the products of desaturase activities and possible further desaturation and elongation result in preferred PUFAs having a higher degree of desaturation, including a further elongation of C 20 to C 22 fatty acids, to fatty acids such as ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, Stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
- Substrates of the desaturases and elongases used in the process of this invention are C 1 6-, C linolenic dihomo- ⁇ -1 8- or C2o fatty acids such as linoleic acid, ⁇ -linolenic acid, ⁇ -linolenic acid, Eicosatetraenoic acid or stearidonic acid.
- Preferred substrates are linoleic acid, v-linolenic acid and / or ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid.
- the synthesized C 2 o- or C 22 -fatty acids with at least two, three, four, five or six double bonds in the fatty acid are obtained in the process according to the invention in the form of the free fatty acid or in the form of their esters, for example in the form of their glycerides.
- glycolide is understood to mean a glycerol esterified with one, two or three carboxylic acid residues (mono-, di- or triglyceride).
- glycolide is also meant a mixture of different glycerides.
- the glyceride or glyceride mixture may contain other additives, e.g. contain free fatty acids, antioxidants, proteins, carbohydrates, vitamins and / or other substances.
- a "glyceride” in the sense of the method according to the invention is also understood to mean derivatives derived from glycerol.
- these also include glycerophospholipids and glyceroglycolipids.
- the glycerophospholipids such as lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and alkylacylglycerophospholipids, may be mentioned by way of example here.
- fatty acids must then be transported to various modification sites and incorporated into the triacylglycerol storage lipid.
- Another important step in lipid synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol-fatty acid acyltransferase (see Frentzen, 1998, Lipid, 100 (4-5): 161-166).
- the PUFAs produced in the process comprise a group of molecules that are no longer able to synthesize, and therefore need to take up, higher animals, or that can no longer sufficiently produce higher animals themselves, and thus have to additionally take up, even though they are readily synthesized by other organisms, such as bacteria For example, cats can no longer synthesize arachidonic acid.
- phospholipids are to be understood as meaning phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and / or phosphatidylinositol, advantageously phosphatidylcholine.
- production or productivity are known in the art and include the concentration of the fermentation product (compounds of formula I) formed in a given period of time and fermentation volume (e.g., kg of product per hour per liter). It also includes productivity within a plant cell or plant, that is, the content of the desired fatty acids produced in the process based on the content of all fatty acids in that cell or plant.
- the term efficiency of production includes the time required to achieve a certain amount of production (e.g., how long the cell takes to establish a given throughput rate of a fine chemical).
- yield or product / carbon yield is known in the art and includes the efficiency of converting the carbon source into the product (i.e., the fine chemical). This is usually expressed, for example, as kg of product per kg of carbon source.
- biosynthesis or biosynthetic pathway are known in the art and involve the synthesis of a compound, preferably an organic compound, by a cell from intermediates, for example in a multi-step and highly regulated process.
- degradation or degradation pathway are well known in the art and involve the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), for example in a multi-step and highly regulated process.
- metabolism is known in the art and includes the entirety of the biochemical reactions that take place in an organism.
- the metabolism of a particular compound e.g., the metabolism of a fatty acid
- the metabolism of a particular compound then comprises all of the biosynthetic, modification, and degradation pathways of that compound in the cell that affect that compound.
- the yield, production and / or efficiency of the production of the polyunsaturated fatty acids in a plant, preferably in an oil crop, or a microorganism can be influenced.
- the number or activity of the polypeptides or polynucleotides according to the invention can be increased, so that larger amounts of the gene products and thus ultimately larger amounts of the compounds of general formula I are produced. Also, a de novo synthesis in an organism lacking the activity and ability to biosynthesize the compounds before introducing the gene (s) of interest is possible.
- a polynucleotide according to the invention By introducing a polynucleotide according to the invention into an organism alone or in combination with other genes into a cell, not only can the biosynthesis flux to the end product be increased, but also the corresponding triacylglycerol composition can be increased or created de novo. Likewise, the number or activity of other genes necessary for the import of nutrients necessary for the biosynthesis of one or more fatty acids, oils, polar and / or neutral lipids may be increased, such that the concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment, thereby further increasing the ability of the cells to produce PUFAs.
- fatty acids obtained in the process are suitable as starting material for the chemical synthesis of other valuable products. They may be used, for example, in combination with each other or solely for the manufacture of pharmaceuticals, foods, animal feed or cosmetics.
- the invention also encompasses a process for preparing an oil, lipid or fatty acid composition. send relates to the steps of the method according to the invention and the further step of formulating the substance as an oil, lipid or fatty acid composition.
- the oil, lipid or fatty acid composition is further formulated into a medicament, a cosmetic, a food, a feed, preferably a fish feed, or a dietary supplement.
- the invention generally relates to the use of the polynucleotide, the vector, the host cell, the polypeptide or the transgenic, non-human organism of the present invention for the preparation of an oil, lipid or fatty acid composition. This is then preferably used as pharmaceuticals, cosmetics, foods, animal feed, preferably fish feed, or dietary supplements.
- FIG. 1 shows a sequence comparison of the ⁇ 5 and ⁇ 6 elongase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
- FIG. 2 shows a sequence comparison of the ⁇ 4-desaturase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
- FIG. 3 shows a sequence comparison of the ⁇ 5-desaturase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
- FIG. 4 shows a sequence comparison of the ⁇ 6-desaturase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
- FIG. 5 shows a sequence comparison of the ⁇ 12-desaturase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
- 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: 4 ⁇ 5, 8, 1, 14 was fed (B, C).
- 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-D ⁇ EIo (OI) (D, E).
- the fatty acids 18: 3 ⁇ 6,9,12 and 18: 4 ⁇ 6,9, 12,15 were fed (B, D) and (C, E), respectively.
- 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 acids 20: 3 ⁇ 5, 8, 1, 14 were fed into (B) and (C).
- 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).
- FIG. 10 shows the gas chromatographic determination of the fatty acids from yeast.
- the cloning methods e.g. Restriction cleavage, 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, culture of bacteria and sequence analysis of recombinant DNA were performed as described in Sambrook et al. (1989) (CoId Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
- Example 2 Sequence Analysis of Recombinant DNA
- the sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad. See, USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
- the effect of genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound may be determined by cultivating the modified microorganism or modified plant under suitable conditions (such as those described above), and Medium and / or the cellular components on the increased production of the desired product (ie of lipids or a fatty acid) is examined.
- suitable conditions such as those described above
- These analytical techniques are well known to those skilled in the art and include spectroscopy, thin layer chromatography, staining methods of various types, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.
- FAME fatty acid methyl ester
- GC-MS gas-liquid chromatography-mass spectrometry
- TAG triacylglycerol
- TLC thin-layer chromatography
- the unambiguous evidence for the presence of fatty acid products can be obtained by analysis of recombinant organisms by standard analytical methods: GC, GC-MS or TLC as variously described by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Edition. Christie, Oliver Press, Dundee, 119-169, 1998, Gas Chromatography Mass Spectrometry Method, Lipids 33: 343-353).
- the material to be analyzed may be broken up by sonication, milling in the glass mill, liquid nitrogen and milling or other applicable methods.
- the material must be centrifuged after rupture.
- the sediment is distilled in aqua. re-suspended, heated at 100 ° C. for 10 minutes, cooled on ice and recentrifuged, followed by extraction into 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90 ° C., resulting in hydrolyzed oil and lipid compounds, which give transmethylated lipids.
- FIG. 1 shows the sequence similarities to other algae (Ostreococcus tauri, Thalassiosira pseudonana) for the different elongase amino acid sequences in the ClustalW sequence comparison. Surprisingly, the sequences of O. lucimarinus differ significantly in their amino acid sequence from the other algae.
- the cloning was carried out as follows:
- 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 extension step at 72 ° C for 10 minutes.
- the open reading frames of the respective DNAs are cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1A / 5-His-TOPO (Invitrogen) to yield pOLE1 and pOLE2.
- the Saccharomyces cerev / s / ae strain 334 is transformed by electroporation (1500 V) with the vector pOLE1 or pOLE2, respectively.
- a yeast is used, which is transformed with the empty vector pYES2.
- the selection of the transformed yeasts is carried out on complete minimal medium (CMdum) agar plates with 2% glucose, but without uracil. After selection, three transformants each are selected for further functional expression.
- CMdum liquid medium For the expression of the oil elongases, first precultures each of 5 ml of CMdum liquid medium with 2% (w / v) raffinose but without uracil are inoculated with the selected transformants and incubated for 2 days at 30 ° C., 200 rpm. 5 ml of CMdum liquid medium (without uracil) with 2% raffinose and 300 ⁇ M of various fatty acids are then inoculated with the precultures to an OD 6 oo of 0.05. Expression is induced by the addition of 2% (w / v) galactose. The cultures were incubated for a further 96 h at 20 ° C.
- Yeasts transformed with plasmids pYES2, pOLE1 and pOLE2 are analyzed as follows:
- the yeast cells from the major cultures are harvested by centrifugation (100 xg, 5 min, 20 ° C) and washed with 100 mM NaHCO 3 , pH 8.0 to remove residual medium and fatty acids.
- fatty acid methyl esters (FAMEs) are produced by acid methanolysis.
- the cell sediments are incubated with 2 ml of 1N methanolic sulfuric acid and 2% (v / v) dimethoxypropane for 1 h at 80 ° C.
- Extraction of the FAMES is carried out by extracting twice with petroleum ether (PE).
- the organic phases are distilled once each with 2 ml of 100 mM NaHCO 3 , pH 8.0 and 2 ml of distilled water. washed. Subsequently, the PE phases were dried with Na 2 SO 4 , evaporated under argon and taken up in 100 ⁇ l of PE. The samples are separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 ⁇ m, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector.
- the conditions for the GLC analysis are as follows: The oven temperature was programmed from 50 ° C to 250 ° C at a rate of 5 ° C / min and finally 10 min at 250 ° C (hold). The signals are identified by comparison of 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.
- d5Elo various fatty acids were fed (Table 3).
- the lined substrates are to be detected in large quantities in all transgenic yeasts.
- the transgenic yeasts show the synthesis of new fatty acids, the products of the d5Elo (OI) reaction. This means that the gene d5Elo (OI) has been functionally expressed.
- FIG. 6 shows the chromatograms of the individual experiments.
- yeasts transformed with pYES without addition of fatty acids were analyzed in FIG. 6A.
- Fig. 1B the yeast was transformed with pYES the fatty acid 20: 4 ⁇ 5, 8, 1, 14 fed. In this case, the fed fatty acid can be detected in large quantities.
- Fig. 6C yeasts transformed with the plasmid pYES-D5Elo (OI).
- OI plasmid pYES-D5Elo
- an additional fatty acid can be detected in the yeasts with pYES-D5Elo (OI), which must be due to the activity of the D5Elo (OI). Based on the activity, D5Elo (OI) can be characterized as a ⁇ 5 elongase.
- D5Elo (OI) SEQ ID12 was functionally expressed and possesses elongase activity. Based on the fed fatty acid, D5Elo (OI) can be characterized as a ⁇ 5 elongase, ie C20 fatty acids with a ⁇ 5 double bond are specifically extended. Activity and Substrate Determination of D6Elo (OI):
- D6Elo D6Elo
- various fatty acids were fed (Table 4).
- the lined substrates are to be detected in large quantities in all transgenic yeasts.
- the transgenic yeasts show the synthesis of new fatty acids, the products of the D6Elo (OI) reaction. This means that the gene D6Elo (OI) has been functionally expressed.
- FIG. 7 shows the chromatograms of the individual experiments.
- yeasts transformed with pYES without addition of fatty acids were analyzed in FIG. 7A.
- Figs. 7B and 7C the yeasts transformed with pYES were added with the fatty acid 18: 3 ⁇ 6, 9, 12 (B) and 18: 4 ⁇ 6, 9, 12, 15 (C), respectively.
- the fed fatty acid can be detected in large quantities.
- Figs. 7C and 7D for yeasts transformed with the plasmid pYES-D ⁇ EIo (OI).
- OI EIo
- an additional fatty acid can be detected in the yeasts with pYES-D ⁇ EIo (OI), which must be due to the activity of the D6Elo (OI). Based on the activity D6Elo (OI) can be characterized as ⁇ 6 elongase.
- D6Elo (OI) SEQ ID16 was functionally expressed and possesses elongase activity. Based on the fed fatty acid, D6Elo (OI) can be characterized as a ⁇ 6 elongase, ie C18 fatty acids with a ⁇ 6 double bond are specifically extended.
- the Saccharomyces cerev / s / ae strain 334 is transformed by electroporation (1500 V) with the vector pYES2.1-d6Elo (OI).
- a yeast was used, which is transformed with the empty vector pYES2.
- the selection of the transformed yeasts is carried out on complete minimal medium (CMdum) agar plates with 2% glucose, but without uracil. After selection, three transformants each are selected for further functional expression.
- d6Elo (OI) desaturase first precultures from 5 ml of CMdum liquid medium with 2% (w / v) raffinose but without uracil are inoculated with the selected transformants and incubated for 2 days at 30 0 C, 200 rpm. 5 ml CMdum liquid medium (without uracil) with 2% raffinose and 300 ⁇ M different fatty acids are then inoculated with the precultures to an OD 6 oo of 0.05. Expression is induced by the addition of 2% (w / v) galactose. The cultures are incubated for a further 96 h at 20 ° C.
- Figures 2 to 5 show the sequence similarities to other algae (Ostreococcus tauri, Thalassiosira pseudonana) for the different desaturase amino acid sequences in ClustalW sequence comparison. Surprisingly, the sequences of O. lucimarinus differ significantly in their amino acid sequence from the other algae.
- yeast cells For expression of the desaturases in yeast cells, they are harvested from the main cultures by centrifugation (100 ⁇ g, 5 min, 20 ° C.) and washed with 100 mM NaHCO 3 , pH 8.0 to remove residual medium and fatty acids. The yeast cell pellets are extracted with chloroform / methanol (1: 1) for 4 h. The resulting organic phase is extracted with 0.45% NaCl, dried with Na 2 SC> 4 and evaporated under vacuum.
- the lipid extract is further purified by thin-layer chromatography (horizontal tank, chloroform: methanol: acetic acid 65: 35: 8) into the lipid classes phosphatidylcholine (PC), phosphatidiylinositol (PI), phosphatidyserine (PS), phosphatidylethanolamine (PE) and neutral Lipids (NL) separated.
- PC phosphatidylcholine
- PI phosphatidiylinositol
- PS phosphatidyserine
- PE phosphatidylethanolamine
- NL neutral Lipids
- the cell sediments are incubated with 2 ml of 1N methanolic sulfuric acid and 2% (v / v) dimethoxypropane for 1 h at 80 ° C.
- the extraction of the FAMES was carried out by extraction twice with petroleum ether (PE).
- PE petroleum ether
- the organic phase is distilled once each with 2 ml of 100 mM NaHCO 3, pH 8.0 and 2 ml of distilled water. washed.
- the PE phases are dried with Na 2 SO 4 , evaporated under argon and taken up in 100 ⁇ l of PE.
- the samples are separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 ⁇ m, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector.
- the conditions for the GLC analysis are as follows: The oven temperature is from 50 0 C to 250 ° C at a rate of 5 ° C / min and finally 10 min at 250 ° C (hold) programmed.
- the signals are identified by comparison of 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.
- D5Des_2 (Ol) SEQ ID 24 To determine the activity and substrate specificity of D5Des_2 (Ol) SEQ ID 24, various fatty acids were fed (Table 7). The lined substrates are to be detected in large quantities in all transgenic yeasts. The transgenic yeasts show the synthesis of new fatty acids, the products of the D5Des_2 (Ol) reaction. This means that the gene D5Des_2 (Ol) has been functionally expressed.
- Table 7 Administration / Implementation of Different Fatty Acids by D5Des OI 2).
- FIG. 8 shows the chromatograms of the individual experiments.
- yeasts transformed with pYES were analyzed in Fig. 8A without addition of fatty acids.
- Fig. 8B the yeasts transformed with pYES were added the fatty acid 20: 3 ⁇ 8, 11, 14. The fed fatty acid can be detected in large quantities.
- Fig. 8C for yeasts transformed with the plasmid pYES-D5Des (OI_2).
- OI_2 yeasts transformed with the plasmid pYES-D5Des
- an additional fatty acid can be detected in the yeasts with pYES-D5Des (OI_2), which must be due to the activity of D5Des (OI_2).
- D5Des (OI_2) can be characterized as ⁇ 5-desaturase.
- D5Des_2 (Ol) SEQ ID 24 was functionally expressed and possesses desaturase activity. From the fed fatty acid, D5Des_2 (Ol) can be characterized as ⁇ 5-desaturase, i. C20 fatty acids with a ⁇ 8 double bond are specifically dehydrogenated at the ⁇ 5 position.
- D12Des (OI) SEQ ID 18 To determine the activity and substrate specificity of D12Des (OI) SEQ ID 18, various fatty acids were fed (Table 8). The lined substrates are to be detected in large quantities in all transgenic yeasts. The transgenic yeasts show the synthesis of new fatty acids, the products of the D12Des (OI) reaction. This means that the gene D12Des (OI) has been functionally expressed.
- Table 8 Feeding / conversion of various fatty acids by D12Des (OI).
- FIG. 9 shows the chromatograms of the individual experiments.
- yeasts transformed with pYES were analyzed in Fig. 9A without addition of fatty acids.
- yeasts transformed with pYES-D12Des (OI) were analyzed.
- an additional fatty acid can be detected in the yeasts with pYES-D12Des (OI), which must be due to the activity of the D12Des (OI).
- D12Des (OI) can be characterized as ⁇ 12-desaturase.
- D12Des (OI) SEQ ID 18 was functionally expressed and possesses desaturase activity. From the fatty acid spectrum, D12Des (OI) can be characterized as ⁇ 12-desaturase, i. C18 fatty acids with a ⁇ 9 double bond are specifically dehydrogenated at the ⁇ 12 position.
- D5Des (OI) SEQ ID 26 To determine the activity and substrate specificity of D5Des (OI) SEQ ID 26, various fatty acids were fed (Table 9). The lined substrates are to be detected in large quantities in all transgenic yeasts. The transgenic yeasts show the synthesis of new fatty acids, the products of the D5Des (OI) reaction. This means that the gene D5Des (OI) has been functionally expressed.
- FIG. 10 shows the gas chromatographic analysis of yeast feeding experiments. After expression of pYes-d5Des (OI_1) in yeast strain InvSc without addition of fatty acids ( Figure 10A), no conversion of the fatty acids present was found.
- pYes-d5Des (OI_1) expression in yeast strain InvSc after addition of fatty acid 20: 3n-6 (B) leads to the specific conversion of 20: 3n-6 to 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) for specific conversion of 20: 4n-3 to 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).
- D5Des (OI) SEQ ID 26 was functionally expressed and has desaturase activity.
- D5Des can be characterized as ⁇ 5-desaturase, i. C20 fatty acids with a ⁇ 8 double bond are bound specifically to the ⁇ 5-
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Abstract
The present invention relates to polynucleotides from Ostreococcus lucimarinus which encode desaturases and elongases, and can be used for the recombinant production of polyunsaturated fatty acids. In addition, the invention relates to vectors, host cells and transgenic non-human organisms which contain the polynucleotides, and also 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.
Description
Verfahren zur Herstellung mehrfach ungesättigter Fettsäuren in transgenen Organismen Process for producing polyunsaturated fatty acids in transgenic organisms
Beschreibungdescription
Die vorliegende Erfindung betrifft Polynucleotide aus Ostreococcus lucimarinus, die Desaturasen und Elongasen kodieren und zur rekombinanten Herstellung von mehrfach ungesättigten Fettsäuren eingesetzt werden können. Weiterhin betrifft die Erfindung Vektoren, Wirtszellen und transgene nicht-humane Organismen, die die Polynucleotide enthalten, sowie die von den Polynucleotiden kodierten Polypeptide. Schließlich betrifft die Erfindung noch Herstellungsverfahren für die mehrfach ungesättigten Fettsäuren und für Öl-, Lipid- und Fettsäurezusammensetzungen.The present invention relates to polynucleotides from Ostreococcus lucimarinus which encode desaturases and elongases and can be used for the recombinant production of polyunsaturated fatty acids. Furthermore, the invention relates to vectors, host cells and transgenic non-human organisms containing the polynucleotides, as well as 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.
Fettsäuren und Triacylglyceride haben eine Vielzahl von Anwendungen in der Lebensmittelindustrie, der Tierernährung, der Kosmetik und im Pharmabereich. Je nachdem, ob es sich um freie gesättigte und ungesättigte Fettsäuren oder um Triacylglyceride mit einem erhöhten Gehalt an gesättigten oder ungesättigten Fettsäuren handelt, sind sie für die unterschiedlichsten Anwendungen geeignet. Mehrfach- ungesättigte Fettsäuren wie Linol- und Linolensäure sind für Säugetiere essentiell, da sie nicht von diesen selbst hergestellt werden können. Deshalb stellen mehrfach ungesättigte ω-3-Fettsäuren und ω-6-Fettsäuren einen wichtigen Bestandteil der tierischen und menschlichen Nahrung dar.Fatty acids and triacylglycerides have a variety of uses in the food, animal nutrition, cosmetics and pharmaceutical industries. Depending on whether they are free saturated and unsaturated fatty acids or triacylglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a wide variety of applications. Polyunsaturated fatty acids such as linoleic and linolenic acid are essential for mammals because they can not be produced by them. Therefore, polyunsaturated ω-3 fatty acids and ω-6 fatty acids are an important component of animal and human food.
Mehrfach ungesättigte langkettige ω-3-Fettsäuren wie Eicosapentaensäure (= EPA, C20:5Δ5'8'11'14'17) oder Docosahexaensäure (= DHA, C22:6Δ4'7'10'13'16'19) sind wichtige Komponenten der menschlichen Ernährung aufgrund ihrer verschiedenen Rollen in der Gesundheit, die Aspekte wie die Entwicklung des kindlichen Gehirns, der Funktionalität des Auges, der Synthese von Hormonen und anderer Signalstoffe, sowie die Vorbeu- gung von Herz-Kreislauf-Beschwerden, Krebs und Diabetes umfassen (Poulos, A Lipids 30:1-14, 1995; Horrocks, LA und Yeo YK Pharmacol Res 40:211-225, 1999). Es besteht aus diesem Grund ein Bedarf an der Produktion mehrfach ungesättigter langkettiger Fettsäuren.Polyunsaturated long-chain ω-3 fatty acids such as eicosapentaenoic acid (= EPA, C20: 5 Δ5 ' 8 ' 11 ' 14 ' 17 ) or docosahexaenoic acid (= DHA, C 22: 6 Δ4 ' 7 ' 10 ' 13 ' 16 '19 ) important components of human nutrition due to their different roles in health, such as the development of the child's brain, the functionality of the eye, the synthesis of hormones and other signaling substances, as well as the prevention of cardiovascular disease, cancer and diabetes (Poulos, A Lipids 30: 1-14, 1995, Horrocks, LA and Yeo YK Pharmacol Res 40: 211-225, 1999). There is therefore a need for the production of polyunsaturated long-chain fatty acids.
Aufgrund der Heute üblichen Zusammensetzung der menschlichen Nahrung ist ein Zusatz von mehrfach ungesättigten ω-3-Fettsäuren, die bevorzugt in Fischölen vorkommen, zur Nahrung besonders wichtig. So werden beispielsweise mehrfach ungesättigte Fettsäuren wie Docosahexaensäure (= DHA, C22:6Δ4'7'10'13'16'19) oder Eisosapentaensäure (= EPA, C20:5A5'8'11'14'17) Babynahrung zur Erhöhung des
Nährwertes zugesetzt. Der ungesättigten Fettsäure DHA wird dabei ein positiver Effekt auf die Entwicklung und Aufrechterhaltung von Gehirnfunktionen zugeschrieben. Im folgenden werden mehrfach ungesättigte Fettsäuren als PUFA, PUFAs, LCPUFA oder LCPUFAs bezeichnet (p_oly unsaturated fatty acids, PUFA, mehrfach ungesättigte Fettsäuren ;_long chain p_oly unsaturated fatty acids, LCPUFA, langkettige mehrfach ungesättigte Fettsäuren).Due to the customary composition of human food today, addition of polyunsaturated ω-3 fatty acids, which are preferred in fish oils, is particularly important for food. For example, polyunsaturated fatty acids such as docosahexaenoic acid (= DHA, C22: 6 Δ4 ' 7 ' 10 ' 13 ' 16 '19 ) or icosapentaenoic acid (= EPA, C20: 5 A5 ' 8 ' 11 ' 14 '17 ) are used to increase baby food Nutritional value added. The unsaturated fatty acid DHA is thereby attributed a positive effect on the development and maintenance of brain functions. In the following, polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA, polyunsaturated fatty acids, long chain poly unsaturated fatty acids, LCPUFA, long-chain polyunsaturated fatty acids).
Hauptsächlich werden die verschiedenen Fettsäuren und Triglyceride aus Mikroorganismen wie Mortierella oder Schizochytrium oder aus Öl-produzierenden Pflanzen wie Soja, Raps, Algen wie Crypthecodinium oder Phaeodactylum und weiteren gewonnen, wobei sie in der Regel in Form ihrer Triacylglyceride (= Triglyceride = Triglycerole) anfallen. Sie können aber auch aus Tieren wie z.B. Fischen gewonnen werden. Die freien Fettsäuren werden vorteilhaft durch Verseifung hergestellt. Sehr langkettige mehrfach ungesättigte Fettsäuren wie DHA, EPA, Arachidonsäure (= ARA, C20:4Δ5'8'11'14), Dihomo-γ-linolensäure (C20:3Δ8'11'14) oder Docosapentaensäure (DPA, C22:5A7'10'13'16'19) werden in Ölfruchtpflanzen wie Raps, Soja, Sonnenblume, Färber- saflor nicht synthetisiert. Übliche natürliche Quellen für diese Fettsäuren sind Fische wie Hering, Lachs, Sardine, Goldbarsch, Aal, Karpfen, Forelle, Heilbutt, Makrele, Zander oder Thunfisch oder Algen.Mainly the various fatty acids and triglycerides are obtained from microorganisms such as Mortierella or Schizochytrium or from oil-producing plants such as soybean, oilseed rape, algae such as Crypthecodinium or Phaeodactylum and others, where they are usually in the form of their triacylglycerides (= triglycerides = triglycerols). But they can also be obtained from animals such as fish. The free fatty acids are advantageously prepared by saponification. Very long-chain polyunsaturated fatty acids such as DHA, EPA, arachidonic acid (= ARA, C20: 4 Δ5 ' 8 ' 11 '14 ), dihomo-γ-linolenic acid (C20: 3 Δ8 ' 11 '14 ) or docosapentaenoic acid (DPA, C22: 5 A7 ' 10 ' 13 ' 16 ' 19 ) are not synthesized in oilseed crops such as oilseed rape, soy, sunflower, dyer's safflower. Common natural sources of these fatty acids are fish such as herring, salmon, sardine, perch, eel, carp, trout, halibut, mackerel, zander or tuna or algae.
Je nach Anwendungszweck werden Öle mit gesättigten oder ungesättigten Fettsäuren bevorzugt. So werden z.B. in der humanen Ernährung Lipide mit ungesättigten Fettsäuren speziell mehrfach ungesättigten Fettsäuren bevorzugt. Den mehrfach ungesättigten ω-3-Fettsäuren wird dabei ein positiver Effekt auf den Cholesterinspiegel im Blut und damit auf die Möglichkeit der Prävention einer Herzerkrankung zugeschrieben. Durch Zugabe dieser ω-3-Fettsäuren zur Nahrung kann das Risiko einer Herzerkrankung, eines Schlaganfalls oder von Bluthochdruck deutlich verringert werden. Auch entzündliche speziell chronisch entzündliche Prozesse im Rahmen immunologischer Erkrankungen wie rheumatroider Arthritis lassen sich durch ω-3-Fettsäuren positiv beeinflussen. Sie werden deshalb Lebensmitteln speziell diätischen Lebensmitteln zugegeben oder finden in Medikamenten Anwendung. ω-6-Fettsäuren wie Arachidonsäure haben bei diesen rheumatischen Erkrankungen aufgrund unserer üblichen Nahrungsmittelzusammensetzung eher einen negativen Effekt auf diese Krankheiten. ω-3- und ω-6-Fettsäuren sind Vorläufer von Gewebshormonen, den sogenannten Eicosanoiden wie den Prostaglandinen, die sich von der Dihomo-γ-linolensäure, der Arachidonsäure und der Eicosapentaensäure ableiten, den Thromoxanen und Leukotrienen, die sich von der Arachidonsäure und der Eicosapentaensäure ableiten. Eicosanoide (sog. PG2-Serie), die aus ω-6-Fettsäuren gebildet werden fördern in der
Regel Entzündungsreaktionen, während Eicosanoide (sog. PG3-Serie) aus ω-3- Fettsäuren geringe oder keine entzündungsfördernde Wirkung haben.Depending on the application, oils with saturated or unsaturated fatty acids are preferred. For example, lipids with unsaturated fatty acids, especially polyunsaturated fatty acids, are preferred in the human diet. The polyunsaturated ω-3 fatty acids thereby a positive effect on the cholesterol level in the blood and thus the possibility of preventing heart disease is attributed. By adding these ω-3 fatty acids to the diet, the risk of heart disease, stroke or hypertension can be significantly reduced. Also, inflammatory, especially chronic inflammatory processes in the context of immunological diseases such as rheumatoid arthritis can be positively influenced by ω-3 fatty acids. They are therefore added to foods especially dietary foods or found in medicines application. ω-6 fatty acids such as arachidonic acid tend to have a negative effect on these diseases in these rheumatic diseases due to our usual food composition. ω-3- and ω-6 fatty acids are precursors of tissue hormones, the so-called eicosanoids such as the prostaglandins derived from dihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, the thromoxanes and leukotrienes derived from arachidonic acid and Derive the eicosapentaenoic acid. Eicosanoids (so-called PG 2 series), which are formed from ω-6 fatty acids promote in the Usually inflammatory reactions, while eicosanoids (so-called PG3 series) of ω-3 fatty acids have little or no proinflammatory effect.
Aufgrund ihrer positiven Eigenschaften hat es in der Vergangenheit nicht an Ansätzen gefehlt, Gene, die an der Synthese von Fettsäuren bzw. Triglyceriden beteiligt sind, für die Herstellung von Ölen in verschiedenen Organismen mit geändertem Gehalt an ungesättigten Fettsäuren verfügbar zu machen. So wird in WO 91/13972 und seinem US-Äquivalent eine Δ-9-Desaturase beschrieben. In WO 93/1 1245 wird eine Δ-15- Desaturase in WO 94/11516 wird eine Δ-12-Desaturase beansprucht. Weitere Desaturasen werden beispielsweise in EP-A-O 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-O 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 oder Huang et al., Lipids 34, 1999: 649-659 beschrieben. Die biochemische Charakterisierung der verschiedenen Desaturasen ist jedoch bisher nur unzureichend erfolgt, da die Enzyme als membrangebundene Proteine nur sehr schwer zu isolieren und zu charakterisieren sind (McKeon et al., Methods in Enzymol. 71 , 1981 : 12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792). In der Regel erfolgt die Charakterisierung membrangebundener Desaturasen durch Einbringung in einen geeigneten Organismus, der anschließend auf Enzymaktivität mittels Edukt- und Produktanalyse unter- sucht wird. Δ-6-Desatu rasen werden in WO 93/06712, US 5,614,393, US5614393, WO 96/21022, WO00/21557 und WO 99/2711 1 beschrieben und auch die Anwendung zur Produktion in transgenen Organismen beschrieben wie in WO98/46763 WO98/46764, WO9846765. Dabei wird auch die Expression verschiedener Desaturasen wie in WO99/64616 oder WO98/46776 und Bildung polyungesättigter Fettsäuren beschrieben und beansprucht. Bzgl. der Effektivität der Expression von Desaturasen und ihren Einfluss auf die Bildung polyungesättigter Fettsäuren ist anzumerken, dass durch Expression einer einzelnen Desaturase wie bisher beschrieben lediglich geringe Gehalte an ungesättigten Fettsäuren/Lipiden wie z.B. γ-Linolensäure und Stearidon- säure erreicht wurden. Weiterhin wurde in der Regel ein Gemisch aus ω-3- und ω-6- Fettsäuren erhalten.Due to their positive properties, there have been no shortage of attempts in the past to make genes involved in the synthesis of fatty acids or triglycerides available for the production of oils in various organisms with modified levels of unsaturated fatty acids. Thus, WO 91/13972 and its US equivalent describe a Δ-9-desaturase. WO 93/1 1245 claims a Δ15-desaturase in WO 94/11516 a Δ12-desaturase is claimed. Further desaturases are described, for example, in EP-AO 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265 , 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. However, the biochemical characterization of the various desaturases has hitherto been inadequate, since the enzymes are very difficult to isolate and characterize as membrane-bound proteins (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al. , Plant Physiol. Biochem., 26, 1988: 777-792). As a rule, the characterization of membrane-bound desaturases takes place by introduction into a suitable organism, which is subsequently examined for enzyme activity by means of reactant and product analysis. Δ-6 desaturases are described in WO 93/06712, US Pat. No. 5,614,393, US Pat. No. 5,614,393, WO 96/21022, WO00 / 21557 and WO 99/2711 1 and also the application for production in transgenic organisms as described in WO98 / 46763 WO98 / 46764, WO9846765. The expression of various desaturases as described in WO99 / 64616 or WO98 / 46776 and formation of polyunsaturated fatty acids is also described and claimed. Concerning. It should be noted that the expression of desaturases and their influence on the formation of polyunsaturated fatty acids has an effect on expression of a single desaturase, as described so far, only low levels of unsaturated fatty acids / lipids such as e.g. γ-linolenic acid and stearidonic acid were achieved. Furthermore, a mixture of ω-3 and ω-6 fatty acids was obtained as a rule.
Besonders geeignete Mikroorganismen zur Herstellung von PUFAs sind Mikroorganismen wie Mikroalgen wie Phaeodactylum tricornutum, Porphiridium-Arten, Thrausto- chytrien-Arten, Schizochytrien-Arten oder Crypthecodinium-Arten, Ciliaten, wie Stylonychia oder Colpidium, Pilze, wie Mortierella, Entomophthora oder Mucor und/oder Moosen wie Physcomitrella, Ceratodon und Marchantia (R. Vazhappilly & F. Chen (1998) Botanica Marina 41 : 553-558; K. Totani & K. Oba (1987) Lipids 22: 1060- 1062; M. Akimoto et al. (1998) Appl. Biochemistry and Biotechnology 73: 269-278). Durch Stammselektion ist eine Anzahl von Mutantenstämmen der entsprechenden Mikroorganismen entwickelt worden, die eine Reihe wünschenswerter Verbindungen,
einschließlich PUFAs, produzieren. Die Mutation und Selektion von Stämmen mit verbesserter Produktion eines bestimmten Moleküls wie den mehrfach ungesättigten Fettsäuren ist jedoch ein zeitraubendes und schwieriges Verfahren. Deshalb werden, wann immer möglich wie oben beschrieben gentechnologische Verfahren bevorzugt. Mit Hilfe der vorgenannten Mikroorganismen lassen sich jedoch nur begrenzte Mengen der gewünschten mehrfach ungesättigten Fettsäuren wie DPA, EPA oder ARA herstellen. Wobei diese in der Regel je nach verwendeten Mikroorganismus als Fettsäuregemische aus beispielsweise EPA, DPA und ARA anfallen.Particularly suitable microorganisms for the production of PUFAs are microorganisms such as microalgae such as Phaeodactylum tricornutum, Porphiridium species, Thraustochytria species, Schizochytria species or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor and / or mosses such as Physcomitrella, Ceratodon and Marchantia (R. Vazhappilly & F. Chen (1998) Botanica Marina 41: 553-558; K. Totani & K. Oba (1987) Lipids 22: 1060-1062; Akimoto, M. et al. (1998) Appl. Biochemistry and Biotechnology 73: 269-278). Through strain selection, a number of mutant strains of the corresponding microorganisms have been developed, containing a number of desirable compounds, including PUFAs, produce. However, mutation and selection of strains with improved production of a particular molecule, such as polyunsaturated fatty acids, is a time-consuming and difficult procedure. Therefore, whenever possible, genetic engineering techniques are preferred as described above. With the aid of the aforementioned microorganisms, however, only limited amounts of the desired polyunsaturated fatty acids such as DPA, EPA or ARA can be produced. These are usually obtained depending on the microorganism used as fatty acid mixtures of, for example, EPA, DPA and ARA.
Für die Synthese von Arachidonsäure, Eicosapentaensäure (EPA) und Docosahe- xaensäure (DHA) werden verschiedene Synthesewege diskutiert. So erfolgt die Produktion von EPA bzw. DHA in marinen Bakterien wie Vibrio sp. oder Shewanella sp. nach dem Polyketid-Weg (Yu, R. et al. Lipids 35:1061-1064, 2000; Takeyama, H. et al. Microbiology 143:2725-2731 , 1997).For the synthesis of arachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), various synthetic routes are discussed. Thus, the production of EPA or DHA in marine bacteria such as Vibrio sp. or Shewanella sp. according to the polyketide pathway (Yu, R. et al., Lipids 35: 1061-1064, 2000; Takeyama, H. et al., Microbiology 143: 2725-2731, 1997).
Ein alternative Strategie verläuft über die wechselnde Aktivität von Desaturasen und Elongasen (Zank, T.K. et al. Plant Journal 31 :255-268, 2002; Sakuradani, E. et al. Gene 238:445-453, 1999). Eine Modifikation des beschriebenen Weges über Δ6- Desaturase, Δ6-Elongase, Δ5-Desaturase, Δ5-Elongase, Δ4-Desaturase ist der Sprecher-Syntheseweg (Sprecher 2000, Biochim. Biophys. Acta 1486:219-231 ) in Säugetieren. Anstelle der Δ4-Desaturierung erfolgt hier ein weiterer Elongationsschritt auf C24, eine weitere Δ6-Desaturierung und abschliessend eine ß-Oxidation auf die C22- Kettenlänge. Für die Herstellung in Pflanzen und Mikroorganismen ist der sogenannte Sprecher-Syntheseweg allerdings nicht geeignet, da die Regulationsmechanismen nicht bekannt sind.An alternative strategy involves the changing activity of desaturases and elongases (Zank, TK et al., Plant Journal 31: 255-268, 2002, Sakuradani, E. et al., Gene 238: 445-453, 1999). A modification of the described pathway via Δ6-desaturase, Δ6-elongase, Δ5-desaturase, Δ5-elongase, Δ4-desaturase is the speaker-synthetic pathway (Sprecher 2000, Biochim Biophys Acta 1486: 219-231) in mammals. Chain length - instead of the Δ4-desaturation, a further elongation step at 24 C, a further Δ6-desaturation and finally beta-oxidation to the C 22 is carried out here. For the production in plants and microorganisms, however, the so-called spokesman synthesis route is not suitable because the regulatory mechanisms are not known.
Die polyungesättigten Fettsäuren können entsprechend ihrem Desaturierungsmuster in zwei große Klassen, in ω-6- oder ω-3-Fettsäuren eingeteilt werden, die metabolisch und funktionell unterschiedlich Aktivitäten haben .The polyunsaturated fatty acids can be divided according to their desaturation pattern into two major classes, ω-6 or ω-3 fatty acids, which have metabolically and functionally different activities.
Als Ausgangsprodukt für den ω-6-Stoffwechselweg fungiert die Fettsäure Linolsäure (18:2Δ9 12), während der ω-3-Weg über Linolensäure (18:3Λ9 12/I5) abläuft. Linolensäure wird dabei durch Aktivität einer ω-3-Desaturase gebildet (Tocher et al. 1998, Prog. Lipid Res. 37, 73-117 ; Domergue et al. 2002, Eur. J. Biochem. 269, 4105-4113). Säugetiere und damit auch der Mensch verfügen über keine entsprechende Desatura- seaktivität (Δ-12- und ω-3-Desaturase) und müssen diese Fettsäuren (essentielle Fettsäuren) über die Nahrung aufnehmen. Über die Abfolge von Desaturase- und Elongase-Reaktionen werden dann aus diesen Vorstufen die physiologisch wichtigen polyungesättigten Fettsäuren Arachidonsäure (= ARA, 20:4A5'8'11'14), eine ω-6-Fettsäure und die beiden ω-3-Fettsäuren Eicosapentaen- (= EPA, 20:5A5'8'11'14'17) und Docosahe-
O xaensäure (DHA, 22:6Δ4'7'10'13'17'19) synthetisiert. Die Applikation von ω-3-Fettsäuren zeigt dabei die wie oben beschrieben therapeutische Wirkung bei der Behandlung von Herz-Kreislaufkrankheiten (Shimikawa 2001 , World Rev. Nutr. Diet. 88, 100-108), Entzündungen (Calder 2002, Proc. Nutr. Soc. 61 , 345-358) und Arthridis (Cleland und James 2000, J. Rheumatol. 27, 2305-2307).The starting material for the ω-6 pathway is the fatty acid linoleic acid (18: 2 Δ9 12 ), while the ω-3 pathway is via linolenic acid (18: 3 Λ9 12 / I5 ). Linolenic acid is formed by the activity of an ω-3-desaturase (Tocher et al., 1998, Prog. Lipid Res., 37, 73-117, Domergue et al., 2002, Eur. J. Biochem., 269, 4105-4113). Mammals, and therefore also humans, have no corresponding desaturase activity (Δ-12 and ω-3-desaturase) and must ingest these fatty acids (essential fatty acids) through the diet. From the sequence of desaturase and elongase reactions, the physiologically important polyunsaturated fatty acids arachidonic acid (= ARA, 20: 4 A5 ' 8 ' 11 '14 ), an ω-6 fatty acid and the two ω-3 are then converted from these precursors. Fatty acids eicosapentaen (= EPA, 20: 5 A5 ' 8 ' 11 ' 14 ' 17 ) and docosahe- O xaensäure (DHA, 22: 6 Δ4 ' 7 ' 10 ' 13 ' 17 '19 ) synthesized. The application of ω-3 fatty acids shows the therapeutic effect as described above in the treatment of cardiovascular diseases (Shimikawa 2001, World Rev. Nutr., Diet, 88, 100-108), inflammations (Calder 2002, Proc. Soc., 61, 345-358) and Arthridis (Cleland and James 2000, J. Rheumatol., 27, 2305-2307).
Die Verlängerung von Fettsäuren durch Elongasen um 2 bzw. 4 C-Atome ist für die Produktion von C20- bzw. C22-PUFAS von entscheidender Bedeutung. Dieser Prozess verläuft über 4 Stufen. Der erste Schritt stellt die Kondensation von Malonyl-CoA an das Fettsäure-Acyl-CoA durch die Ketoacyl-CoA-Synthase (KCS, im weiteren Text als Elongase bezeichnet). Es folgt dann ein Reduktionschritt (Ketoacyl-CoA-Reduktase, KCR), ein Dehydratationsschritt (Dehydratase) und ein abschliessender Reduktionsschritt (enoyl-CoA-Reduktase). Es wurde postuliert, dass die Aktivität der Elongase die Spezifität und Geschwindigkeit des gesamten Prozesses beeinflussen (Miliar and Kunst, 1997 Plant Journal 12:121-131 ).The elongation of fatty acids by elongases by 2 or 4 C atoms is of crucial importance for the production of C 2 O or C 22 PUFAs. This process runs over 4 stages. The first step is the condensation of malonyl-CoA on the fatty acyl-CoA by ketoacyl-CoA synthase (KCS, hereinafter referred to as elongase). This is followed by a reduction step (ketoacyl-CoA reductase, KCR), a dehydration step (dehydratase) and a final reduction step (enoyl-CoA reductase). It has been postulated that the activity of elongase affects the specificity and speed of the entire process (Miliar and Kunst, 1997 Plant Journal 12: 121-131).
In der Vergangenheit wurden zahlreiche Versuche unternommen, Elongase Gene zu erhalten. Miliar and Kunst, 1997 (Plant Journal 12:121-131 ) und Miliar et al. 1999, (Plant Cell 1 1 :825-838) beschreiben die Charakterisierung von pflanzlichen Elongasen zur Synthese von einfachungesättigten langkettigen Fettsäuren (C22:1 ) bzw. zur Synthese von sehr langkettigen Fettsäuren für die Wachsbildung in Pflanzen (C2S-C32). Beschreibungen zur Synthese von Arachidonsäure und EPA finden sich beispielsweise in WO0159128, WO0012720, WO02077213 und WO0208401. Die Synthese von mehrfachungesättigter C24 Fettsäuren ist beispielsweise in Tvrdik et al 2000, JCB 149:707-717 oder WO0244320 beschrieben.In the past, numerous attempts have been made to obtain elongase genes. Miliar and Art, 1997 (Plant Journal 12: 121-131) and Miliar et al. 1999, (Plant Cell 1 1: 825-838) describe the characterization of plant elongases for the synthesis of monounsaturated long-chain fatty acids (C22: 1) or for the synthesis of very long-chain fatty acids for wax formation in plants (C 2 SC 32 ). Descriptions of the synthesis of arachidonic acid and EPA can be found, for example, in WO0159128, WO0012720, WO02077213 and WO0208401. The synthesis of polyunsaturated C24 fatty acids is described, for example, in Tvrdik et al 2000, JCB 149: 707-717 or WO0244320.
Zur Herstellung von DHA (C22:6 n-3) in Organismen, die diese Fettsäure natürlicherweise nicht produzieren, wurde bisher keine spezifische Elongase beschrieben. Bisher wurden nur Elongasen beschrieben, die C2o- bzw. C24-Fettsäuren bereitstellen. Eine Δ- 5-Elongase-Aktivität wurde bisher noch nicht beschrieben.To produce DHA (C22: 6n-3) in organisms that do not naturally produce this fatty acid, no specific elongase has yet been described. So far, only elongases have been described which provide C 2 O or C 24 fatty acids. Δ5-elongase activity has not been described previously.
Höhere Pflanzen enthalten mehrfach ungesättigte Fettsäuren wie Linolsäure (C18:2) und Linolensäure (C18:3). ARA, EPA und DHA kommen im Samenöl höherer Pflanzen gar nicht oder nur in Spuren vor (E. Ucciani: Nouveau Dictionnaire des Huiles Vegeta- les. Technique & Documentation - Lavoisier, 1995. ISBN: 2-7430-0009-0). Es wäre jedoch vorteilhaft, in höheren Pflanzen, bevorzugt in Ölsaaten wie Raps, Lein, Sonnenblume und Soja, LCPUFAs herzustellen, da auf diese Weise große Mengen qualitativ hochwertiger LCPUFAs für die Lebensmittelindustrie, die Tierernährung und für pharmazeutische Zwecke kostengünstig gewonnen werden können. Hierzu müssen vorteilhaft über gentechnische Methoden Gene kodierend für Enzyme der Biosynthese
von LCPUFAs in Ölsaaten eingeführt und exprimiert werden. Dies sind Gene, die beispielsweise für Δ-6-Desaturasen, Δ-6-Elongasen, Δ-5-Desaturasen oder Δ-4- Desaturasen codieren. Diese Gene können vorteilhaft aus Mikroorganismen und niederen Pflanzen isoliert werden, die LCPUFAs herstellen und in den Membranen oder Triacylglyceriden einbauen. So konnten bereits Δ-6-Desaturase-Gene aus dem Moos Physcomitrella patens und Δ-6-Elongase-Gene aus P. patens und dem Nematoden C. elegans isoliert.Higher plants contain polyunsaturated fatty acids such as linoleic acid (C18: 2) and linolenic acid (C18: 3). ARA, EPA and DHA are absent or only found in the seed oil of higher plants (E. Ucciani: Nouveau Dictionnaire des Huiles Vegetales, Technique & Documentation - Lavoisier, 1995. ISBN: 2-7430-0009-0). However, it would be advantageous to produce LCPUFAs in higher plants, preferably in oilseeds such as oilseed rape, linseed, sunflower and soybeans, as this will enable large quantities of high quality LCPUFAs to be obtained inexpensively for the food, animal and pharmaceutical industries. For this purpose, gene coding for enzymes of biosynthesis must be advantageous via genetic engineering methods introduced and expressed by LCPUFAs in oilseeds. These are genes encoding for example Δ6-desaturases, Δ6-elongases, Δ5-desaturases or Δ4-desaturases. These genes can be advantageously isolated from microorganisms and lower plants that produce LCPUFAs and incorporate them into the membranes or triacylglycerides. For example, Δ6-desaturase genes from the moss Physcomitrella patens and Δ6 elongase genes from P. patens and the nematode C. elegans have been isolated.
Erste transgene Pflanzen, die Gene kodierend für Enzyme der LCPUFA-Biosynthese enthalten und exprimieren und LCPUFAs produzieren wurden beispielsweise in DE 102 19 203 (Verfahren zur Herstellung mehrfach ungesättigter Fettsäuren in Pflanzen) erstmals beschrieben. Diese Pflanzen produzieren allerdings LCPUFAs in Mengen, die für eine Aufarbeitung der in den Pflanzen enthaltenen Öle noch weiter optimiert werden müssen.First transgenic plants which contain and express genes encoding enzymes of LCPUFA biosynthesis and produce LCPUFAs have been described for the first time in, for example, DE 102 19 203 (Process for the Production of Polyunsaturated Fatty Acids in Plants). However, these plants produce LCPUFAs in quantities that need to be further optimized for processing the oils contained in the plants.
Um eine Anreicherung der Nahrung und des Futters mit diesen mehrfach ungesättigten Fettsäuren zu ermöglichen, besteht daher ein großer Bedarf an Mitteln und Maßnahmen für eine einfache, kostengünstige Herstellung dieser mehrfach ungesättigten Fettsäuren speziell in eukaryontischen Systemen. Die der vorliegende Erfindung zugrunde liegende Aufgabe wäre also die ereitstellung von solchen Mitteln und Maßnahmen. Die Aufgabe wird durch die Ausführungsformen gelöst, die in den Patentansprüchen und im Folgenden beschrieben werden.Therefore, in order to facilitate fortification of food and feed with these polyunsaturated fatty acids, there is a great need for means and measures for simple, inexpensive production of these polyunsaturated fatty acids, especially in eukaryotic systems. The object underlying the present invention would therefore be the provision of such means and measures. The object is achieved by the embodiments which are described in the patent claims and in the following.
Die vorliegende Erfindung betrifft somit ein Polynucleotid umfassend eine Nukleinsäu- resequenz, die ausgewählt ist aus der Gruppe bestehend aus:The present invention thus relates to a polynucleotide comprising a nucleic acid sequence which is selected from the group consisting of:
(a) Nucleinsäuresequenz wie in einer der SEQ ID No. 1 , 3, 5, 7, 9, 1 1 , 13, oder 15 gezeigt;(a) Nucleic acid sequence as in one of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, or 15;
(b) Nucleinsäuresequenz, die ein Polypeptid kodiert, das eine Aminosäuresequenz wie in einer der SEQ ID No. 2, 4, 6, 8, 10, 12, 14 oder 16 gezeigt aufweist;(b) nucleic acid sequence encoding a polypeptide having an amino acid sequence as in any one of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16;
(c) Nucleinsäuresequenz, die ein Polypeptid kodiert, das mindestens 70% identisch zu einem Polypeptid ist, das von der Nucleinsäurese- quenzen aus (a) oder (b) kodiert wird, wobei das Polypeptid Desatu- rase oder Elongase Aktivität aufweist; und
(d) Nucleinsäuresequenz für ein Fragment einer Nucleinsäure aus (a), (b) oder (c), woei das Fragment ein Polypeptid mit einer Desaturase oder Elongase Aktivität kodiert.(c) a nucleic acid sequence encoding a polypeptide that is at least 70% identical to a polypeptide encoded by the nucleic acid sequences of (a) or (b), the polypeptide having desaturase or elongase activity; and (d) a nucleic acid sequence for a fragment of a nucleic acid of (a), (b) or (c), wherein the fragment encodes a polypeptide having a desaturase or elongase activity.
Der Begriff "Polynucleotid" betrifft erfindungsgemäß Polynucleotide, die Nukleinsäure- sequenzen umfassen, die Polypeptide mit Desaturase oder Elongase Aktivität kodieren. Die Desaturase oder Elongase Aktivitäten werden vorzugsweise für die Biosynthese von Lipiden oder Fettsäuren benötigt. Besonders bevorzugt handelt es sich um die folgenden Desaturase bzw. Elongase Aktivitäten: Δ-4-Desaturase, Δ-5-Desaturase, Δ- 5-Elongase, Δ-6-Desaturase, Δ-6-Elongase oder Δ-12-Desaturase. Die Desaturasen und/oder Elongasen sind bevorzugt an der Synthese mehrfach ungesättigter Fettsäuren (PUFAs) und besonders bevorzugt an der Synthese lankettiger PUFAs (LCPUFAs) beteiligt. Geeignete Nachweissysteme für diese Desaturase bzw. Elongase Aktivitäten sind in den Beispielen oder in WO2005/083053 beschrieben. Besonders bevorzugt handelt es sich bei den zuvor genannten Aktivitäten hinsichtlich Substratspezifitäten und Umsetzungsraten um die der jeweiligen Enzyme aus Ostereococcus lucimarinus. Die erfindungsgemäßen spezifischen Polynucleotide, d.h. die Polynucleotide mit einer Nukleinsäuresequenz gemäß SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13 oder 15, wurden aus Ostereococcus lucimarinus gewonnen.The term "polynucleotide" according to the invention relates to polynucleotides comprising nucleic acid sequences encoding polypeptides having desaturase or elongase activity. The desaturase or elongase activities are preferably required for the biosynthesis of lipids or fatty acids. Particularly preferred are the following desaturase or elongase activities: Δ-4-desaturase, Δ-5-desaturase, Δ-5 elongase, Δ-6-desaturase, Δ-6 elongase or Δ-12-desaturase. The desaturases and / or elongases are preferably involved in the synthesis of polyunsaturated fatty acids (PUFAs) and more preferably in the synthesis of long-chain PUFAs (LCPUFAs). Suitable detection systems for these desaturase or elongase activities are described in the examples or in WO2005 / 083053. With regard to substrate specificities and conversion rates, the abovementioned activities are particularly preferably those of the respective enzymes from Ostereococcus lucimarinus. The specific polynucleotides of the invention, i. the polynucleotides having a nucleic acid sequence according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 were obtained from Ostereococcus lucimarinus.
Insbesondere sind also erfindungsgemäße Polynucleotide:In particular, therefore, polynucleotides according to the invention are:
Polynucleotide, die ein Polypeptid mit Δ-12-Desaturase Aktivität kodieren und (i) eine Nucleinsäuresequenz umfassen, wie in SEQ ID NO: 1 oder 3 gezeigt, (ii) eine Nuclein- süresequenz umfassen, die für ein Polypeptid kodiert wie in SEQ ID NO: 2 oder 4 gezeigt, (iii) eine Nucleinsäuresequenz umfassen, die mindestens 83% identisch zu einer der Nucleinsäuresequenzen aus (i) oder (ii) ist, oder (iv) eine Nucleinsäuresequenz eines Fragments einer Nucleinsäure aus (i), (ii) oder (iii).Polynucleotides which encode a polypeptide having Δ-12-desaturase activity and (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 1 or 3, (ii) a nucleic acid sequence encoding a polypeptide as in SEQ ID NO: 2 or 4, (iii) comprise a nucleic acid sequence which is at least 83% identical to any of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acid of (i), (ii ) or (iii).
Polynucleotide, die ein Polypeptid mit Δ-4-Desaturase Aktivität kodieren und (i) eine Nucleinsäuresequenz umfassen, wie in SEQ ID NO: 5 gezeigt, (ii) eine Nucleinsürese- quenz umfassen, die für ein Polypeptid kodiert wie in SEQ ID NO: 6 gezeigt, (iii) eine Nucleinsäuresequenz umfassen, die mindestens 72% identisch zu einer der Nuclein- säuresequenzen aus (i) oder (ii) ist, oder (iv) eine Nucleinsäuresequenz eines Fragments einer Nucleinsäure aus (i), (ii) oder (iii).Polynucleotides which encode a polypeptide having Δ-4-desaturase activity and (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 5, (ii) comprise a nucleic acid sequence coding for a polypeptide as shown in SEQ ID NO: 6, (iii) comprise a nucleic acid sequence which is at least 72% identical to one of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acid of (i), (ii) or (iii).
Polynucleotide, die ein Polypeptid mit Δ-5-Desaturase Aktivität kodieren und (i) einePolynucleotides encoding a Δ5-desaturase activity polypeptide; and (i) a
Nucleinsäuresequenz umfassen, wie in SEQ ID NO: 7 oder 9 gezeigt, (ii) eine Nuclein- süresequenz umfassen, die für ein Polypeptid kodiert wie in SEQ ID NO: 8 oder 10
gezeigt, (iii) eine Nucleinsäuresequenz umfassen, die mindestens 72% identisch zu einer der Nucleinsäuresequenzen aus (i) oder (ii) ist, oder (iv) eine Nucleinsäuresequenz eines Fragments einer Nucleinsäure aus (i), (ii) oder (iii).Nucleic acid sequence comprise, as shown in SEQ ID NO: 7 or 9, (ii) a nucleic acid sequence encoding a polypeptide as in SEQ ID NO: 8 or 10 (iii) comprise a nucleic acid sequence which is at least 72% identical to any one of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acid of (i), (ii) or (iii) ,
Polynucleotide, die ein Polypeptid mit Δ-5-Elongase Aktivität kodieren und (i) eine Nucleinsäuresequenz umfassen, wie in SEQ ID NO: 11 gezeigt, (ii) eine Nucleinsüre- sequenz umfassen, die für ein Polypeptid kodiert wie in SEQ ID NO: 12 gezeigt, (iii) eine Nucleinsäuresequenz umfassen, die mindestens 78% identisch zu einer der Nucleinsäuresequenzen aus (i) oder (ii) ist, oder (iv) eine Nucleinsäuresequenz eines Fragments einer Nucleinsäure aus (i), (ii) oder (iii).Polynucleotides which encode a Δ5-elongase activity polypeptide and (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 11, (ii) comprise a nucleic acid sequence encoding a polypeptide as shown in SEQ ID NO: 12, (iii) comprise a nucleic acid sequence at least 78% identical to any one of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a nucleic acid fragment of (i), (ii) or (iii ).
Polynucleotide, die ein Polypeptid mit Δ-6-Desaturase Aktivität kodieren und (i) eine Nucleinsäuresequenz umfassen, wie in SEQ ID NO: 13 gezeigt, (ii) eine Nucleinsüre- sequenz umfassen, die für ein Polypeptid kodiert wie in SEQ ID NO: 14 gezeigt, (iii) eine Nucleinsäuresequenz umfassen, die mindestens 72% identisch zu einer der Nucleinsäuresequenzen aus (i) oder (ii) ist, oder (iv) eine Nucleinsäuresequenz eines Fragments einer Nucleinsäure aus (i), (ii) oder (iii).Polynucleotides which encode a polypeptide having Δ-6-desaturase activity and (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 13, (ii) comprise a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 14, (iii) comprise a nucleic acid sequence which is at least 72% identical to any one of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a fragment of a nucleic acid of (i), (ii) or (iii ).
Polynucleotide, die ein Polypeptid mit Δ-6-Elongase Aktivität kodieren und (i) eine Nucleinsäuresequenz umfassen, wie in SEQ ID NO: 15 gezeigt, (ii) eine Nucleinsüre- sequenz umfassen, die für ein Polypeptid kodiert wie in SEQ ID NO: 16 gezeigt, (iii) eine Nucleinsäuresequenz umfassen, die mindestens 71 % identisch zu einer der Nucleinsäuresequenzen aus (i) oder (ii) ist, oder (iv) eine Nucleinsäuresequenz eines Fragments einer Nucleinsäure aus (i), (ii) oder (iii).Polynucleotides which encode a polypeptide having Δ-6 elongase activity and (i) comprise a nucleic acid sequence as shown in SEQ ID NO: 15, (ii) comprise a nucleic acid sequence which codes for a polypeptide as shown in SEQ ID NO: 16, (iii) comprise a nucleic acid sequence that is at least 71% identical to any of the nucleic acid sequences of (i) or (ii), or (iv) a nucleic acid sequence of a nucleic acid fragment of (i), (ii) or (iii ).
Es versteht sich, dass die zuvor genannten spezifischen Sequenzen angesichts des degenerierten genetischen Codes auch verändert werden können wobei von den veränderten Polynucleotiden immer noch Polypeptide mit einer Aminosäuresequenz gemäß einer der SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 oder 16 kodiert werden, die die zuvor genannten Desaturase bzw. Elongase Aktivitäten aufweisen.It will be appreciated that the aforementioned specific sequences may also be altered in view of the degenerate genetic code, wherein of the altered polynucleotides, polypeptides having an amino acid sequence according to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 or 16, which have the abovementioned desaturase or elongase activities.
Der Begriff "Polynucleotid" umfasst auch Varianten der zuvor genannten spezifischen Polynucleotide. Bei diesen kann es sich um Homologe, Orthologe oder Paraloge Sequenzen handeln. Solche Varainaten umfassen Nukleinsäuresequenzen, die mindesten einen Basenaustausch, eine Basenaddition oder eine Basendeletion aufweisen, wobei die Varaianten immer noch ein Polypeptid mit der zuvor genannten biologischen Aktivität der jeweiligen Ausgangssequenz kodieren sollen. Varaianten umfassen Polynucleotide, die mit den zuvor genannten Polynucleotiden hybridisieren können, bevorzugt unter stringenten Bedingungen. Besonders bevorzugte stringente Bedingungen sind dem Fachmann bekannt und lassen sich in Current Protocols in
Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6., finden. Ein bevorzugtes Beispiel für stringente Hybridisierungsbedingungen sind Hybridisierungen in 6 x Natriumchlorid/Natriumcitrat (sodium chloride/sodiumcitrate = SSC) bei etwa 45°C, gefolgt von einem oder mehreren Waschschritten in 0,2 x SSC, 0,1 % SDS bei 50 bis 65°C. Dem Fachmann ist bekannt, dass diese Hybridisierungsbedingungen sich je nach dem Typ der Nukleinsäure und, wenn beispielsweise organische Lösungsmittel vorliegen, hinsichtlich der Temperatur und der Konzentration des Puffers unterscheiden. Die Temperatur unterscheidet sich beispielsweise unter "Standard- Hybridisierungsbedingungen" je nach dem Typ der Nukleinsäure zwischen 42°C und 58°C in wässrigem Puffer mit einer Konzentration von 0,1 bis 5 x SSC (pH 7,2). Falls organisches Lösungsmittel im obengenannten Puffer vorliegt, zum Beispiel 50 % Formamid, ist die Temperatur unter Standardbedingungen etwa 42°C. Vorzugsweise sind die Hybridisierungsbedingungen für DNA:DNA-Hybride zum Beispiel 0,1 x SSC und 200C bis 45°C, vorzugsweise zwischen 300C und 45°C. Vorzugsweise sind die Hybridisierungsbedingungen für DNA:RNA-Hybride zum Beispiel 0,1 x SSC und 300C bis 55°C, vorzugsweise zwischen 45°C und 55°C. Die vorstehend genannten Hybridi- sierungstemperaturen sind beispielsweise für eine Nukleinsäure mit etwa 100 bp (= Basenpaare) Länge und einem G + C-Gehalt von 50 % in Abwesenheit von Formamid bestimmt. Der Fachmann weiß, wie die erforderlichen Hybridisierungsbedingungen anhand von Lehrbüchern, wie dem vorstehend erwähnten oder aus den folgenden Lehrbüchern Sambrook et al., "Molecular Cloning", CoId Spring Harbor Laboratory, 1989; Harnes und Higgins (Hrsgb.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Hrsgb.) 1991 , "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford, bestimmt werden können. Alternativ können Varaianten der erfindungsgemäßen spezifischen Polynucleotide auch durch Polymerasekettenreaktion (PCR) basierte Verfahren bereitgestellt werden. Hierzu können zunächst Primer von konservierten Sequenzen (z.B. Sequenzen, die funktionelle Domänen im Polypeptid kodieren) abgeleitet werden. Konserviere Sequenzen können durch Sequenzvergleiche mit Polynucleotiden, die Polypeptide ähnlicher Aktivität kodieren, ermittelt werden. Als Matritze kann DNA oder cDNA aus Bakterien, Pilzen, Pflanzen oder Tieren vedrwendet werden. DNA Fragmente, die durch die PCR erhalten wurde, können zum Screening von entsprechenden genomischen oder cDNA Bibliotheken verwendet werden um, - falls erforderlich - den kompletten offenen Leserahmen des Polynucleotids zu islieren und durch Sequenzierung zu ermitteln. Weitere Varianten umfassen Polynucleotide, die eine Nukleinsäuresequnz umfassen, die mindestens 70%, mindestens 75%, mindestens 80%, mindesten 81%, mindestens 82%, mindestens 83%, mindestens 84%, mindestens 85%, mindestens 86%, mindestens 87%, mindestens 88%, mindestens 89%, mindestens 90%, mindestens 91%, mindestens 92%, mindestens 93%, mindestens 94%, mindestens 95%, mindestens 96%, mindestens 97%, mindestens
98% oder mindestens 99% oder zu einem anderen hierin genannten Prozentsatz identisch ist mit einer der zuvor genannten spezifischen Nukleinsäuresequenzen und ein Polypeptid mit der jeweiligen biologischen Aktivität kodiert.. Ebenso umfasst sind Polynucleotide, die Nukleinsäuresequenzen umfassen, die ein Polypeptid mit einer Aminosäuresequenz kodieren, die mindestens 70%, mindestens 75%, mindestens 80%, mindesten 81%, mindestens 82%, mindestens 83%, mindestens 84%, mindestens 85%, mindestens 86%, mindestens 87%, mindestens 88%, mindestens 89%, mindestens 90%, mindestens 91 %, mindestens 92%, mindestens 93%, mindestens 94%, mindestens 95%, mindestens 96%, mindestens 97%, mindestens 98% oder mindestens 99% oder zu einem anderen hierin genannten Prozentsatz identisch ist mit einer der zuvor genannten spezifischen Aminosäuresequenzen und wobei das Polypeptid die jeweilige biologische Aktivität der Ausgangssequenz aufweist. Der Prozentsatz identischer Nucleotide oder Aminosäuren bezieht sich vorzugsweise auf einen Sequenzabschnitt von mindestens 50% der zu vergleichenden Sequenzen und besonders bevorzugt überdie gesamte Länge der zu vergleichenden Sequenzen. Eine Vielzahl von Programmen, die Algorithmen für solche vergleiche implementieren ist im Stand der Technik eschrieben und kommerziell erhältlich. Insbesondere sei auf die Algorithmen von Needleman und Wunsch oder Smith und Waterman verwiesen, die besonders zuverlässige Ergebnisse liefern. Diese Algorithmen können vorzugsweise durch die folgenden Programme implementiert werden: PiIeUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153), Gap und BestFit (Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) und Smith und Waterman (Adv. Appl. Math. 2; 482-489 (1981 ))), als Teil der GCG Software [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 (1991 )]. Besonders bevorzugt wird der Prozentsatz (%) der Sequenzidentität im Rahmen der vorliegenden Erfindung mit dem GAP Programm über die gesammte Sequenz mit folgenden festgelegten Größen bestimmt: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 und Average Mismatch: 0.000.The term "polynucleotide" also encompasses variants of the aforementioned specific polynucleotides. These may be homologs, orthologues or paralogue sequences. Such varainates comprise nucleic acid sequences which have at least one base exchange, one base addition or one base deletion, wherein the variants should still encode a polypeptide having the aforementioned biological activity of the respective starting sequence. Varieties include polynucleotides that can hybridize with the aforementioned polynucleotides, preferably under stringent conditions. Particularly preferred stringent conditions are known in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A preferred example of stringent hybridization conditions are hybridizations in 6x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 50-65 ° C. It is known to those skilled in the art that these hybridization conditions differ with respect to the type of nucleic acid and, for example, when organic solvents are present, with respect to the temperature and the concentration of the buffer. The temperature differs, for example under "standard hybridization conditions" depending on the type of nucleic acid between 42 ° C and 58 ° C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2). If organic solvent is present in the above buffer, for example 50% formamide, the temperature is about 42 ° C under standard conditions. Preferably, the hybridization conditions for DNA: DNA hybrids are, for example, 0.1 x SSC and 20 0 C to 45 ° C, preferably between 30 0 C and 45 ° C. Preferably, the hybridization conditions for DNA: RNA hybrids are, for example, 0.1 x SSC and 30 0 C to 55 ° C, preferably between 45 ° C and 55 ° C. The abovementioned hybridization temperatures are determined, for example, for a nucleic acid of about 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. Those skilled in the art will understand how to obtain the required hybridization conditions from textbooks such as the aforementioned or the following textbooks, Sambrook et al., "Molecular Cloning", CoId Spring Harbor Laboratory, 1989; Harnes and Higgins (Eds.) 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed.) 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford. Alternatively, variants of the specific polynucleotides of the invention may also be provided by polymerase chain reaction (PCR) based methods. For this purpose, primers can first be derived from conserved sequences (eg sequences encoding functional domains in the polypeptide). Conserved sequences can be determined by sequence comparisons with polynucleotides encoding polypeptides of similar activity. As a template, DNA or cDNA from bacteria, fungi, plants or animals can be used. DNA fragments obtained by the PCR can be used to screen appropriate genomic or cDNA libraries to isolate, if necessary, the complete open reading frame of the polynucleotide and to determine by sequencing. Other variants include polynucleotides comprising a nucleic acid sequence comprising at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%. , at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or at any other percentage referred to herein, is identical to any one of the aforementioned specific nucleic acid sequences and a polypeptide encodes the particular biological activity. Also included are polynucleotides comprising nucleic acid sequences encoding a polypeptide having an amino acid sequence, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or at any other percentage referred to herein is identical to one of aforementioned specific amino acid sequences and wherein the polypeptide has the respective biological activity of the starting sequence. The percentage of identical nucleotides or amino acids preferably refers to a sequence segment of at least 50% of the sequences to be compared and most preferably the full length of the sequences to be compared. A variety of programs that implement algorithms for such comparisons are known in the art and commercially available. In particular, reference is made to the algorithms of Needleman and Wunsch or Smith and Waterman, which provide particularly reliable results. These algorithms may preferably be implemented by the following programs: PiIeUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153), Gap and Bestfit (Needleman and Wunsch Biol. 48: 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2: 482-489 (1981)), as part of the GCG Software [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 (1991)]. More preferably, the percentage (%) of sequence identity within the scope of the present invention is determined with the GAP program over the entire sequence with the following fixed sizes: Gap Weight: 50, Length Weight: 3, Average Match: 10,000, and Average Mismatch: 0.000.
Ein Polynucleotid, das lediglich ein Fragment der zuvor genannten Nukleinsäuresequenzen umfasst, ist auch ein erfindungsgemäßes Polynucleotid. Das Fragment soll hierbei ein Polypeptid kodieren, das die biologische Aktivität der Ausgangssequenz bzw. des davon kodierten Polypeptids aufweist. Polypeptide, die von solchen Polynuc- leotiden kodiert werden, umfassen oder bestehen daher aus Domänen der zuvor genannten spezifischen Polypeptide (Ausgangspolypeptide), die die biologische Aktivität vermitteln. Ein Fragment im Sinne der Erfindung umfasst vorzugsweise mindestens 50, mindestens 100, mindestens 250 oder mindestens 500 aufeinanderfolgende Nucleotide der zuvor genannten spezifischen Sequenzen oder kodiert eine Aminosäuresequenz umfassend mindestens 20, mindestens 30, mindestens 50,
mindestens 80, mindestens 100 oder mindestens 150 aufeinanderfolgende Aminosäuren einer der zuvor genannten spezifischen Aminosäuresequenzen.A polynucleotide comprising only a fragment of the aforementioned nucleic acid sequences is also a polynucleotide of the invention. The fragment is intended to encode a polypeptide which has the biological activity of the starting sequence or of the polypeptide encoded therefrom. Polypeptides encoded by such polynucleotides therefore comprise or consist of domains of the aforementioned specific polypeptides (parent polypeptides) which mediate biological activity. A fragment according to the invention preferably comprises at least 50, at least 100, at least 250 or at least 500 consecutive nucleotides of the abovementioned specific sequences or encodes an amino acid sequence comprising at least 20, at least 30, at least 50, at least 80, at least 100, or at least 150 consecutive amino acids of any one of the aforementioned specific amino acid sequences.
Die erfindungsgemäßen Polynucleotid- Varianten weisen vorzugsweise mindestens 10%, mindestens 20%, mindestens 30%, mindestens 40%, mindestens 50%, mindestens 60%, mindestens 70%, mindestens 80% oder mindestens 90% der jeweiligen biologischen Aktivität des Polypeptids, das von der Ausgangssequenz kodiert wird, auf. D.h. die Polypeptide, die von den erfindungsgemäßen Polynucleotiden kodiert werden, können am Stoffwechsel von zum Aufbau von Fettsäuren, Fettsäureester wie Dia- cylglyceride und/oder Triacylglyceride in einem Organismus, bevorzugt in einer Pflanze oder Pflanzenzelle, notwendigen Verbindungen oder am Transport von Molekülen über Membranen teilnehmen, wobei Ciβ-, C20- oder C22-Kohlenstoffketten im Fettsäuremolekül mit Doppelbindungen an mindestens zwei, vorteilhaft drei, vier, fünf oder sechs Stellen gemeint sind.The polynucleotide variants of the invention preferably have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the respective biological activity of the polypeptide is encoded by the starting sequence. That is, the polypeptides encoded by the polynucleotides of the invention may be involved in the metabolism of compounds necessary for building fatty acids, fatty acid esters such as di-glycylglycerides and / or triacylglycerides in an organism, preferably in a plant or plant cell, or transporting molecules through membranes participate, wherein Ciβ-, C 2 0 or C 22 carbon chains in the fatty acid molecule with double bonds at least two, preferably three, four, five or six positions are meant.
Die erfindungsgemäßen Polynucleotide umfassen entweder die zuvor genannten spezifischen Nukleinsäuresequenzen oder bestehen daraus. D.h. die erfindungsgemäßen Polynucleotide können grundsätzlich auch noch weitere Nukleotide umfassen. Dabei kann es sich vorzugsweise um 3' oder 5' untranslatierte Bereiche der genomi- sehen Nukleinsäuresequenz handeln. Diese bestehen vorzugsweise aus mindestens 100, 200 oder 500 Nukleotiden am 5' Terminus und mindestens 20, 50 oder 100 Nukleotiden am 3' Terminus des kodierenden Bereichs. Weitere Polynucleotide, die zusätzliche Nukleinsäuresequenzen umfassen, sind solche, die für Fusionsproteine kodieren. Solche Fusionsproteine können zusätzlich zu den zuvor genannten Polypep- tiden weiter Polypeptid bzw. Polypeptidanteile kodieren. Bei dem zusätzlichen Polypeptid bzw. Polypeptidanteil kann es sich um weitere Enzyme der Lipid- oder Fettsäure Biosynthese handeln. Auch denkbar sind Polypeptide, die als Marker für die Expression dienen können (Grün-, Gelb-, Rot-, Blau-fluoreszierende Proteine, alkalische Phosphatase u.a.) bzw. sogenannte "tags" als Marker oder Hilfe für die Aufreinigung (z.B. FLAG-tags, 6-Histidin-tags, MYC-tags u.a.).The polynucleotides of the invention either comprise or consist of the aforementioned specific nucleic acid sequences. That is to say, the polynucleotides according to the invention may in principle also comprise further nucleotides. These may preferably be 3 ' or 5 ' untranslated regions of the genomic nucleic acid sequence. These preferably consist of at least 100, 200 or 500 nucleotides at the 5 'terminus and at least 20, 50 or 100 nucleotides at the 3' terminus of the coding region. Other polynucleotides comprising additional nucleic acid sequences are those encoding fusion proteins. Such fusion proteins may further encode polypeptide or polypeptide portions in addition to the aforementioned polypeptides. The additional polypeptide or polypeptide portion may be other enzymes of lipid or fatty acid biosynthesis. Also conceivable are polypeptides which can serve as markers for expression (green, yellow, red, blue-fluorescent proteins, alkaline phosphatase, etc.) or so-called "tags" as markers or aid for purification (eg FLAG tags , 6-histidine tags, MYC tags, etc.).
Polynucleotid- Varianten können von verschiedenen natürlichen oder künstlichen Quellen isoliert werden. Beispielsweise können sie künstlich durch in vitro oder in vivo Mutagenese erzeugt werden. Homologe oder Orthologe der spezifischen Sequenzen können aus den verschiedensten Tieren, Pflanzen oder Mikroorganismen gewonnen werden. Vorzugsweise werden sie aus Algen gewonnen. Besonders bevorzugt sind Algen der Familie der Prasinophyceae wie aus den Gattungen Heteromastix, Mammel- Ia, Mantoniella, Micromonas, Nephroselmis, Ostreococcus, Prasinocladus, Prasinococ- cus, Pseudoscourfielda, Pycnococcus, Pyramimonas, Scherffelia oder Tetraselmis wie den Gattungen und Arten Heteromastix longifillis, Mamiella gilva, Mantoniella squama-
ta, Micromonas pusilla, Nephroselmis olivacea, Nephroselmis pyriformis, Nephroselmis rotunda, Ostreococcus tauri, Ostreococcus sp. Prasinocladus ascus, Prasinocladus lubricus, Pycnococcus provasolii, Pyramimonas amylifera, Pyramimonas disomata, Pyramimonas obovata, Pyramimonas orientalis, Pyramimonas parkeae, Pyramimonas spinifera, Pyramimonas sp., Tetraselmis apiculata, Tetraselmis carteriaformis, Tetra- selmis chui, Tetraselmis convolutae, Tetraselmis desikacharyi, Tetraselmis gracilis, Tetraselmis hazeni, Tetraselmis impellucida, Tetraselmis inconspicua, Tetraselmis levis, Tetraselmis maculata, Tetraselmis marina, Tetraselmis striata, Tetraselmis subcordiformis, Tetraselmis suecica, Tetraselmis tetrabrachia, Tetraselmis tetrathele, Tetraselmis verrucosa, Tetraselmis verrucosa fo. Rubens oder Tetraselmis sp. Vorteilhafterweise stammen die Polynucleotide aus Algen der Gattungen Mantonielle oder Ostreococcus. Ebenso bevorzugt sind Algen wie Isochrysis oder Crypthecodini- um, Algen/Diatomeen wie Thalassiosira, Phaeodactylum oder Thraustochytrium, Moose wie Physcomitrella oder Ceratodon, ganz besonders bevorzugt sind die Algen der Gattungen Mantonielle oder Ostreococcus oder die Diatomeen der Gattungen Thalassiosira oder Crypthecodinium. Die Polynucleotide können ebenfalls bevorzugt aus höheren Pflanzen gewonnen werden, wie den Primulaceae wie Aleuritia, Calendula stellata, Osteospermum spinescens oder Osteospermum hyoseroides, Mikroorganismen wie Pilzen wie Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor oder Mortierella, Bakterien wie Shewanella, Hefen oder Tieren wie Nematoden z.B. Caenorhabditis, Insekten oder Fischen. Die Polynucleotid- Varianten stammen ebenfalls bevorzugt aus einem Tier aus der Ordnung der Vertebraten. Besonders bevorzugt stammen die Polynucleotide aus der Klasse der Vertebrata; Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae bzw. Oncorhynchus und, ganz besonders bevorzugt, aus der Ordnung der Salmoniformes wie der Familie der Salmonidae wie der Gattung Salmo beispielsweise aus den Gattungen und Arten Oncorhynchus mykiss, Trutta trutta oder Salmo trutta fario. Die erfindungsgemäßen Polynucleotide können hierbei mittels molekularbiologischer Standardtechniken und der hier bereitgestellten Sequenzinformation isoliert werden. Auch kann Mithilfe von Vergleichsalgorithmen beispielsweise eine homologe Sequenz oder homologe, konservierte Sequenzbereiche auf DNA oder Aminosäureebene identifiziert werden. Diese können als Hybridisierungssonde sowie Standard- Hybridisierungstechniken (wie z.B. beschrieben in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2. Aufl., CoId Spring Harbor Laboratory, CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY, 1989) zur Isolierung weiterer im Verfahren nützlicher Nukleinsäuresequenzen verwendet werden. Überdies lassen sich Polynucleotide oder Fragmente davon, durch Polymerasekettenreaktion (PCR) isolieren, wobei Oligonukleotidprimer, die auf der Basis dieser Sequenz oder von Teilen davon, verwendet werden (z.B. kann ein Nukleinsäuremolekül, umfassend die vollständigen Sequenz oder einen Teil davon, durch Polymerasekettenreaktion unter Verwendung
von Oligonukleotidprimern isoliert werden, die auf der Basis dieser gleichen Sequenz erstellt worden sind). Zum Beispiel lässt sich mRNA aus Zellen isolieren (z.B. durch das Guanidiniumthiocyanat-Extraktionsverfahren von Chirgwin et al. (1979) Bioche- mistry 18:5294-5299) und cDNA mittels Reverser Transkriptase (z.B. Moloney-MLV- Reverse-Transkriptase, erhältlich von Gibco/BRL, Bethesda, MD, oder AMV-Reverse- Transkriptase, erhältlich von Seikagaku America, Inc., St. Petersburg, FL) herstellen. Synthetische Oligonukleotidprimer zur Amplifizierung mittels Polymerasekettenreaktion lassen sich auf der Basis der in den SEQ ID Nummern dargestellten Aminosäuresequenzen erstellen. Eine erfindungsgemäße Nukleinsäure kann unter Verwendung von cDNA oder alternativ von genomischer DNA als Matrize und geeigneten Oligonukleotidprimern gemäß Standard-PCR-Amplifikationstechniken amplifiziert werden. Die so amplifizierte Nukleinsäure kann in einen geeigneten Vektor kloniert werden und mittels DNA-Sequenzanalyse charakterisiert werden. Oligonukleotide, die einer Desaturase- Nukleotidsequenz entsprechen, können durch Standard-Syntheseverfahren, beispiels- weise mit einem automatischen DNA-Synthesegerät, hergestellt werden.Polynucleotide variants can be isolated from a variety of natural or artificial sources. For example, they can be generated artificially by in vitro or in vivo mutagenesis. Homologs or orthologues of the specific sequences can be obtained from a wide variety of animals, plants or microorganisms. Preferably, they are obtained from algae. Particularly preferred are algae of the family Prasinophyceae as from the genera Heteromastix, Mammiella, Mantoniella, Micromonas, Nephroselmis, Ostreococcus, Prasinocladus, Prasinococcus, Pseudoscourfielda, Pycnococcus, Pyramimonas, Scherffelia or Tetraselmis as the genera and species Heteromastix longifillis, Mamiella gilva, Mantoniella squama- ta, Micromona pusilla, Nephroselmis olivacea, Nephroselmis pyriformis, Nephroselmis rotunda, Ostreococcus tauri, Ostreococcus sp. Prasinocladus ascus, Prasinocladus lubricus, Pycnococcus provasolii, Pyramimonas amylifera, Pyramimonas disomata, Pyramimonas obovata, Pyramimonas orientalis, Pyramimonas parkeae, Pyramimonas spinifera, Pyramimonas sp., Tetraselmis apiculata, Tetraselmis carteriaformis, Tetraselmis chui, convolutae Tetraselmis, Tetraselmis desikacharyi, Tetraselmis gracilis , Tetraselmis hazeni, Tetraselmis impellucida, Tetraselmis inconspicua, Tetraselmis levis, Tetraselmis maculata, Tetraselmis marina, Tetraselmis striata, Tetraselmis subcordiformis, Tetraselmis suecica, Tetraselmis tetrabrachia, Tetraselmis tetrathele, Tetraselmis verrucosa, Tetraselmis verrucosa fo. Rubens or Tetraselmis sp. Advantageously, the polynucleotides are derived from algae of the genera Mantonielle or Ostreococcus. Likewise preferred are algae such as Isochrysis or Crypthecodinium, algae / diatoms such as Thalassiosira, Phaeodactylum or Thraustochytrium, mosses such as Physcomitrella or Ceratodon, most preferably the algae of the genera Mantonielle or Ostreococcus or the diatoms of the genera Thalassiosira or Crypthecodinium. The polynucleotides may also preferably be obtained from higher plants such as the primulaceae such as Aleuritia, Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides, microorganisms such as fungi such as Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella, bacteria such as Shewanella, yeasts or animals such as Nematodes eg Caenorhabditis, insects or fish. The polynucleotide variants are also preferably derived from an animal of the vertebrate order. Most preferably, the polynucleotides are of the vertebrate class; Euteleostomi, Actinopterygii; Neopterygii; Teleostei; Euteleostei, Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus and, most preferably, from the order of Salmoniformes such as the family Salmonidae such as the genus Salmo, for example, from the genera and species Oncorhynchus mykiss, Trutta trutta or Salmo trutta fario. The polynucleotides according to the invention can in this case be isolated by means of standard molecular biological techniques and the sequence information provided here. Also, by comparison algorithms, for example, a homologous sequence or homologous, conserved sequence regions at the DNA or amino acid level can be identified. These may be used as a hybridization probe as well as standard hybridization techniques (such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CoId Spring Harbor Laboratory, ColD Spring Harbor Laboratory Press, ColD Spring Harbor, NY, 1989) Isolation of other useful in the process of nucleic acid sequences can be used. Moreover, polynucleotides or fragments thereof can be isolated by polymerase chain reaction (PCR) using oligonucleotide primers based on this sequence or portions thereof (eg, a nucleic acid molecule comprising the complete sequence or a portion thereof can be polymerase chain-reaction utilized isolated from oligonucleotide primers prepared on the basis of this same sequence). For example, mRNA can be isolated from cells (eg, by the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA by reverse transcriptase (eg, Moloney MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for amplification by polymerase chain reaction can be prepared on the basis of the amino acid sequences shown in the SEQ ID numbers. A nucleic acid of the invention may be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis. Oligonucleotides corresponding to a desaturase nucleotide sequence can be prepared by standard synthetic methods, for example with an automated DNA synthesizer.
Die erfindungsgemäßen Polynucleotide können entweder als isolierte Polynucleotide (d.h. isoliert aus ihrem natürlichen Ursprung, z.B. dem genomischen Locus) oder in genetisch veränderter Form bereitgestellt werden (d.h. die Polynucleotide können auch an ihrem natürlichen genetischen Locus vorliegen, müssen dann aber genetisch verändert werden). Ein isoliertes Polynucleotid umfasst vorzugsweise weniger als 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0,5 kb oder 0,1 kb an Nukleinsäuresequenz, die natürlicherweise in seiner Nachbarschaft vorkommt. Das erfindugsgemäße Polynucleotid kann als einzelsträngiges oder doppelsträngiges Nukleinsäuremolekül vorliegen und genomi- sehe DNA, cDNA oder RNA sein. Die erfindungsgemäßen Polynucleotide ufassen alle Orientierungen der in den SEQ ID Nummern aufgezeigten Sequenzen, d.h. auch komplementäre Stränge sowie reverse oder revers-komplementäre Orientierungen. Ferner umfasst der Begriff auch chemisch modifizierte Nucleinsäuren, wie die natürlich vorkommenden methylierten DNA Moleküle, oder artifizielle Nucleinsäuren z.B. biotinylierte Nukleinsären.The polynucleotides of the invention may be provided either as isolated polynucleotides (i.e., isolated from their natural origin, e.g., the genomic locus) or in genetically altered form (i.e., the polynucleotides may also be present at their natural genetic locus, but must then be genetically engineered). An isolated polynucleotide preferably comprises less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequence naturally present in its vicinity. The polynucleotide according to the invention can be in the form of a single-stranded or double-stranded nucleic acid molecule and can be genomic DNA, cDNA or RNA. The polynucleotides of the invention include all orientations of the sequences shown in the SEQ ID Nos., I. also complementary strands and reverse or reverse-complementary orientations. Further, the term also includes chemically modified nucleic acids, such as the naturally occurring methylated DNA molecules, or artificial nucleic acids, e.g. biotinylated nucleic acids.
Die Erfindung umfasst auch Oligonucleotide von mindestens 15 bp, bevorzugt mindestens 20 bp, mindestens 25 bp, mindestens 30 bp, mindestens 35 bp, oder mindestens 50 bp, die spezifisch mit einem der zuvor genannten Polynucleotide unter stringenten Bedingungen hybridisieren können. Die Oliguncleotide können aus DNA oder RNA oder beidem bestehen. Solche Oligonucleotide können als Primer für die PCR, als die Expression hemmende, antisense Oligonucleotide, für RNA Interferenz (RNAi) oder für Chimero- oder Genoplastie Ansätze verwendet werden. RNAi Verfahren sind beispielsweise in Fire et al., Nature (1998) 391 :806-811 ; Fire, Trends Genet. 15, 358-363 (1999); Sharp, RNA interference 2001. Genes Dev. 15,485-490 (2001 ); Hammond et
al. Nature Rev. Genet. 2, 11 10-11 19 (2001 ); Tuschl, Chem. Biochem. 2, 239-245 (2001 ); Hamilton et al., Science 286, 950-952 (1999); Hammond et al., Nature 404, 293-296 (2000); Zamore et al., Cell 101 , 25-33 (2000); Bernstein et al., Nature 409, 363-366 (2001 ); Elbashir et al., Genes Dev. 15, 188-200 (2001 ); WO 01/29058; WO 99/32619; oder Elbashir et al., 2001 Nature 41 1 : 494-498 beschrieben und dienen der Inhibierung der Genexpression durch Abbau der mRNA. Chimero- oder Genoplastie Ansätze dienen der in vivo Modifizierung (z.B. dem Einfügen von Punktmutationen) in Genen an deren endogenen Loci. Entsprechende Verfahren sind offenbart in US5,565,350, US5,756,325, US5,871 ,984, US5,731 ,181 , US5,795,972, US6,573,046, US6,21 1 ,351 , US6,586,184, US6,271 ,360 und US6,479,292.The invention also encompasses oligonucleotides of at least 15 bp, preferably at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, or at least 50 bp, which can specifically hybridize with one of the aforementioned polynucleotides under stringent conditions. The oliguncleotides may consist of DNA or RNA or both. Such oligonucleotides can be used as primers for PCR, as expression-inhibiting, antisense oligonucleotides, for RNA interference (RNAi) or for chimeric or genomic approaches. RNAi methods are described, for example, in Fire et al., Nature (1998) 391: 806-811; Fire, Trends Genet. 15, 358-363 (1999); Sharp, RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond et al. Nature Rev. Genet. 2, 11 10-11 19 (2001); Tuschl, Chem. Biochem. 2, 239-245 (2001); Hamilton et al., Science 286, 950-952 (1999); Hammond et al., Nature 404, 293-296 (2000); Zamore et al., Cell 101, 25-33 (2000); Bernstein et al., Nature 409, 363-366 (2001); Elbashir et al., Genes Dev. 15, 188-200 (2001); WO 01/29058; WO 99/32619; or Elbashir et al., 2001 Nature 41 1: 494-498, and serve to inhibit gene expression by degrading the mRNA. Chimeric or genomic approaches are used for in vivo modification (eg insertion of point mutations) in genes at their endogenous loci. Corresponding methods are disclosed in US5,565,350, US5,756,325, US5,871,984, US5,731,1181, US5,795,972, US6,573,046, US6,211,351, US6,586,184, US6,271,360 and US Pat US6,479,292.
Vorteilhafterweise hat sich gezeigt, dass die erfindungsgemäßen Polynucleotide zur rekombinanten Herstellung mehrfach ungesättigter Fettsäuren in Wirtszellen und transgenen Organismen besonders effektiv eingesetzt weden können. Insbesondere können die von den erfindungsgemäßen Polynucleotiden kodierten Polypeptide mit Δ- 12-Desaturase, Δ-4-Desaturase, Δ-5-Desaturase, Δ-5-Elongase, Δ-6-Desaturase und Δ-6-Elongase Aktivität Ci8-, C2o- und C22-Fettsäuren mit ein, zwei, drei, vier oder fünf Doppelbindungen und bevorzugt mehrfach ungesättigte Ci8-Fettsäuren mit ein, zwei oder drei Doppelbindungen wie C18:1Δ9, C18:2Δ9'12oder C18:3 Δ9'12'15 , mehrfach ungesättigte C2o-Fettsäuren mit drei oder vier Doppelbindungen wie C20:3Δ8'11'14 oder C20:4Δ8'11'14'17 oder mehrfach ungesättigte C22-Fettsäuren mit vier oder fünf Doppelbindungen wie C22:4Δ7'10'13'16 oder C22:5Δ7'10'13'16'19 umsetzen. Bevorzugt werden die Fettsäuren in Phospholipiden oder CoA-Fettsäureestern desaturiert, vorteilhaft in den CoA-Fettsäureester. Somit ist eine einfache, kostengünstige Herstellung dieser mehrfach ungesättigten Fettsäuren speziell in eukaryontischen Systemen möglich. Die mittels der erfindungsgemäßen Polynucleotide hergestellten ungesättigten Fettsäuren können dann als Öl-, Lipid- und Fettsäurezusammensetzungen formuliert und entsprechend eingesetzt werden.Advantageously, it has been shown that the polynucleotides according to the invention can be used particularly effectively for the recombinant production of polyunsaturated fatty acids in host cells and transgenic organisms. In particular, the proteins encoded by the polynucleotides of the invention encode polypeptides with Δ- 12 desaturase, Δ-4-desaturase, Δ-5 desaturase, Δ-5-elongase, Δ-6-desaturase and Δ-6-elongase activity can Ci 8 -, C 2 O and C 22 fatty acids with one, two, three, four or five double bonds, and preferably polyunsaturated Ci 8 fatty acids with one, two or three double bonds, such as C18: 1 Δ9, C18: 2 Δ9 '12 or C18: 3 Δ9 ' 12 ' 15 , polyunsaturated C 2 o fatty acids having three or four double bonds such as C20: 3 Δ8 ' 11 ' 14 or C20: 4 Δ8 ' 11 ' 14 '17 or polyunsaturated C 22 fatty acids with four or five Double bonds such as C22: 4 Δ7 ' 10 ' 13 '16 or C22: 5 Δ7 ' 10 ' 13 ' 16 '19 . Preferably, the fatty acids are desaturated in phospholipids or CoA fatty acid esters, advantageously in the CoA fatty acid esters. Thus, a simple, inexpensive production of these polyunsaturated fatty acids is possible especially in eukaryotic systems. The unsaturated fatty acids prepared by means of the polynucleotides of the invention can then be formulated as oil, lipid and fatty acid compositions and used accordingly.
Die vorliegende Erfindung betrifft weiterhin einen Vektor, der das erfindungsgemäße Polynucleotid umfasst.The present invention further relates to a vector comprising the polynucleotide of the invention.
Der Begriff "Vektor" bezeichnet ein Nukleinsäuremolekül, das ein anderes Nukleinsäu- remolekül, wie die erfindungsgemäßen Polynucleotide, transportieren kann, an welche es gebunden ist. Ein Vektortyp ist ein Plasmid, das für eine zirkuläre doppelsträngigeThe term "vector" denotes a nucleic acid molecule which can transport another nucleic acid molecule, such as the polynucleotides according to the invention, to which it is bound. One type of vector is a circular double-stranded plasmid
DNA-Schleife steht, in die zusätzlichen DNA-Segmente ligiert werden können. Ein weiterer Vektortyp ist ein viraler Vektor, wobei zusätzliche DNA-Segmente in das viraleDNA loop can be ligated into the additional DNA segments. Another vector type is a viral vector, with additional DNA segments inserted into the viral vector
Genom ligiert werden können. Bestimmte Vektoren können in einer Wirtszelle, in die sie eingebracht worden sind, autonom replizieren (z.B. Bakterienvektoren mit bakteriel-
lern Replikationsursprung). Andere Vektoren werden vorteilhaft beim Einbringen in die Wirtszelle in das Genom einer Wirtszelle integriert und dadurch zusammen mit dem Wirtsgenom repliziert. Zudem können bestimmte Vektoren die Expression von Genen, mit denen sie funktionsfähig verbunden sind, steuern. Diese Vektoren werden hier auch als Expressionsvektoren bezeichnet. Gewöhnlich haben Expressionsvektoren, die für DNA-Rekombinationstechniken geeignet sind, die Form von Plasmiden. In der vorliegenden Beschreibung können die Begriffe Plasmid und Vektor austauschbar verwendet werden, da das Plasmid die am häufigsten verwendete Vektorform ist. Die Erfindung soll jedoch diese anderen Expressionsvektorformen, wie virale Vektoren, die ähnliche Funktionen ausüben, umfassen. Ferner soll der Begriff Vektor auch andere Vektoren, die dem Fachmann bekannt sind, wie Phagen, Viren, wie SV40, CMV, TMV, Transposons, IS-Elemente, Phasmide, Phagemide, Cosmide, lineare oder zirkuläre DNA, künstliche Chromosomen, umfassen. Der Begriff umfasst schließlich auch Konstrukte für zielgerichtete, d.h. homologe Rekombination, oder heterologe Insertion von Polynucleotiden.Genome can be ligated. Certain vectors can autonomously replicate in a host cell into which they have been introduced (eg bacterial vectors with bacterial learn replication origin). Other vectors are advantageously integrated into the genome of a host cell upon introduction into the host cell and thereby replicated together with the host genome. In addition, certain vectors may direct the expression of genes to which they are operably linked. These vectors are also referred to herein as expression vectors. Usually, expression vectors suitable for recombinant DNA techniques are in the form of plasmids. In the present specification, the terms plasmid and vector can be used interchangeably since the plasmid is the most commonly used vector form. However, the invention is intended to encompass these other forms of expression vectors, such as viral vectors that perform similar functions. Further, the term vector is also intended to encompass other vectors known to those skilled in the art, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA, artificial chromosomes. Finally, the term also includes constructs for targeted, ie homologous recombination, or heterologous insertion of polynucleotides.
Vektoren lassen sich in prokaryotische oder eukaryotische Zellen über herkömmliche Transformations- oder Transfektionstechniken einbringen. Die Begriffe "Transformation" und "Transfektion", Konjugation und Transduktion, wie hier verwendet, sollen eine Vielzahl von im Stand der Technik bekannten Verfahren zum Einbringen fremder Nukleinsäure (z.B. DNA) in eine Wirtszelle, einschließlich Calciumphosphat- oder Calciumchlorid-Copräzipitation, DEAE-Dextran-vermittelte Transfektion, Lipofektion, natürliche Kompetenz, chemisch vermittelter Transfer, Elektroporation oder Teilchen- beschuss, umfassen. Geeignete Verfahren zur Transformation oder Transfektion von Wirtszellen, einschließlich Pflanzenzellen, lassen sich finden in Sambrook et al. (Molecular Cloning: A Laboratory Manual., 2. Aufl., CoId Spring Harbor Laboratory, CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY, 1989) und anderen Labor-Handbüchern, wie Methods in Molecular Biology, 1995, Bd. 44, Agrobacterium protocols, Hrsgb: Gartland und Davey, Humana Press, Totowa, New Jersey.Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection", conjugation and transduction are intended to encompass a variety of methods known in the art for introducing foreign nucleic acid (eg DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE- Dextran-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for transforming or transfecting host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., CoId Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColD Spring Harbor, NY, 1989) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44 , Agrobacterium protocols, Eds: Gartland and Davey, Humana Press, Totowa, New Jersey.
Geeignete Klonierungsvektoren sind dem Fachmann allgemein bekannt. Hierzu gehören insbesondere Vektoren, die in mikrobiellen Systemen replizierbar sind, also vor allem Vektoren, die eine effiziente Klonierung in Hefen oder Pilze gewährleisten, und die stabile Transformation von Pflanzen ermöglichen. Zu nennen sind insbesonde- re verschiedene für die T-DNA-vermittelte Transformation geeignete, binäre und co- integrierte Vektorsysteme. Derartige Vektorsysteme sind in der Regel dadurch gekennzeichnet, dass sie zumindest die für die Agrobakterium-vermittelte Transformation benötigten vir-Gene sowie die T-DNA begrenzenden Sequenzen (T-DNA-Border) beinhalten. Vorzugsweise umfassen diese Vektorsysteme auch weitere cis- regulatorische Regionen wie Promotoren und Terminatoren und/oder Selektionsmar-
ker, mit denen entsprechend transformierte Organismen identifiziert werden können. Während bei co-integrierten Vektorsystemen vir-Gene und T-DNA-Sequenzen auf demselben Vektor angeordnet sind, basieren binäre Systeme auf wenigstens zwei Vektoren, von denen einer vir-Gene, aber keine T-DNA und ein zweiter T-DNA, jedoch kein vir-Gen trägt. Dadurch sind letztere Vektoren relativ klein, leicht zu manipulieren und sowohl in E. -coli als auch in Agrobacterium zu replizieren. Zu diesen binären Vektoren gehören Vektoren der Serien pBI B-HYG, pPZP, pBecks, pGreen. Erfindungsgemäß bevorzugt verwendet werden Bin19, pB1101 , pBinAR, pGPTV und pCAMBIA. Eine Übersicht über binäre Vektoren und ihre Verwendung gibt Hellens et al, Trends in Plant Science (2000) 5, 446-451. Die Vektoren mit den inseriernten erfindungsgemäßen Polynucleotiden lassen sich in Mikroorganismen, insbesondere Escherichia coli und Agrobacterium tumefaciens, unter selektiven Bedingungen stabil propagieren und ermöglichen einen Transfer von heterologer DNA in Pflanzen oder Mikroorganismen. Mittels der Klonierungsvektoren können die erfindungsgemäßen Polynucleotide in Organismen wie Mikroorganismen oder Pflanzen eingebracht werden und damit zur Pflanzentransformation verwendet werden. Geeignet Vektoren dafür sind veröffentlicht in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Kapitel 6/7, S. 71-1 19 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Bd. 1 , Engineering and Utilization, Hrsgb.: Kung und R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Bd. 1 , Engineering and Utilization, Hrsgb.: Kung und R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991 ), 205-225)).Suitable cloning vectors are well known to those skilled in the art. These include, in particular, vectors which can be replicated in microbial systems, ie in particular vectors which ensure efficient cloning in yeasts or fungi, and which enable stable transformation of plants. Particular mention should be made of various binary and co-integrated vector systems suitable for T-DNA-mediated transformation. Such vector systems are usually characterized in that they contain at least the vir genes required for the Agrobacterium-mediated transformation as well as the T-DNA limiting sequences (T-DNA border). Preferably, these vector systems also comprise further cis-regulatory regions such as promoters and terminators and / or selection markers. ker, with which appropriately transformed organisms can be identified. Whereas in co-integrated vector systems vir genes and T-DNA sequences are located on the same vector, binary systems are based on at least two vectors, one of them vir genes, but no T-DNA and a second T-DNA, but no carries vir gene. As a result, the latter vectors are relatively small, easy to manipulate and replicate in both E.coli and Agrobacterium. These binary vectors include vectors of the series pBI B-HYG, pPZP, pBecks, pGreen. Bin19, pB1101, pBinAR, pGPTV and pCAMBIA are preferably used according to the invention. For a review of binary vectors and their use, see Hellens et al, Trends in Plant Science (2000) 5, 446-451. The vectors with the inserted polynucleotides according to the invention can be stably propagated in microorganisms, in particular Escherichia coli and Agrobacterium tumefaciens, under selective conditions and enable a transfer of heterologous DNA into plants or microorganisms. By means of the cloning vectors, the polynucleotides according to the invention can be introduced into organisms such as microorganisms or plants and thus be used for plant transformation. Suitable vectors for this are published in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-1 19 (1993); FF White, Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, 15-38; Genes Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)).
Bevorzugt handelt es sich bei dem Vektor um einen Expressionsvektor. Bei dem erfindungsgemäßen Expressionsvektor liegt das Polynucleotid in operativer (d.h. funktioneller) Verbindung zu einer Expressionskontrollsequenz vor. Die Expressionskontrollsequenz zusammen mit dem Polynucleotid und optional weiteren Sequenzelementen des Vektors wird auch als Expressionskassette bezeichnet. Durch die Expres- sionskotrollsequenz wird sichergestellt, dass das Polynucleotid nach Transformation oder Transfektion in eine Wirtszelle exprimiert werden kann. Die zu verwendende Expressionskontrollsequenz enthält vorzugsweise cis-regulatorische Elemente wie Promotor und/oder Enhancer Nukleinsäuresequenzen, die von der Transkriptionsmaschinerie der Wirtszellen erkannt werden. Der Begriff umfasst zudem andere Expressi- onskontrollelemente z.B. Polyadenylierungssignale und RNA stabilisierende Sequenzen. Diese Regulationssequenzen sind z.B. beschrieben in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), oder siehe: Gruber und Crosby, in: Methods in Plant Molecular Biology and Biotech- nolgy, CRC Press, Boca Raton, Florida, Hrsgb.: Glick und Thompson, Kapitel 7, 89- 108, einschließlich der Literaturstellen darin. Expressionskontrollsequenzen umfassen
solche, welche die konstitutive Expression einer Nukleotidsequenz in vielen Wirtszelltypen steuern, und solche, welche die direkte Expression der Nukleotidsequenz nur in bestimmten Wirtszellen unter bestimmten Bedingungen steuern. Der Fachmann weiß, dass die Gestaltung des Expressionsvektors von Faktoren, wie der Auswahl der zu transformierenden Wirtszelle, dem Ausmaß der Expression des gewünschten Proteins usw., abhängen kann. Die erfindungsgemäßen Polynucleotide können in einer oder mehreren Kopien in der Expressionskassette oder dem erfindungsgemäßen Expressionsvektor vorliegen (z.B. in Form mehrerer Expressionskassetten). Die regulatorischen Sequenzen bzw. Faktoren können dabei wie oben beschrieben vorzugsweise die Genexpression der eingeführten Gene positiv beeinflussen und dadurch erhöhen. So kann eine Verstärkung der regulatorischen Elemente vorteilhafterweise auf der Transkriptionsebene erfolgen, indem starke Transkriptionssignale wie Promotoren und/oder "Enhancer" verwendet werden. Daneben ist aber auch eine Verstärkung der Translation möglich, indem beispielsweise die Stabilität der mRNA verbessert wird. Weitere Expressionskontrollsequenzen im Sinne der vorliegenden Erfindung sind Translationsterminatoren am 3'-Ende der zu translatierenen Polynucleotide. Verwendet werden kann hier z.B. der OCS1 Terminator. Wie auch für die Promotoren, so sollte hier für jedes zu exprimierende Polynucleotid eine unterschiedliche Terminatorsequenz verwendet werden.Preferably, the vector is an expression vector. In the expression vector of the present invention, the polynucleotide is in operative (ie, functional) connection to an expression control sequence. The expression control sequence together with the polynucleotide and optionally further sequence elements of the vector is also referred to as expression cassette. The expression co-rolling sequence ensures that the polynucleotide can be expressed in a host cell after transformation or transfection. The expression control sequence to be used preferably contains cis-regulatory elements such as promoter and / or enhancer nucleic acid sequences which are recognized by the transcription machinery of the host cells. The term also includes other expression control elements such as polyadenylation signals and RNA stabilizing sequences. These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotecholgy, CRC Press, Boca Raton, Florida, Ed .: Glick and Thompson, Chapter 7, 89-108, including references therein. Include expression control sequences those which direct the constitutive expression of a nucleotide sequence in many types of host cells and those which direct the direct expression of the nucleotide sequence only in certain host cells under certain conditions. One skilled in the art will appreciate that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the level of expression of the desired protein, etc. The polynucleotides according to the invention may be present in one or more copies in the expression cassette or the expression vector according to the invention (for example in the form of several expression cassettes). The regulatory sequences or factors can, as described above, preferably positively influence the gene expression of the introduced genes and thereby increase them. Thus, enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers. In addition, however, an enhancement of the translation is possible by, for example, the stability of the mRNA is improved. Further expression control sequences for the purposes of the present invention are translation terminators at the 3 'end of the polynucleotides to be translated. For example, the OCS1 terminator can be used here. As with the promoters, a different terminator sequence should be used here for each polynucleotide to be expressed.
Bevorzugte Expressionskontrollsequenzen bzw. Regulationssequenzen liegen in Promotoren vor, wie dem cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, Ipp-lac-, laclq-, T7- , T5-, T3-, gal-, trc-, ara-, SP6-, λ-PR- oder λ-PL-Promotor und werden vorteilhafterweise in Gram-negativen Bakterien angewendet. Weitere vorteilhafte Regulationsse- quenzen liegen beispielsweise in den Gram-positiven Promotoren amy und SPO2, in den Hefe- oder Pilzpromotoren ADC1 , MFa, AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH oder in den Pflanzenpromotoren CaMV/35S [Franck et al., Cell 21 (1980) 285- 294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1 , B33, nos oder im Ubiquitin- oder Phaseolin-Promotor vor. In diesem Zusammenhang vorteilhaft sind ebenfalls induzierbare Promotoren, wie die in EP-A-O 388 186 (Benzylsulfonamid-induzierbar), Plant J. 2, 1992:397-404 (Gatz et al., Tetracyclin- induzierbar), EP-A-O 335 528 (Abzisinsäure-induzierbar) oder WO 93/21334 (Ethanol- oder Cyclohexenol-induzierbar) beschriebenen Promotoren. Weitere geeignete Pflanzenpromotoren sind der Promotor von cytosolischer FBPase oder der ST-LSI- Promotor der Kartoffel (Stockhaus et al., EMBO J. 8, 1989, 2445), der Phosphoribo- sylprophosphatamidotransferase-Promotor aus Glycine max (Genbank-Zugangsnr. U87999) oder der in EP-A-O 249 676 beschriebene nodienspezifische Promotor. Besonders vorteilhafte Promotoren sind Promotoren, welche die Expression in Geweben ermöglichen, die an der Fettsäurebiosynthese beteiligt sind. Ganz besonders vorteilhaft sind samenspezifische Promotoren, wie der ausführungsgemäße USP
Promotor aber auch andere Promotoren wie der LeB4-, DC3, Phaseolin- oder Napin- Promotor. Weitere besonders vorteilhafte Promotoren sind samenspezifische Promotoren, die für monokotyle oder dikotyle Pflanzen verwendet werden können und in US 5,608,152 (Napin-Promotor aus Raps), WO 98/45461 (Oleosin-Promotor aus Arobidopsis), US 5,504,200 (Phaseolin-Promotor aus Phaseolus vulgaris), WO 91/13980 (Bce4-Promotor aus Brassica), von Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4-Promotor aus einer Leguminose) beschrieben sind, wobei sich diese Promotoren für Dikotyledonen eignen. Die folgenden Promotoren eignen sich beispielsweise für Monokotyledonen lpt-2- oder lpt-1 -Promotor aus Gerste (WO 95/15389 und WO 95/23230), Hordein-Promotor aus Gerste und andere, in WO 99/16890 beschriebene geeignete Promotoren. Es ist im Prinzip möglich, alle natürlichen Promotoren mit ihren Regulationssequenzen, wie die oben genannten, als Expressionskontrollsequenzen zu verwenden. Es ist ebenfalls möglich, zusätzlich oder alleine synthetische Promotoren zu verwenden, besonders wenn sie eine Samen- spezifische Expression vermitteln, wie z.B. beschrieben in WO 99/16890.Preferred expression control sequences or regulatory sequences are present in promoters, such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3 , gal, trc, ara, SP6, λ-PR or λ-PL promoter and are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are, 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 CaMV / 35S [ Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Biol. 22 (1993)], SSU, OCS, Iib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter. Also advantageous in this context are inducible promoters, such as those in EP-AO 388 186 (benzylsulfonamide-inducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracycline inducible), EP-AO 335 528 ( Abzisinic inducible) or WO 93/21334 (ethanol or cyclohexenol inducible) promoters. Further suitable plant promoters are the promoter of cytosolic FBPase or the ST LSI promoter of the potato (Stockhaus et al., EMBO J. 8, 1989, 2445), the glycine max phosphoribosylprophosphate amidotransferase promoter (Genbank Accession No. U87999) or the nodia-specific promoter described in EP-A-0 249 676. Particularly advantageous promoters are promoters which allow expression in tissues involved in fatty acid biosynthesis. Very particularly advantageous are seed-specific promoters, such as the USP Promoter but also other promoters such as the LeB4, DC3, phaseolin or napin promoter. Further particularly advantageous promoters are seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and in US Pat. No. 5,608,152 (napin promoter from rapeseed), WO 98/45461 (oleosin promoter from Arobidopsis), US Pat. No. 5,504,200 (Phaseolin promoter from Phaseolus vulgaris ), WO 91/13980 (Bce4 promoter from Brassica), Baeumlein et al., Plant J., 2, 2, 1992: 233-239 (LeB4 promoter from a legume), these promoters being for dicotyledons suitable. The following promoters are suitable, for example, for barley monocotylone lpt-2 or lpt-1 promoter (WO 95/15389 and WO 95/23230), barley hordein promoter and other suitable promoters described in WO 99/16890. It is possible in principle to use all natural promoters with their regulatory sequences, such as those mentioned above, as expression control sequences. It is also possible to use synthetic promoters in addition or alone, especially if they mediate seed-specific expression, as described, for example, in WO 99/16890.
Um einen besonders hohen Gehalt an PUFAs vor allem in transgenen Pflanzen zu erzielen, sollten die Polynucleotide der vorliegenden Erfindung bevorzugt samenspezifisch in Ölsaaten exprimiert werden. Hierzu können Samen-spezifische Promotoren verwendet werden, bzw. solche Promotoren die im Embryo und/oder im Endosperm aktiv sind. Samen-spezifische Promotoren können prinzipiell sowohl aus dikotolydonen als auch aus monokotolydonen Pflanzen isoliert werden. Im Folgenden sind vorteilhafte bevorzugte Promotoren aufgeführt: USP (= unknown seed protein) und Vicilin (Vicia faba) [Bäumlein et al., Mol. Gen Genet, 1991 , 225(3)], Napin (Raps) [US 5,608,152], Acyl-Carrier Protein (Raps) [US 5,315,001 und WO 92/18634], Oleosin (Arabidopsis thaliana) [WO 98/45461 und WO 93/20216], Phaseolin (Phaseolus vulgaris) [US 5,504,200], Bce4 [WO 91/13980], Leguminosen B4 (LegB4-Promotor) [Bäumlein et al., Plant J., 2,2, 1992], Lpt2 und lpt1 (Gerste) [WO 95/15389 u. WO95/23230], Samenspezifische Promotoren aus Reis, Mais u. Weizen [WO 99/16890], Amy32b, Amy 6-6 und Aleurain [US 5,677,474], Bce4 (Raps) [US 5,530,149], Glycinin (Soja) [EP 571 741], Phosphoenol-Pyruvatcarboxylase (Soja) [JP 06/62870], ADR12-2 (Soja) [WO 98/08962], Isocitratlyase (Raps) [US 5,689,040] oder α-Amylase (Gerste) [EP 781 849].In order to achieve a particularly high content of PUFAs, especially in transgenic plants, the polynucleotides of the present invention should preferably be seed-specifically expressed in oilseeds. For this purpose, seed-specific promoters can be used, or such promoters that are active in the embryo and / or in the endosperm. In principle, seed-specific promoters can be isolated from both dicotolydone and monocotolydone plants. Preferred preferred promoters are listed below: USP (= unknown seed protein) and Vicilin (Vicia faba) [Baumlein et al., Mol. Gen Genet, 1991, 225 (3)], Napin (rapeseed) [US Pat. No. 5,608,152], acyl Carrier protein (rapeseed) [US 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO 98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [US 5,504,200], Bce4 [WO 91/13980] , Legumes B4 (LegB4 promoter) [Bäumlein et al., Plant J., 2,2, 1992], Lpt2 and lpt1 (barley) [WO 95/15389 u. WO95 / 23230], seed-specific promoters from rice, maize and the like. Wheat [WO 99/16890], Amy32b, Amy 6-6 and Aleurain [US 5,677,474], Bce4 (rape) [US 5,530,149], glycinin (soybean) [EP 571 741], phosphoenol pyruvate carboxylase (soybean) [JP 06 / 62870], ADR12-2 (soybean) [WO 98/08962], isocitrate lyase (rapeseed) [US 5,689,040] or α-amylase (barley) [EP 781 849].
Die Pflanzengenexpression lässt sich auch über einen chemisch induzierbaren Promotor erleichtern (siehe eine Übersicht in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemisch induzierbare Promotoren eignen sich besonders, wenn gewünscht wird, dass die Genexpression auf zeitspezifische Weise erfolgt. Beispiele für solche Promotoren sind ein Salicylsäure-induzierbarer Promotor
(WO 95/19443), ein Tetracyclin-induzierbarer Promotor (Gatz et al. (1992) Plant J. 2, 397-404) und ein Ethanol-induzierbarer Promotor.Plant gene expression can also be facilitated by a chemically inducible promoter (see review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108). Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.
Um eine stabile Integration der verschiedenen Biosynthesegene in die transgene Pflanze über mehrere Generation sicherzustellen, sollte jedes der erfindungsgemäßen Polynucleotide unter der Kontrolle eines eigenen bevorzugt eines unterschiedlichen Promotors exprimiert werden, da sich wiederholende Sequenzmotive zu Instabilität der T-DNA bzw. zu Rekombinationsereignissen führen können. Die Expressionskassette ist dabei vorteilhaft so aufgebaut, dass einem Promotor eine geeignete Schnittstelle zur Insertion der zu exprimierenden Nukleinsäure folgt vorteilhaft in einem Polylinker anschließend gegebenenfalls ein Terminator hinter dem Polylinker liegt. Diese Abfolge wiederholt sich mehrfach bevorzugt drei-, vier- oder fünfmal, so dass bis zu fünf Gene in einem Konstrukt zusammengeführt werden und so zur Expression in die transgene Pflanze eingebracht werden können. Vorteilhaft wiederholt sich die Abfolge bis zu dreimal. Die Nukleinsäuresequenzen werden zur Expression über die geeignete Schnittstelle, beispielsweise im Polylinker, hinter den Promotor inseriert. Vorteilhaft hat jede Nukleinsäuresequenz ihren eigenen Promotor und gegebenenfalls ihren eigenen Terminator. Derartige vorteilhafte Konstrukte werden beispielsweise in DE 10102337 oder DE 10102338 offenbart. Es ist aber auch möglich mehrere Nukleinsäuresequen- zen hinter einem Promotor und ggf. vor einem Terminator zu inserieren. Dabei ist die Insertionsstelle bzw. die Abfolge der inserierten Nukleinsäuren in der Expressionskassette nicht von entscheidender Bedeutung, das heißt eine Nukleinsäuresequenz kann an erster oder letzter Stelle in der Kassette inseriert sein, ohne dass dadurch die Expression wesentlich beeinflusst wird. Es können in der Expressionskassette vorteilhaft unterschiedliche Promotoren wie beispielsweise der USP-, LegB4 oder DC3- Promotor und unterschiedliche Terminatoren verwendet werden. Es ist aber auch möglich nur einen Promotortyp in der Kassette zu verwenden. Dies kann jedoch zu unerwünschten Rekombinationsereignissen führen.In order to ensure stable integration of the various biosynthetic genes into the transgenic plant over several generations, each of the polynucleotides of the invention should be expressed under the control of their own preferred, a different promoter, since repeating sequence motifs can lead to instability of the T-DNA or to recombination events. The expression cassette is advantageously constructed in such a way that a promoter is followed by a suitable interface for insertion of the nucleic acid to be expressed, advantageously followed by a terminator behind the polylinker in a polylinker. This sequence is repeated several times, preferably three, four or five times, so that up to five genes can be brought together in one construct and thus introduced into the transgenic plant for expression. Advantageously, the sequence is repeated up to three times. The nucleic acid sequences are inserted for expression via the appropriate interface, for example in the polylinker, behind the promoter. Advantageously, each nucleic acid sequence has its own promoter and optionally its own terminator. Such advantageous constructs are disclosed for example in DE 10102337 or DE 10102338. However, it is also possible to insert several nucleic acid sequences downstream of a promoter and optionally in front of a terminator. In this case, the insertion site or the sequence of the inserted nucleic acids in the expression cassette is not of decisive importance, that is to say a nucleic acid sequence can be inserted at the first or last position in the cassette, without this significantly influencing the expression. It is advantageous to use different promoters, for example the USP, LegB4 or DC3 promoter and different terminators, in the expression cassette. But it is also possible to use only one type of promoter in the cassette. However, this can lead to unwanted recombination events.
Die verwendeten rekombinanten Expressionsvektoren können zur Expression in prokaryotischen oder eukaryotischen Zellen gestaltet sein. Dies ist vorteilhaft, da häufig Zwischenschritte der Vektorkonstruktion der Einfachheit halber in Mikroorganismen durchgeführt werden. Beispielsweise können die Δ-12-Desaturase-, Δ-6-Desaturase-, Δ-6-Elongase-, Δ-5-Desaturase-, Δ-5-Elongase- und/oder Δ-4-Desaturase-Gene in bakteriellen Zellen, Insektenzellen (unter Verwendung von Baculovirus- Expressionsvektoren), Hefe- und anderen Pilzzellen (siehe Romanos, M.A., et al. (1992) "Foreign gene expression in yeast: a review", Yeast 8:423-488; van den Hondel, C.A.M.J.J., et al. (1991 ) "Heterologous gene expression in filamentous fungi", in: More Gene Manipulations in Fungi, J.W. Bennet & L. L. Lasure, Hrsgb., S. 396-428: Acade- mic Press: San Diego; und van den Hondel, C.A.M.J.J., & Punt, PJ. (1991 ) "Gene
transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F., et al., Hrsgb., S. 1-28, Cambridge University Press: Cambridge), Algen (Falciatore et al., 1999, Marine Biotechnology.1 , 3:239-251 ), Ciliaten der Typen: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Desaturaseudocohni- lembus, Euplotes, Engelmaniella und Stylonychia, insbesondere der Gattung Stylony- chia lemnae, mit Vektoren nach einem Transformationsverfahren, wie beschrieben in WO 98/01572, sowie bevorzugt in Zellen vielzelliger Pflanzen (siehe Schmidt, R. und Willmitzer, L. (1988) "High efficiency Agrobacterium tumefaciens-mediated transforma- tion of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep.:583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Florida, Kapitel 6/7, S.71- 1 19 (1993); F.F. White, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Bd. 1 , Engineering and Utilization, Hrsgb.: Kung und R. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991 ), 205- 225 (und darin zitierte Literaturstellen)) exprimiert werden. Geeignete Wirtszellen werden ferner erörtert in Goeddel, Gene Expression Technology: Methods in Enzymo- logy 185, Academic Press, San Diego, CA (1990). Der rekombinante Expressionsvektor kann alternativ, zum Beispiel unter Verwendung von T7-Promotor- Regulationssequenzen und T7-Polymerase, in vitro transkribiert und translatiert werden.The recombinant expression vectors used may be designed for expression in prokaryotic or eukaryotic cells. This is advantageous since intermediate steps of the vector construction are often carried out in microorganisms for the sake of simplicity. For example, the Δ12-desaturase, Δ6-desaturase, Δ6-elongase, Δ5-desaturase, Δ5-elongase and / or Δ-4-desaturase genes can be expressed in bacterial cells , Insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA, et al., 1992 "Foreign gene expression in yeast: a review", Yeast 8: 423-488, van den Hondel, CAMJJ, et al. (1991) "Heterologous gene expression in filamentous fungi", in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, eds., Pp. 396-428: Acadic Press: San Diego, and van Hondel, CAMJJ, & Punt, PJ. (1991) Gene Transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, JF, et al., Eds., pp. 1-28, Cambridge University Press: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology.1, 3: 239-251), ciliates of the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Desaturaseudocohniblemus, Euplotes, Engelmaniella and Stylonychia, especially the genus Stylony chia lemnae, with vectors according to a transformation method as described in WO 98/01572, and preferably in cells of multicellular plants (see Schmidt, R. and Willmitzer, L. (1988) "High-efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants "Plant Cell Rep.: 583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Fla., Chapter 6/7, pp. 71-119 (1993); FF White, B. Jenes et al., Techniques for Gene Transfer, in: Tran Sgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein). Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). The recombinant expression vector may alternatively be transcribed and translated in vitro using, for example, T7 promoter regulatory sequences and T7 polymerase.
Die Expression von Proteinen in Prokaryoten erfolgt meist mit Vektoren, die konstituti- ve oder induzierbare Promotoren enthalten, welche die Expression von Fusions- oder nicht-Fusionsproteinen steuern. Typische Fusions-Expressionsvektoren sind u.a. pGEX (Pharmacia Biotech Ine; Smith, D. B., und Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) und pRIT5 (Pharmacia, Piscataway, NJ), bei denen Glutathion-S-Transferase (GST), Maltose E-bindendes Protein bzw. Protein A an das rekombinante Zielprotein fusioniert wird. Beispiele für geeignete induzierbare nicht-Fusions-E. coli-Expressionsvektoren sind u.a. pTrc (Amann et al. (1988) Gene 69:301-315) und pET 11 d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Kalifornien (1990) 60-89). Die Zielgenexpression vom pTrc-Vektor beruht auf der Transkription durch Wirts-RNA- Polymerase von einem Hybrid-trp-lac-Fusionspromotor. Die Zielgenexpression aus dem pET 11 d-Vektor beruht auf der Transkription von einem T7-gn10-lac-Fusions- Promotor, die von einer coexprimierten viralen RNA-Polymerase (T7 gn1 ) vermittelt wird. Diese virale Polymerase wird von den Wirtsstämmen BL21 (DE3) oder HMS174 (DE3) von einem residenten λ-Prophagen bereitgestellt, der ein T7 gn1-Gen unter der Transkriptionskontrolle des lacUV 5-Promotors birgt. Andere in prokaryotischen Organismen geeignete Vektoren sind dem Fachmann bekannt, diese Vektoren sind beispielsweise in E. coli pLG338, pACYC184, die pBR-Reihe, wie pBR322, die pUC-
Reihe, wie pUC18 oder pUC19, die M1 13mp-Reihe, pKC30, pRep4, pHS1 , pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-1111 13-B1 , λgtl 1 or pBdCI, in Streptomyces plJ101 , plJ364, plJ702 oder plJ361 , in Bacillus pUB1 10, pC194 oder pBD214, in Corynebacterium pSA77 oder pAJ667.The expression of proteins in prokaryotes is usually carried out with vectors which contain constitutive or inducible promoters which control the expression of fusion or non-fusion proteins. Typical fusion expression vectors include pGEX (Pharmacia Biotech Ine, Smith, DB, and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ). in which glutathione S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990). 60-89). Target gene expression from the pTrc vector is based on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11 d vector is based on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident λ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter. Other suitable vectors in prokaryotic organisms are known to those skilled in the art, these vectors are for example in E. coli pLG338, pACYC184, the pBR series, such as pBR322, the pUC Series, such as pUC18 or pUC19, the M1 13mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pN-1111 13-B1, λgt11 or pBdCl, in Streptomyces plJ101, pIJ364, pIJ702 or pIJ361 , in Bacillus pUB1 10, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667.
Bei einer weiteren Ausführungsform ist der Expressionsvektor ein Hefe- Expressionsvektor. Beispiele für Vektoren zur Expression in der Hefe S. cerevisiae umfassen pYeDesaturased (Baldari et al. (1987) Embo J. 6:229-234), pMFa (Kurjan und Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113- 123) sowie pYES2 (Invitrogen Corporation, San Diego, CA). Vektoren und Verfahren zur Konstruktion von Vektoren, die sich zur Verwendung in anderen Pilzen, wie den filamentösen Pilzen, eignen, umfassen diejenigen, die eingehend beschrieben sind in: van den Hondel, C.A.M.J.J., & Punt, PJ. (1991 ) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J. F. Peberdy et al., Hrsgb., S. 1-28, Cambridge University Press: Cambridge, oder in: More Gene Manipulations in Fungi [J.W. Bennet & L. L. Lasure, Hrsgb., S. 396-428: Acade- mic Press: San Diego]. Weitere geeignete Hefevektoren sind beispielsweise pAG-1 , YEp6, YEp13 oder pEMBLYe23.In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisiae include pYeDesaturased (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 pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for constructing vectors suitable for use in other fungi, such as filamentous fungi, include those described in detail in: van den Hondel, C.A.M.J.J., & Punt, PJ. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, JF Peberdy et al., Eds., Pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi [JW Bennet & LL Lasure, eds., Pp. 396-428: Acadic Press: San Diego] Other suitable yeast vectors are, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.
Alternativ können die Polynucleotide der vorliegenden Erfindung auch in Insektenzellen unter Verwendung von Baculovirus-Expressionsvektoren exprimiert werden. Baculovi- rus-Vektoren, die zur Expression von Proteinen in gezüchteten Insektenzellen (z.B. Sf9-Zellen) verfügbar sind, umfassen die pAc-Reihe (Smith et al. (1983) Mol. Cell Biol.. 3:2156-2165) und die pVL-Reihe (Lucklow und Summers (1989) Virology 170:31-39).Alternatively, the polynucleotides of the present invention may also be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors that are available for expression of proteins in cultured insect cells (eg, Sf9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and U.S. Pat pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
Bevorzugte Pflanzen-Expressionsvektoren umfassen solche, die eingehend beschrieben sind in: Becker, D., Kemper, E., Schell, J., und Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20:1 195-1 197; und Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12:871 1-8721 ; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Bd. 1 , Engineering and Utilization, Hrsgb.: Kung und R. Wu, Academic Press, 1993, S. 15-38. Eine Pflanzen-Expressionskassette enthält vorzugsweise Expressionskontrollsequenzen, welche die Genexpression in Pflanzenzellen steuern können und funktionsfähig verbunden sind, so dass jede Sequenz ihre Funktion, wie Termination der Transkription, erfüllen kann, beispielsweise Polyadeny- lierungssignale. Bevorzugte Polyadenylierungssignale sind diejenigen, die aus Agrobacterium tumefaciens-T-DNA stammen, wie das als Octopinsynthase bekannte Gen 3 des Ti-Plasmids pTiACHδ (Gielen et al., EMBO J. 3 (1984) 835ff.) oder funktionelle Äquivalente davon, aber auch alle anderen in Pflanzen funktionell aktiven Terminatoren sind geeignet. Da die Pflanzengenexpression sehr oft nicht auf
Transkriptionsebenen beschränkt ist, enthält eine Pflanzen-Expressionskassette vorzugsweise andere funktionsfähig verbunden Sequenzen, wie Translationsenhancer, beispielsweise die Overdrive-Sequenz, welche die 5'-untranslatierte Leader-Sequenz aus Tabakmosaikvirus, die das Protein/RNA-Verhältnis erhöht, enthält (Gallie et al., 1987, Nucl. Acids Research 15:8693-871 1 ). Die Pflanzengenexpression muss wie oben beschrieben funktionsfähig mit einem geeigneten Promotor verbunden sein, der die Genexpression auf rechtzeitige, zell- oder gewebespezifische Weise durchführt. Nutzbare Promotoren sind konstitutive Promotoren (Benfey et al., EMBO J. 8 (1989) 2195-2202), wie diejenigen, die von Pflanzenviren stammen, wie 35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (siehe auch US 5352605 und WO 84/02913) oder Pflanzenpromotoren, wie der in US 4,962,028 beschriebene der kleinen Untereinheit der Rubisco. Andere bevorzugte Sequenzen für die Verwendung zur funktionsfähigen Verbindung in Pflanzengenexpressions-Kassetten sind Targeting-Sequenzen, die zur Steuerung des Genproduktes in sein entsprechendes Zellkompartiment notwendig sind (siehe eine Übersicht in Kermode, Crit. Rev. Plant Sei. 15, 4 (1996) 285-423 und darin zitierte Literaturstellen), beispielsweise in die Vakuole, den Zellkern, alle Arten von Piastiden, wie Amyloplasten, Chloroplasten, Chromoplasten, den extrazellulären Raum, die Mitochondrien, das Endoplasmatische Retikulum, Ölkörper, Peroxiso- men und andere Kompartimente von Pflanzenzellen.Preferred plant expression vectors include those described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border Biol. 20: 1 195-1 197; and Bevan, MW (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 871 1-8721; Vectors for Gene Transfer to Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds .: Kung and R. Wu, Academic Press, 1993, pp. 15-38. A plant expression cassette preferably contains expression control sequences which can direct gene expression in plant cells and are operably linked so that each sequence can fulfill its function, such as termination of transcription, for example polyadenylation signals. Preferred polyadenylation signals are those derived from Agrobacterium tumefaciens T-DNA, such as the gene 3 of the Ti plasmid pTiACHδ known as octopine synthase (Gielen et al., EMBO J. 3 (1984) 835ff.) Or functional equivalents thereof, as well all other terminators functionally active in plants are suitable. As the plant gene expression very often not on Transcriptional levels, a plant expression cassette preferably contains other operably linked sequences such as translation enhancers such as the overdrive sequence containing the tobacco mosaic virus 5 'untranslated leader sequence which increases the protein / RNA ratio (Gallie et al , 1987, Nucl. Acids Research 15: 8693-871 1). Plant gene expression, as described above, must be operably linked to a suitable promoter that performs gene expression in a timely, cell or tissue-specific manner. Useful promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those derived from plant viruses, such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5352605 and WO 84/02913) or plant promoters, such as the Rubisco small subunit described in US 4,962,028. Other preferred sequences for use in the functional compound in plant gene expression cassettes are targeting sequences necessary to direct the gene product into its corresponding cell compartment (see review in Kermode, Crit., Plant, 15, 4 (1996) 285) -423 and references cited therein), for example to the vacuole, the nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes, and other compartments of plant cells.
Die Pflanzengenexpression lässt sich auch wie oben beschrieben über einen chemisch induzierbaren Promotor erleichtern (siehe eine Übersicht in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemisch induzierbare Promotoren eignen sich besonders, wenn gewünscht wird, dass die Genexpression auf zeitspezifische Weise erfolgt. Beispiele für solche Promotoren sind ein Salicylsäure-induzierbarer Promotor (WO 95/19443), ein Tetracyclin-induzierbarer Promotor (Gatz et al. (1992) Plant J. 2, 397-404) und ein Ethanol-induzierbarer Promotor. Auch Promotoren, die auf biotische oder abiotische Stressbedingungen reagieren, sind geeignete Promotoren, beispielsweise der pathogeninduzierte PRP1 -Gen-Promotor (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), der hitzeinduzierbare hspδO-Promotor aus Tomate (US 5,187,267), der kälteinduzierbare Alpha-Amylase-Promotor aus Kartoffel (WO 96/12814) oder der durch Wunden induzierbare pinl I-Promotor (EP-A-O 375 091 ).Plant gene expression can also be facilitated by a chemically inducible promoter as described above (see review in Gatz 1997, Annu Rev. Plant Physiol Plant Mol. Biol., 48: 89-108). Chemically inducible promoters are particularly useful when it is desired that gene expression be in a time-specific manner. Examples of such promoters are a salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter. Promoters which react to biotic or abiotic stress conditions are also suitable promoters, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant Mol. Biol. 22 (1993) 361-366), the heat-inducible hspδO promoter Tomato (US 5,187,267), the potato inducible alpha-amylase promoter (WO 96/12814) or the wound inducible pinI I promoter (EP-A-0 375 091).
Es sind insbesondere solche Promotoren bevorzugt, welche die Genexpression in Geweben und Organen herbeiführen, in denen die Fettsäure-, Lipid- und Ölbiosynthe- se stattfindet, in Samenzellen, wie den Zellen des Endosperms und des sich entwickelnden Embryos. Geeignete Promotoren sind der Napingen-Promotor aus Raps (US 5,608,152), der USP-Promotor aus Vicia faba (Baeumlein et al., Mol Gen Genet, 1991 , 225 (3):459-67), der Oleosin-Promotor aus Arabidopsis (WO 98/45461 ), der Phaseolin-Promotor aus Phaseolus vulgaris (US 5,504,200), der Bce4-Promotor aus
Brassica (WO 91/13980) oder der Legumin-B4-Promotor (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9) sowie Promotoren, welche die samenspezifische Expression in Monokotyledonen-Pflanzen, wie Mais, Gerste, Weizen, Roggen, Reis usw. herbeiführen. Geeignete beachtenswerte Promotoren sind der lpt2- oder lpt1 - Gen-Promotor aus Gerste (WO 95/15389 und WO 95/23230) oder die in WO 99/16890 beschriebenen (Promotoren aus dem Gersten-Hordein-Gen, dem Reis-Glutelin-Gen, dem Reis-Oryzin-Gen, dem Reis-Prolamin-Gen, dem Weizen-Gliadin-Gen, Weizen- Glutelin-Gen, dem Mais-Zein-Gen, dem Hafer-Glutelin-Gen, dem Sorghum-Kasirin- Gen, dem Roggen-Secalin-Gen). Ebenfalls besonders geeignet sind Promotoren, welche die plastidenspezifische Expression herbeiführen, da Piastiden dasKomparti- ment sind, in dem die Vorläufer sowie einige Endprodukte der Lipidbiosynthese synthetisiert werden. Geeignete Promotoren, wie der virale RNA-Polymerase- Promotor, sind beschrieben in WO 95/16783 und WO 97/06250, und der cIpP- Promotor aus Arabidopsis, beschrieben in WO 99/46394.Particularly preferred are those promoters which induce gene expression in tissues and organs in which the fatty acid, lipid and oil biosynthesis takes place in sperm cells such as the cells of the endosperm and the developing embryo. Suitable promoters are the rapeseed napkin promoter (US 5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), the Arabidopsis oleosin promoter (US Pat. WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US Pat. No. 5,504,200), the Bce4 promoter Brassica (WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9) as well as promoters expressing seed-specific expression in monocotyledonous plants, such as maize To bring in barley, wheat, rye, rice, etc. Suitable noteworthy promoters are the lpt2 or lpt1 gene promoter from barley (WO 95/15389 and WO 95/23230) or those described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene , the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene). Also particularly suitable are promoters which induce plastid-specific expression, since plastids are the compart- ment in which the precursors as well as some end products of lipid biosynthesis are synthesized. Suitable promoters, such as the viral RNA polymerase promoter, are described in WO 95/16783 and WO 97/06250, and the cIpP promoter from Arabidopsis described in WO 99/46394.
Die oben genannten Vektoren bieten nur einen kleinen Überblick über mögliche geeignete Vektoren. Weitere Plasmide sind dem Fachmann bekannt und sind zum Beispiel beschrieben in: Cloning Vectors (Hrsgb. Pouwels, P. H., et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Weitere geeignete Expres- sionssysteme für prokaryotische und eukaryotische Zellen siehe in den Kapiteln 16 und 17 von Sambrook, J., Fritsch, E. F., und Maniatis, T., Molecular Cloning: A Laboratory Manual, 2. Auflage, CoId Spring Harbor Laboratory, CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY, 1989.The above vectors provide only a brief overview of possible suitable vectors. Other plasmids are known in the art and are described, for example, in: Cloning Vectors (Eds. Pouwels, P.H., et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). For other suitable expression systems for prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Ed., CoId Spring Harbor Laboratory, CoId Spring Harbor Laboratory Press, Spring Coast, NY, 1989.
Der Expressionsvektor kann, wie oben beschrieben, auch weitere Gene umfassen, die in die Organismen eingebracht werden sollen. Es ist möglich und bevorzugt, in die Wirtsorganismen Regulationsgene, wie Gene für Induktoren, Repressoren oder Enzyme, welche durch ihre Enzymaktivität in die Regulation eines oder mehrerer Gene eines Biosynthesewegs eingreifen, einzubringen und darin zu exprimieren. Diese Gene können heterologen oder homologen Ursprungs sein. Heterologe Gene oder Polynuc- leotide stammen aus einem Ursprungsorganismus, der sich von dem Zielorganismus unterscheidet, in den die Gene oder Polynucleotide eingebracht werden sollen. Bei homologen Gene oder Polynucleotide sind Zielorganismus und Ursprungsorganismus gleich. Vorzugsweise umfasst der Vektor daher mindestens ein weiteres Polynucleotid, das ein weiteres Enzym kodiert, das in die Biosynthese von Lipiden oder Fettsäuren eingebunden ist. Das Enzym ist vorzugsweise ausgewählt aus der Gruppe bestehend aus: Acyl-CoA-Dehydrogenase(n), Acyl-ACP[= acyl carrier protein]-Desaturase(n), Acyl-ACP-Thioesterase(n), Fettsäure-Acyl-Transferase(n), Acyl-The expression vector may, as described above, also include other genes to be introduced into the organisms. It is possible and preferred to introduce into the host organisms regulatory genes, such as genes for inducers, repressors or enzymes, which intervene by their enzyme activity in the regulation of one or more genes of a biosynthetic pathway, and to express therein. These genes may be of heterologous or homologous origin. Heterologous genes or polynucleotides are derived from a parent organism that differs from the target organism into which the genes or polynucleotides are to be introduced. For homologous genes or polynucleotides, the target organism and the parent organism are the same. Preferably, therefore, the vector comprises at least one further polynucleotide encoding a further enzyme involved in the biosynthesis of lipids or fatty acids. The enzyme is preferably selected from the group consisting of: acyl-CoA-dehydrogenase (s), acyl-ACP [= acyl carrier protein] desaturase (s), acyl-ACP-thioesterase (s), fatty acid-acyl-transferase ( n), acyl
CoA:Lysophospholipid-Acyltransferase(n), Fettsäure-Synthase(n), Fettsäure- Hydroxylase(n), Acetyl-Coenzym A-Carboxylase(n), Acyl-Coenzym A-Oxidase(n),
Fettsäure-Desaturase(n), Fettsäure-Acetylenase(n), Lipoxygenase(n), Triacylglycerol- Lipase(n), Allenoxid-Synthase(n), Hydroperoxid-Lyase(n), Fettsäure-Elongase(n), Δ4- Desaturase(n), Δ5-Desaturase(n), Δ6-Desaturase(n), Δ8-Desaturase(n), Δ9- Desaturase(n), Δ12-Desaturase(n), Δ5-Elongase(n), Δ6-Elongase(n) und Δ9- Elongase(n).CoA: lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (s), Fatty acid desaturase (s), fatty acid acetylenase (s), lipoxygenase (s), triacylglycerol lipase (s), allene oxide synthase (s), hydroperoxide lyase (s), fatty acid elongase (s), Δ4-desaturase (n), Δ5-desaturase (s), Δ6-desaturase (s), Δ8-desaturase (s), Δ9-desaturase (s), Δ12-desaturase (s), Δ5-elongase (s), Δ6-elongase ( n) and Δ9 elongase (s).
Die Erfindung betrifft auch eine Wirtszelle, die das erfindungsgemäße Polynucleotid oder den erfindungsgemäßen Vektor umfasst.The invention also relates to a host cell comprising the polynucleotide or vector of the invention.
Wirtszellen im Sinne der vorliegenden Erfindung können prinzipiell alle eukaryotischen oder prokaryotischn Zellen sein. Es können primäre Zellen aus Tieren, Pflanzen oder mehrzelligen Mikroorganismen sein, z.B. aus denen, die in der Beschreibung an anderer Stelle genannt sind. Ferner umfasst der Begriff auch Zelllinien, die aus diesen Organismen gewonnen werden können.Host cells according to the present invention may in principle be any eukaryotic or prokaryotic cells. They may be primary cells from animals, plants or multicellular microorganisms, e.g. from those mentioned elsewhere in the description. Furthermore, the term also includes cell lines that can be obtained from these organisms.
Wirtszellen im Sinne der Erfindung können aber auch einzellige Mikroorganismen sein, z.B. Bakterien oder Pilze. Besonders bevorzugte Mikroorganismen sind Pilze ausgewählt aus der Gruppe der Familien Chaetomiaceae, Choanephoraceae, Cryptococca- ceae, Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae oder Tuberculariaceae. Weitere evorzugte Mikroorganismen sind ausgewählt aus der Gruppe: Choanephoraceae wie den Gattungen Blakeslea, Choanephora z.B. die Gattungen und Arten Blakeslea trispora, Choanephora cucurbi- tarum, Choanephora infundibulifera var. cucurbitarum, Mortierellaceae wie der Gattung Mortierella z.B. die Gattungen und Arten Mortierella isabellina, Mortierella polycephala , Mortierella ramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae wie den Gattungen Phytium, Phytophthora z.B. die Gattungen und Arten Pythium debaryanum, Pythium intermedium, Pythium irreguläre, Pythium megalacanthum, Pythium paroe- candrum, Pythium sylvaticum, Pythium ultimum, Phytophthora cactorum, Phytophthora cinnamomi, Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, Phytophthora erythroseptica, Phytophthora lateralis, Phytophthora megasperma, Phytophthora nicotianae, Phytophthora nicotianae var. parasitica, Phytophthora palmivora, Phytophthora parasitica, Phytophthora syringae, Saccharomycetaceae wie den Gattungen Hansenula, Pichia, Saccharomyces, Saccharomycodes, Yarrowia z.B. die Gattungen und Arten Hansenula anomala, Hansenula californica, Hansenula canadensis, Hansenula capsulata, Hansenula ciferrii, Hansenula glucozyma, Hansenula henricii, Hansenula holstii, Hansenula minuta, Hansenula nonfermentans, Hansenula philodendri, Hansenula polymorpha, Hansenula saturnus, Hansenula subpelliculosa, Hansenula wickerhamii, Hansenula
wingei, Pichia alcoholophila, Pichia angusta, Pichia anomala, Pichia bispora, Pichia burtonii, Pichia canadensis, Pichia capsulata, Pichia carsonii, Pichia cellobiosa, Pichia ciferrii, Pichia farinosa, Pichia fermentans, Pichia finlandica, Pichia glucozyma, Pichia guilliermondii, Pichia haplophila, Pichia henricii, Pichia holstii, Pichia jadinii, Pichia lindnerii, Pichia membranaefaciens, Pichia methanolica, Pichia minuta var. minuta, Pichia minuta var. nonfermentans, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia philodendri, Pichia pini, Pichia polymorpha, Pichia quercuum, Pichia rhodanen- sis, Pichia sargentensis, Pichia stipitis, Pichia strasburgensis, Pichia subpelliculosa, Pichia toletana, Pichia trehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti, Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces bisporυs, Saccharomyces capensis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces cerevisiae var. ellipsoideus, Saccharomyces chevalieri, Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomyces ellipsoideus, Saccharomyces fermentati, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces heterogenicus, Saccharomyces hienipiensis, Saccharomyces inusitatus, Saccharomyces italicus, Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis, Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomyces montanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis, Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum, Saccharomycodes ludwigii, Yarrowia lipolytica, Schizosacharomycetaceae such as the genera Schizosaccharomyces e.g. the species Schizosaccharomyces japonicus var. japonicus, Schizosaccharomyces japonicus var. versatilis, Schizosaccharomyces malidevorans, Schizosaccharomyces octosporus, Schizosaccharomyces pombe var. malidevorans, Schizosaccharomyces pombe var. pombe, Thraustochytriaceae such as the genera Althornia, Aplanochytrium, Japono- chytrium, Schizochytrium, Thraustochytrium e.g. the species Schizochytrium aggrega- tum, Schizochytrium limacinum, Schizochytrium mangrovei, Schizochytrium minutum, Schizochytrium octosporum, Thraustochytrium aggregatum, Thraustochytrium amoe- boideum, Thraustochytrium antacticum, Thraustochytrium arudimentale, Thraustochytrium aureum, Thraustochytrium benthicola, Thraustochytrium globosum, Thraustochytrium indicum, Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytrium multirudimentale, Thraustochytrium pachyder- mum, Thraustochytrium proliferum, Thraustochytrium roseum, Thraustochytrium rossii, Thraustochytrium striatum oder Thraustochytrium visurgense.However, host cells according to the invention may also be unicellular microorganisms, for example bacteria or fungi. Particularly preferred microorganisms are fungi selected from the family of Chaetomiaceae, Choanephoraceae, Cryptococceae, Cunninghamellaceae, Demetiaceae, Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae, Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae or Tuberculariaceae. Further preferred microorganisms are selected from the group: Choanephoraceae such as the genera Blakeslea, Choanephora eg the genera and species Blakeslea trispora, Choanephora cucurbiturum, Choanephora infundibulifera var. Cucurbitarum, Mortierellaceae such as the genus Mortierella eg the genera and species Mortierella isabellina, Mortierella polycephala , Mortierella ramanniana, Mortierella vinacea, Mortierella zonata, Pythiaceae such as the genera Phytium, Phytophthora eg the genera and species Pythium debaryanum, Pythium intermedium, Irregular Pythium, Pythium megalacanthum, Pythium paro-candrum, Pythium sylvaticum, Pythium ultimum, Phytophthora cactorum, Phytophthora cinnamomi , Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, Phytophthora erythroseptica, Phytophthora lateralis, Phytophthora megasperma, Phytophthora nicotianae, Phytophthora nicotianae var. Parasitica, Phytophthora palmivora, Phytophthora parasitica, Phyt ophthora syringae, Saccharomycetaceae such as the genera Hansenula, Pichia, Saccharomyces, Saccharomyces, Yarrowia eg the genera and species Hansenula anomala, Hansenula californica, Hansenula canadensis, Hansenula capsulata, Hansenula ciferrii, Hansenula glucozyma, Hansenula henricii, Hansenula holstii, Hansenula minuta, Hansenula nonfermentans Hansenula philodendri, Hansenula polymorpha, Hansenula saturnus, Hansenula subpelliculosa, Hansenula wickerhamii, Hansenula Pichia anophala, Pichia angusta, Pichia anomala, Pichia bispora, Pichia burtonii, Pichia canadensis, Pichia capsulata, Pichia carsonii, Pichia cellobiosa, Pichia ciferrii, Pichia farinosa, Pichia fermentans, Pichia finlandica, Pichia glucozyma, Pichia guilliermondii, Pichia haplophila, Pichia henricii, Pichia holstii, Pichia jadinii, Pichia lindnerii, Pichia membranaefaciens, Pichia methanolica, Pichia minuta var. Minuta, Pichia minuta var nonfermentans, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia philodendri, Pichia pini, Pichia polymorpha, Pichia quercuum , Pichia rhodanensis, Pichia sargentensis, Pichia stipitis, Pichia strasburgensis, Pichia subpelliculosa, Pichia toletana, Pichia trehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti, Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces bisporus, Saccharomyces capensis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae , Saccharomyces cerevisiae var. Ellipsoideus, Saccharomyces cheva lieri, Saccharomyces delbrueckii, Saccharomyces diastaticus, Saccharomyces drosophilarum, Saccharomyces elegans, Saccharomyces ellipsoideus, Saccharomyces fermentati, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces heterogenicus, Saccharomyces hienipiensis, Saccharomyces inusitatus, Saccharomyces italicus, Saccharomyces kluyveri, Saccharomyces krusei, Saccharomyces lactis, Saccharomyces marxianus, Saccharomyces microellipsoides, Saccharomyces montanus, Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis, Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum, Saccharomyces ludwigii, Yarrowia lipolytica, Schizosacharomycetaceae as the generic Schizosaccharomyces eg the species Schizosaccharomyces japonicus var. Japonicus , Schizosaccharomyces japonicus var. Versatilis, Schizosaccharomyces malidevorans, Schizosaccharomyces octosporus, Schizosaccharomyces pombe var. Malidevorans, Sch izosaccharomyces pombe var. pombe, Thraustochytriaceae as the genera Althornia, Aplanochytrium, Japonochytrium, Schizochytrium, Thraustochytrium eg the species Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium mangrovei, Schizochytrium minutum, Schizochytrium octosporum, Thraustochytrium aggregatum, Thraustochytrium amoeboideum, Thraustochytrium antacticum, arudimentale Thraustochytrium, Thraustochytrium aureum, benthicola Thraustochytrium, Thraustochytrium globosum, Thraustochytrium indicum, Thraustochytrium kerguelense, Thraustochytrium kinnei, Thraustochytrium motivum, Thraustochytrium multi Rudi mental, Thraustochytrium pachyder- mum, Thraustochytrium proliferum, Thraustochytrium roseum, Thraustochytrium rossii, striatum Thraustochytrium or visurgense Thraustochytrium.
Ebenfalls bevorzugt als Mikroorganismen sind Bakterien ausgewählt aus der Gruppe der Familien Bacillaceae, Enterobacteriacae oder Rhizobiaceae. Besonders bevorzugt seien die folgenden Bakterinen genannt ausgewählt aus der Gruppe: Bacillaceae wie die Gattung Bacillus z.B die Gattungen und Arten Bacillus acidocaldarius, Bacillus
acidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus sphaericus subsp. fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillus globisporus subsp. marinus, Bacillus halophilus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacillus psychrosaccha- rolyticus, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis subsp. spizizenii, Bacillus subtilis subsp. subtilis oder Bacillus thuringiensis; Enterobacteriacae wie die Gattungen Citrobacter, Edwardsieila, Enterobacter, Erwinia, Escherichia, Klebsiella, Salmonella oder Serratia z.B die Gattungen und Arten Citrobacter amalonaticus, Citrobacter diversus, Citrobacter freundii, Citrobacter genomospecies, Citrobacter gillenii, Citrobacter intermedium, Citrobacter koseri, Citrobacter murliniae, Citrobacter sp., Edwardsiella hoshinae, Edwardsiella ictaluri, Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwinia ananatis, Erwinia aphidicola, Erwinia billingiae, Erwinia cacticida, Erwinia cancerogena, Erwinia carnegieana, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera, Erwinia carotovora subsp. wasabiae, Erwinia chrysanthemi, Erwinia cypripe- dii, Erwinia dissolvens, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia nimipressuralis, Erwinia persicina, Erwinia psidii, Erwinia pyrifoliae, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia Salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Escherichia adecarboxylata, Escherichia anindolica, Escherichia aurescens, Escherichia blattae, Escherichia coli, Escherichia coli var. communior, Escherichia coli-mutabile, Escherichia fergusonii, Escherichia hermannii, Escherichia sp., Escherichia vulneris, Klebsiella aerogenes, Klebsiella edwardsii subsp. atlantae, Klebsiella ornithinolytica, Klebsiella oxytoca, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae, Klebsiella sp., Klebsiella terrigena, Klebsiella trevisanii, Salmonella abony, Salmonella arizonae, Salmonella bongori, Salmonella choleraesuis subsp. arizonae, Salmonella choleraesuis subsp. bongori, Salmonella choleraesuis subsp. cholereasuis, Salmonella choleraesuis subsp. diarizonae, Salmonella choleraesuis subsp. houtenae, Salmonella choleraesuis subsp. indica, Salmonella choleraesuis subsp. salamae, Salmonella daressalaam, Salmonella enterica subsp. houtenae, Salmonella enterica subsp. salamae, Salmonella enteritidis, Salmonella gallinarum, Salmonella heidelberg, Salmonella panama, Salmonella senftenberg, Salmonella typhimurium, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia marcescens, Serratia marcescens subsp. marcescens, Serratia marinorubra, Serratia odorifera, Serratia plymouthensis, Serratia plymuthica, Serratia proteamacu- lans, Serratia proteamaculans subsp. quinovora, Serratia quinivorans oder Serratia rubidaea; Rhizobiaceae wie die Gattungen Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium z.B. die Gattungen und Arten Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium gelatinovorum, Agrobacterium
larrymoorei, Agrobacterium meteori, Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi, Agrobacterium stellulatum, Agrobacterium tumefa- ciens, Agrobacterium vitis, Carbophilus carboxidus, Chelatobacter heintzii, Ensifer adhaerens, Ensifer arboris, Ensifer fredii, Ensifer kostiensis, Ensifer kummerowiae, Ensifer medicae, Ensifer meliloti, Ensifer saheli, Ensifer terangae, Ensifer xinjiangensis, Rhizobium ciceri Rhizobium etli, Rhizobium fredii, Rhizobium galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium huakuii, Rhizobium huautlense, Rhizobium indigoferae, Rhizobium japonicum, Rhizobium leguminosarum, Rhizobium loessense, Rhizobium loti, Rhizobium lupini, Rhizobium mediterraneum, Rhizobium meliloti, Rhizobium mongolense, Rhizobium phaseoli, Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium sullae, Rhizobium tianshanen- se, Rhizobium trifolii, Rhizobium tropici, Rhizobium undicola, Rhizobium vitis, Sinorhi- zobium adhaerens, Sinorhizobium arboris, Sinorhizobium fredii, Sinorhizobium kostiense, Sinorhizobium kummerowiae, Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium morelense, Sinorhizobium saheli oder Sinorhizobium xinjian- gense.Also preferred as microorganisms are bacteria selected from the group of the families Bacillaceae, Enterobacteriacae or Rhizobiaceae. Particularly preferred are the following bacteria selected from the group: Bacillaceae such as the genus Bacillus eg the genera and species Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylolyticus, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus sphaericus subsp. fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillus globisporus subsp. marinus, Bacillus halophilus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis subsp. spizizenii, Bacillus subtilis subsp. subtilis or Bacillus thuringiensis; Enterobacteriacae such as the genera Citrobacter, Edwardsieila, Enterobacter, Erwinia, Escherichia, Klebsiella, Salmonella or Serratia eg the genera and species Citrobacter amalonaticus, Citrobacter diversus, Citrobacter freundii, Citrobacter genomospecies, Citrobacter gillenii, Citrobacter intermedium, Citrobacter koseri, Citrobacter murliniae, Citrobacter sp , Edwardsiella hoshinae, Edwardsiella ictaluri, Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwiniaananatis, Erwinia aphidicola, Erwinia billingiae, Erwinia cacticida, Erwinia carcinogena, Erwinia carnegieana, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. betavasculorum, Erwinia carotovora subsp. odorifera, Erwinia carotovora subsp. wasabiae, Erwinia chrysanthemi, Erwinia cypripedi, Erwinia dissolvens, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia nimipressuralis, Erwinia persicina, Erwinia psidii, Erwinia pyrifoliae, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Escherichia adecarboxylata, Escherichia anindolica, Escherichia aurescens, Escherichia blattae, Escherichia coli, Escherichia coli var. communior, Escherichia coli mutabile, Escherichia fergusonii, Escherichia hermannii, Escherichia sp., Escherichia vulneris, Klebsiella aerogenes, Klebsiella edwardsii subsp. atlantae, Klebsiella ornithinolytica, Klebsiella oxytoca, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella pneumoniae subsp. pneumoniae, Klebsiella spp., Klebsiella terrigena, Klebsiella trevisanii, Salmonella abony, Salmonella arizonae, Salmonella bongori, Salmonella choleraesuis subsp. arizonae, Salmonella choleraesuis subsp. bongori, Salmonella choleraesuis subsp. cholereasuis, Salmonella choleraesuis subsp. diarizonae, Salmonella choleraesuis subsp. houtenae, Salmonella choleraesuis subsp. indica, Salmonella choleraesuis subsp. Salamae, Salmonella daressalaam, Salmonella enterica subsp. houtenae, Salmonella enterica subsp. salamae, Salmonella enteritidis, Salmonella gallinarum, Salmonella heidelberg, Salmonella panama, Salmonella senftenberg, Salmonella typhimurium, Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia marcescens, Serratia marcescens subsp. marcescens, Serratia marinorubra, Serratia odorifera, Serratia plymouthensis, Serratia plymuthica, Serratia proteamaculans, Serratia proteamaculans subsp. quinovora, Serratia quinivorans or Serratia rubidaea; Rhizobiaceae such as the genera Agrobacterium, Carbophilus, Chelatobacter, Ensifer, Rhizobium, Sinorhizobium eg the genera and species Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium gelatinovorum, Agrobacterium larrymoorei, Agrobacterium meteori, Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium rubi, Agrobacterium stellulatum, Agrobacterium tumefaciens, Agrobacterium vitis, Carbophilus carboxidus, Chelatobacter heintzii, Ensifer adhaerens, Ensifer arboris, Ensifer fredii, Ensifer costiensis, Ensifer kummerowiae, Ensifer medicae, Ensifer meliloti, Ensifer saheli, Ensifer terangae, Ensifer xinjiangensis, Rhizobium ciceri Rhizobium etli, Rhizobium fredii, Rhizobium galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium hainanense, Rhizobium huakuii, Rhizobium huautlense, Rhizobium indigoferae, Rhizobium japonicum, Rhizobium leguminosarum, Rhizobium loessense, Rhizobium loti, Rhizobium lupini, Rhizobium mediterraneum, Rhizobium meliloti, Rhizobium mongolense, Rhizobium phaseoli, Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium sullae, Rhizobium tianshanensis, Rhizobium trifolii, Rhizobium tropici, Rhizobium undicola, Rhizobium vitis, Sinorhizobium adhaerens, Sinorhizobium arboris, Sinorhizobium fredii, Sinorhizobium kostiense, Sinorhizobium kummerowiae, Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium morelense, Sinorhizobium saheli or Sinorhizobium xinjian-gense.
Nutzbare Wirtszellen sind weiterhin genannt in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).Verwendbare Expressionsstämme z.B. solche, die eine geringere Proteaseaktivität aufweisen sind beschrieben in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128. Hierzu gehören Pflanzenzellen und bestimmte Gewebe, Organe und Teile von Pflanzen in all ihren Erscheinungsformen, wie Antheren, Fasern, Wurzelhaare, Stängel, Embryos, KaIIi, Kotelydo- nen, Petiolen, Erntematerial, pflanzliches Gewebe, reproduktives Gewebe und Zellkulturen, das von der eigentlichen transgenen Pflanze abgeleitet ist und/oder dazu verwendet werden kann, die transgene Pflanze hervorzubringen.Useful host cells are also mentioned in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Useful expression strains e.g. those which have lower protease activity are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128. These include plant cells and certain tissues, organs and parts of plants in all their forms such as anthers, fibers, root hairs, stems, embryos, cilia, kotelydones, petioles, crops, plant tissue, reproductive tissue and cell cultures transgenic plant is derived and / or can be used to produce the transgenic plant.
Polynucleotide oder Vektoren können mit im Stand der Technik bekannten Transforma- tions- bzw. Transfektionsverfahren in die Wirtszelle eingebracht werden wie zuvor beschrieben. Bedingungen und Medien für die Kultivierung der Wirtszellen sind dem Fachmann ebenfalls bekannt.Polynucleotides or vectors may be introduced into the host cell using transformation or transfection techniques known in the art, as previously described. Conditions and media for the cultivation of the host cells are also known to the person skilled in the art.
Bevorzugt umfasst die erfindungsgemäße Wirtszelle zusätzlich mindestens ein weiteres Enzym, das in die Biosynthese von Lipiden oder Fettsäuren eingebunden ist.The host cell according to the invention preferably additionally comprises at least one further enzyme, which is involved in the biosynthesis of lipids or fatty acids.
Bevorzugte Enzyme sind bereits an anderer Stelle in der Beschreibung genannt. DasPreferred enzymes are already mentioned elsewhere in the description. The
Enzym kann endogen in der Wirtszelle vorliegen, d.h. die Wirtszelle exprimiert bereits natürlicherweise ein Gen, das für ein entsprechendes Enzym kodiert. Alternativ kann auch ein heterologes Polynucleotid in die Wirtszelle eingebracht werden, das für das Enzym kodiert. Geeignete Verfahren und Maßnahmen für die Expression eines
heterologen Polynucleotids sind im Stand der Technik bekannt und in Zusammenhang mit den efindungsgemäßen Polynucleotiden, Vektoren und Wirtszellen hier beschrieben.Enzyme can be present endogenously in the host cell, ie the host cell already naturally expresses a gene which codes for a corresponding enzyme. Alternatively, a heterologous polynucleotide may be introduced into the host cell encoding the enzyme. Suitable methods and measures for the expression of a Heterologous polynucleotides are known in the art and described herein in connection with the polynucleotides, vectors and host cells of the present invention.
Die Erfindung betrifft auch ein Verfahren zur Herstellung eines Polypeptids mit Desaturase oder Elongase Aktivität umfassend die Schritte:The invention also relates to a method for producing a polypeptide having desaturase or elongase activity comprising the steps:
(a) Exprimieren eines erfindungsgemäßen Polynucleotids in einer Wirtszelle; und (b) Gewinnen des Polypeptids, das von dem Polynucleotid kodiert wird, aus der Wirtszelle.(a) expressing a polynucleotide of the invention in a host cell; and (b) recovering the polypeptide encoded by the polynucleotide from the host cell.
Das Polypeptid kann hierbei durch alle gängigen Verfahren zur Proteinreinigung gewonnen werden. Die Verfahren umfassen beispielseweise Affinitätschroma- tographie, Molsiebchromatographie, Hochdruck Flüssigkeitschromatographie oder auch Proteinpräzipitation ggf. mit spezifischen Antikörpern. Obwohl dies bevorzugt ist, muss das Verfahren nicht notwendigerweise ein reines Präparat des Polypeptids bereitstellen.The polypeptide can be obtained by all common methods for protein purification. The methods include, for example, affinity chromatography, molecular sieve chromatography, high-pressure liquid chromatography or protein precipitation optionally with specific antibodies. Although preferred, the method need not necessarily provide a pure preparation of the polypeptide.
Die Erfindung betrifft somit auch ein Polypeptid, das von dem erfindungsgemäßen Polynucleotid kodiert wird oder das erhätlich ist durch das zuvor genannte erfindungsgemäße Verfahren.The invention thus also relates to a polypeptide which is encoded by the polynucleotide according to the invention or which is obtainable by the abovementioned method according to the invention.
Der Begriff "Polypeptid" bezeichnet sowohl ein im Wesentlichen reines Polypeptid als auch ein Polypeptid Präparat, das noch weitere Komponenten oder Verunreinigungen aufweist. Der Begriff wird auch für Fusionsproteine oder Proteinaggregate verwendet, die das erfindungsgemäße Polypeptid und zusätzlich noch weitere Komponenten umfassen. Der Begriff bezeichnet auch chemisch modifizierte Polypeptide. Chemische Modifikationen beinhalten in diesem Zusammenhang künstliche Modifikationen oder natürlich auftretende Modifikationen, z.B posttranslationale Modifikationen wie Phosphorylierung, Myristylierung, Glykosylierung usw.. Die Begriffe Polypeptid, Peptid oder Protein sind austauschbar und werden entsprechend in der Beschreibung und im Stand der Technik verwendet. Die erfindungsgemäßen Polypeptide haben die zuvor genannten biologischen Aktivitäten, also Desaturase oder Elongase Aktivitäten, und können die Biosynthese von mehrfach ungesättigten Fettsäuren (PUFAs), vorzugsweise den langkettigen PUFAs (LCPUFAs), wie hier beschrieben beeinflussen.
Von der Erfindung umfasst ist auch ein Antikörper, der das erfindungsgemäße Polypeptid spezifisch erkennt.The term "polypeptide" refers to both a substantially pure polypeptide and a polypeptide preparation that contains other components or contaminants. The term is also used for fusion proteins or protein aggregates comprising the polypeptide of the invention and additionally further components. The term also refers to chemically modified polypeptides. Chemical modifications in this context include artificial modifications or naturally occurring modifications, eg, post-translational modifications such as phosphorylation, myristylation, glycosylation, etc. The terms polypeptide, peptide, or protein are interchangeable and are used accordingly in the description and in the prior art. The polypeptides according to the invention have the abovementioned biological activities, ie desaturase or elongase activities, and can influence the biosynthesis of polyunsaturated fatty acids (PUFAs), preferably the long-chain PUFAs (LCPUFAs), as described herein. The invention also encompasses an antibody which specifically recognizes the polypeptide according to the invention.
Antikörper gegen das erfindungsgemäße Polypeptid können mit bekannten Verfahren hergestellt werden, bei denen gereingtes Polypeptid oder Fragmente davon mit geeigneten Epitopen als Antigen verwendet werden. Geeignete Epitope können mittels bekannter Algorithmen zur Antigenizitätsbestimmung basierend auf den hier bereitgestellten Aminosäuresequenzen der efindungsgemäßen Polypeptide ermittelt werden. Die entsprechenden Polypeptide oder Fragmente können dann synthetisiert oder rekombinant gewonnen werden. Nach Immunisierung von Tieren, vorzugsweise von Säugern z.B. Hasen, Ratten oder Mäusen, können die Antikörper dann aus dem Serum mit bekannten Verfahren gewonnen werden. Alternativ können monoklonale Antikörper oder Antikörperfragmente mit den bekannte Verfahren bereitgestellt werden; siehe z.B. Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, CoId Spring Harbor, 1988, oder Köhler und Milstein, Nature 256 (1975), 495, und Galfre, Meth. Enzymol. 73 (1981 ), 3.Antibodies to the polypeptide of the invention may be prepared by known methods using purified polypeptide or fragments thereof with appropriate epitopes as the antigen. Suitable epitopes can be determined by known antigenicity determination algorithms based on the amino acid sequences of the polypeptides of the invention provided herein. The corresponding polypeptides or fragments can then be synthesized or recombinantly recovered. After immunization of animals, preferably mammals e.g. Rabbits, rats or mice, the antibodies can then be recovered from the serum by known methods. Alternatively, monoclonal antibodies or antibody fragments may be provided by known methods; see, e.g. Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Colard Spring Harbor, 1988, or Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3.
Vorzugsweise handelt es sich bei den Antikörpern um monoklonale oder polyklonale Antikörper, "single chain"-Antikörper oder chimäre Antikörper sowie Fragmenten davon wie Fab, Fv oder scFv. Weitere Antikörper im Sinne der Erfindung sind bispezifische Antikörper, synthetische Antikörper oder deren chemisch modifizierte Derivate.The antibodies are preferably monoclonal or polyclonal antibodies, single-chain antibodies or chimeric antibodies and fragments thereof such as Fab, Fv or scFv. Further antibodies within the meaning of the invention are bispecific antibodies, synthetic antibodies or their chemically modified derivatives.
Die erfindugsgemäßen Antikörper sollen die erfindungsgemäßen Polypeptide spezifisch erkennen, d.h. sie sollen nicht in signifikantem Ausmaß mit anderen Proteinen kreuzreagieren. Dies kann mit im Stand der Technik bekannten Verfahren getestet werden. Die Antikörper können beispielsweise zur Immunopräzipitation, zur Immun- histochemie oder zur Proteinreinigung (z.B. Affinitätschromatographie) eingesetzt werden.The antibodies of the invention are intended to specifically recognize the polypeptides of the invention, i. they should not crossreact with other proteins to any significant extent. This can be tested by methods known in the art. The antibodies can be used, for example, for immunoprecipitation, for immunohistochemistry or for protein purification (for example affinity chromatography).
Die Erfindung betrifft ferner einen transgenen, nicht-humanen Organismus, der das Polynucleotid, den Vektor oder die Wirtszelle der vorliegenden Erfindung umfasst. Bevorzugt handelt es sich bei dem transgenen, nicht humanen Organismus um ein Tier, eine Pflanze oder ein multizellulärere Mikroorganismus.The invention further relates to a transgenic non-human organism comprising the polynucleotide, the vector or the host cell of the present invention. Preferably, the transgenic, non-human organism is an animal, a plant or a multicellular microorganism.
Unter dem Begriff „transgen" ist zu verstehen, dass ein heterologes Polynucleotid, also ein in dem jeweiligen Organismus nicht natürlicherweise vorkommendes Polynucleotid, in den Organismus eingebracht wird. Dies kann entweder durch zufällige Insertion des Polynucleotids oder durch homologe Rekombination erreicht werden. Selbstverständ- lieh kann statt des Polynucleotids auch der erfindungsgemäße Vektor eingebracht
werden. Verfahren zum Einbringen von Polynucleotiden oder Vektoren zwecks zufälliger Insertion oder homologer Rekombination sind im Stand der Technik bekannt und auch nachfolgend genauer beschrieben. Wirtszellen, die das Polynucleotid oder den Vektor enthalten, können ebenfalls in einen Organismus eingebracht weden und so einen transgenen Organismus erzeugen. Bei einem solchen Organsimus handelt es sich dann aber um einen Chimären Organismus bei dem lediglich die Zellen, die sich von den eingebrachten Zellen ableiten, transgen sind, d.h. das heterologe Polynucleotid umfassen.The term "transgenic" is to be understood as meaning that a heterologous polynucleotide, ie a polynucleotide which does not naturally occur in the particular organism, is introduced into the organism, which can be achieved either by random insertion of the polynucleotide or by homologous recombination it is also possible to introduce the vector according to the invention instead of the polynucleotide become. Methods for introducing polynucleotides or vectors for random insertion or homologous recombination are known in the art and also described in more detail below. Host cells containing the polynucleotide or vector may also be introduced into an organism to produce a transgenic organism. However, such an organism is then a chimeric organism in which only the cells which are derived from the introduced cells are transgenic, ie comprise the heterologous polynucleotide.
Bevorzugt sind die transgenen, nicht-humanen Organismen Öl-produzierenden Organismen, das heißt die für die Herstellung von Ölen verwendet werden, wie Pilze wie Mortierella oder Thraustochytrium, Algen wie Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylum oder Pflanzen.Preferred are the transgenic non-human organisms oil-producing organisms, that is, used for the production of oils, such as fungi such as Mortierella or Thraustochytrium, algae such as Nephroselmis, Pseudoscourfielda, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus, Crypthecodinium, Phaeodactylum or plants.
Als transgene Pflanzen können grundsätzlich alle Pflanzen verwendet werden, d.h. sowohl zweikeimblättrige als auch einkeimblättrige Pflanzen. Vorzugsweise handelt es sich um Ölfruchtpflanzen, die große Mengen an Lipidverbindungen enthalten, wie Erdnuss, Raps, Canola, Sonnenblume, Saflor (Carthamus tinctoria), Mohn, Senf, Hanf, Rizinus, Olive, Sesam, Calendula, Punica, Nachtkerze, Königskerze, Distel, Wildrosen, Haselnuss, Mandel, Macadamia, Avocado, Lorbeer, Kürbis, Lein, Soja, Pistazien, Borretsch, Bäume (Ölpalme, Kokosnuss oder Walnuss) oder Feldfrüchte, wie Mais, Weizen, Roggen, Hafer, Triticale, Reis, Gerste, Baumwolle, Maniok, Pfeffer, Tagetes, Solanaceen-Pflanzen, wie Kartoffel, Tabak, Aubergine und Tomate, Vicia-Arten, Erbse, Alfalfa oder Buschpflanzen (Kaffee, Kakao, Tee), Salix-Arten sowie ausdauernde Gräser und Futterfeldfrüchte. Bevorzugte erfindungsgemäße Pflanzen sind Ölfruchtpflanzen, wie Erdnuss, Raps, Canola, Sonnenblume, Saflor , Mohn, Senf, Hanf, Rhizinus, Olive, Calendula, Punica, Nachtkerze, Kürbis, Lein, Soja, Borretsch, Bäume (Ölpalme, Kokosnuss). Besonders bevorzugt sind C18:2- und/oder C18:3-Fettsäure reiche Pflanzen wie Sonnenblume, Färberdistel, Tabak, Königskerze, Sesam, Baumwolle, Kürbis, Mohn, Nachtkerze, Walnuss, Lein, Hanf, Distel oder Färberdistel. Ganz besonders bevorzugt sind Pflanzen wie Färberdistel, Sonnenblume, Mohn, Nachtkerze, Walnuss, Lein oder Hanf. Prinzipiell kommen aber alle Pflanzen in Frage, die in der Lage sind Fettsäuren zu synthetisieren wie alle dicotylen oder monokotylen Pflanzen, Algen oder Moose. Vorteilhaft Pflanzen sind ausgewählt aus der Gruppe der Pflanzenfamilien Adelotheciaceae, Anacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae, Cannabaceae, Convolvula- ceae, Chenopodiaceae, Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagna- ceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae, Gramineae, Juglandaceae,
Lauraceae, Leguminosae, Linaceae, Prasinophyceae oder Gemüsepflanzen oder Zierpflanzen wie Tagetes in Betracht.In principle, all plants can be used as transgenic plants, ie both dicotyledonous and monocotyledonous plants. Preferably, they are oilseed crops containing high levels of lipid compounds such as peanut, rapeseed, canola, sunflower, safflower (Carthamus tinctoria), poppy, mustard, hemp, castor, olive, sesame, calendula, punica, evening primrose, mullein, thistle , Wild roses, hazelnut, almond, macadamia, avocado, bay leaf, pumpkin, flax, soy, pistachio, borage, trees (oil palm, coconut or walnut) or crops such as corn, wheat, rye, oats, triticale, rice, barley, cotton , Cassava, pepper, tagetes, solanaceae plants such as potato, tobacco, aubergine and tomato, Vicia species, pea, alfalfa or bush plants (coffee, cocoa, tea), Salix species and perennial grasses and forage crops. Preferred plants according to the invention are oil crop plants, such as peanut, rapeseed, canola, sunflower, safflower, poppy, mustard, hemp, castor, olive, calendula, punica, evening primrose, pumpkin, flax, soy, borage, trees (oil palm, coconut). Particularly preferred are C18: 2 and / or C18: 3 fatty acid rich plants such as sunflower, safflower, tobacco, mullein, sesame, cotton, pumpkin, poppy, evening primrose, walnut, flax, hemp, thistle or safflower. Very particularly preferred are plants such as safflower, sunflower, poppy, evening primrose, walnut, flax or hemp. In principle, however, all plants come into question, which are able to synthesize fatty acids as all dicotyledonous or monocotyledonous plants, algae or mosses. Advantageous plants are selected from the group of the plant families Adelotheciaceae, Anacardiaceae, Asteraceae, Apiaceae, Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae, Cannabaceae, Convolvulceae, Chenopodiaceae, Crypthecodiniaceae, Cucurbitaceae, Ditrichaceae, Elaeagaceae, Ericaceae, Euphorbiaceae, Fabaceae , Geraniaceae, Gramineae, Juglandaceae, Lauraceae, Leguminosae, Linaceae, Prasinophyceae or vegetables or ornamental plants such as Tagetes.
Besonders bevorzugt seien die folgenden Pflanzen genannt ausgewählt aus der Gruppe: Adelotheciaceae wie die Gattungen Physcomitrella z.B. die Gattung und Arten Physcomitrella patens, Anacardiaceae wie die Gattungen Pistacia, Mangifera, Anacar- dium z.B. die Gattung und Arten Pistacia vera [Pistazie], Mangifer indica [Mango] oder Anacardium occidentale [Cashew], Asteraceae wie die Gattungen Calendula, Cartha- mus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana z.B. die Gattung und Arten Calendula officinalis [Garten-Ringelblume], Carthamus tinctorius [Färberdistel, safflower], Centaurea cyanus [Kornblume], Cichorium intybus [Wegwarte], Cynara scolymus [Artichoke], Helianthus annus [Sonnenblume], Lactuca sativa, Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa, Lactuca scariola L. var. integrata, Lactuca scariola L. var. integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta [Salat], Tagetes lucida, Tagetes erecta oder Tagetes tenuifolia [Studentenblume], Apiaceae wie die Gattung Daucus z.B. die Gattung und Art Daucus carota [Karotte], Betulaceae wie die Gattung Corylus z.B. die Gattungen und Arten Corylus avellana oder Corylus colurna [Haselnuss], Boragina- ceae wie die Gattung Borago z.B. die Gattung und Art Borago officinalis [Borretsch], Brassicaceae wie die Gattungen Brassica, Camelina, Melanosinapis, Sinapis, Araba- dopsis z.B. die Gattungen und Arten Brassica napus, Brassica rapa ssp. [Raps], Sinapis arvensis Brassica juncea, Brassica juncea var. juncea, Brassica juncea var. crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassica sinapioides, Camelina sativa, Melanosinapis communis [Senf], Brassica oleracea [Futterrübe] oder Arabi- dopsis thaliana, Bromeliaceae wie die Gattungen Anana, Bromelia (Ananas) z.B. die Gattungen und Arten Anana comosus, Ananas ananas oder Bromelia comosa [Ananas], Caricaceae wie die Gattung Carica wie die Gattung und Art Carica papaya [Papaya], Cannabaceae wie die Gattung Cannabis wie die Gattung und Art Cannabis sative [Hanf], Convolvulaceae wie die Gattungen Ipomea, Convolvulus z.B. die Gattungen und Arten Ipomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba oder Convolvulus panduratus [Süßkartoffel, Batate], Chenopodiaceae wie die Gattung Beta wie die Gattungen und Arten Beta vulgaris, Beta vulgaris var. altissima, Beta vulgaris var. Vulgaris, Beta maritima, Beta vulgaris var. perennis, Beta vulgaris var. conditiva oder Beta vulgaris var. esculenta [Zuckerrübe], Crypthecodiniaceae wie die Gattung Crypthecodinium z.B. die Gattung und Art Cryptecodinium cohnii, Cucurbitaceae wie die Gattung Cucubita z.B. die Gattungen und Arten Cucurbita maxima, Cucurbita mixta, Cucurbita pepo oder Cucurbita moschata [Kürbis], Cymbellaceae wie die Gattungen Amphora, Cymbella, Okedenia, Phaeodactylum, Reimeria z.B. die Gattung und Art Phaeodactylum tricornutum, Ditrichaceae wie die Gattungen Ditrichaceae,
Astomiopsis, Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium, Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon, Skottsbergia z.B. die Gattungen und Arten Ceratodon antarcticus, Ceratodon columbiae, Ceratodon heterophyllus, Ceratodon purpurascens, Ceratodon purpureus, Ceratodon purpureus ssp. convolutus, Ceratodon purpureus ssp. stenocarpus, Ceratodon purpureus var. rotundifolius, Ceratodon ratodon, Ceratodon stenocarpus, Chrysoblastella chilensis, Ditrichum ambiguum, Ditrichum brevisetum, Ditrichum crispatissimum, Ditrichum difficile, Ditrichum falcifolium, Ditrichum flexicaule, Ditrichum giganteum, Ditrichum heteromallum, Ditrichum lineare, Ditrichum lineare, Ditrichum montanum, Ditrichum montanum, Ditrichum pallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillum var. tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichum tortile, Distichium capillaceum, Distichium hagenii, Distichium inclinatum, Distichium macounii, Eccremidium floridanum, Eccremidium whiteleggei, Lophidion strictus, Pleuridium acuminatum, Pleuridium alternifolium, Pleuridium holdridgei, Pleuridium mexicanum, Pleuridium ravenelii, Pleuridium subulatum, Saelania glaucescens, Trichodon borealis, Trichodon cylindricus oder Trichodon cylindricus var. oblongus, Elaeagnaceae wie die Gattung Elaeagnus z.B. die Gattung und Art Olea europaea [Olive], Ericaceae wie die Gattung Kalmia z.B. die Gattungen und Arten Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistus chamaerhodendros oder Kalmia lucida [Berglorbeer], Euphorbiaceae wie die Gattungen Manihot, Janipha, Jatropha, Ricinus z.B. die Gattungen und Arten Manihot utilissima, Janipha manihot,, Jatropha manihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihot melanoba- sis, Manihot esculenta [Manihot] oder Ricinus communis [Rizinus], Fabaceae wie die Gattungen Pisum, Albizia, Cathormion, Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine, Dolichos, Phaseolus, Soja z.B. die Gattungen und Arten Pisum sativum, Pisum arvense, Pisum humile [Erbse], Albizia berteriana, Albizia julibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis, Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuillea berteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobium fragrans, Pithecolobium berterianum, Pseudalbizzia berteriana, Acacia julibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck, Acacia macrophyl- Ia, Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa [Seidenbaum], Medicago sativa, Medicago falcata, Medicago varia [Alfalfa] Glycine max Dolichos soja, Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida oder Soja max [Soja- bohne], Funariaceae wie die Gattungen Aphanorrhegma, Entosthodon, Funaria, Physcomitrella, Physcomitrium z.B. die Gattungen und Arten Aphanorrhegma serra- tum, Entosthodon attenuatus, Entosthodon bolanderi, Entosthodon bonplandii, Entosthodon californicus, Entosthodon drummondii, Entosthodon jamesonii, Entosthodon leibergii, Entosthodon neoscoticus, Entosthodon rubrisetus, Entosthodon spathuli- folius, Entosthodon tucsoni, Funaria americana, Funaria bolanderi, Funaria calcarea,
Funaria californica, Funaria calvescens, Funaria convoluta, Funaria flavicans, Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var. arctica, Funaria hygro- metrica var. calvescens, Funaria hygrometrica var. convoluta, Funaria hygrometrica var. muralis, Funaria hygrometrica var. utahensis, Funaria microstoma, Funaria microstoma var. obtusifolia, Funaria muhlenbergii, Funaria orcuttii, Funaria plano- convexa, Funaria polaris, Funaria ravenelii, Funaria rubriseta, Funaria serrata, Funaria sonorae, Funaria sublimbatus, Funaria tucsoni, Physcomitrella californica, Physco- mitrella patens, Physcomitrella readeri, Physcomitrium austräte, Physcomitrium californicum, Physcomitrium collenchymatum, Physcomitrium coloradense, Physco- mitrium cupuliferum, Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitrium flexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum, Physcomitrium immersum, Physcomitrium kellermanii, Physcomitrium megalocarpum, Physcomitrium pyriforme, Physcomitrium pyriforme var. serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitrium subsphaericum, Physcomitrium washingtoniense, Geraniaceae wie die Gattungen Pelargonium, Cocos, Oleum z.B. die Gattungen und Arten Cocos nucifera, Pelargonium grossularioides oder Oleum cocois [Kokusnuss], Gramineae wie die Gattung Saccharum z.B. die Gattung und Art Saccharum officinarum, Juglandaceae wie die Gattungen Juglans, Wallia z.B. die Gattungen und Arten Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa, Juglans nigra oder Wallia nigra [Walnuss], Lauraceae Wie die Gattungen Persea, Laurus z.B. die Gattungen und Arten Laurus nobilis [Lorbeer], Persea americana, Persea gratissi- ma oder Persea persea [Avocado], Leguminosae wie die Gattung Arachis z.B. die Gattung und Art Arachis hypogaea [Erdnuss], Linaceae wie die Gattungen Linum, Adenolinum z.B. die Gattungen und Arten Linum usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var. lewisii, Linum pratense oder Linum trigynum [Lein], Lythrarieae wie die Gattung Punica z.B. die Gattung und Art Punica granatum [Granatapfel], Malvaceae wie die Gattung Gossypium z.B. die Gattungen und Arten Gossypi- um hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum oder Gossypium thurberi [Baumwolle], Marchantiaceae wie die Gattung Marchantia z.B. die Gattungen und Arten Marchantia berteroana, Marchantia foliacea, Marchantia macropora, Musaceae wie die Gattung Musa z.B. die Gattungen und Arten Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [Banane], Onagraceae wie die Gattungen Camissonia, Oenothera z.B. die Gattungen und Arten Oenothera biennis oder Camissonia brevipes [Nachtkerze], Palmae wie die Gattung Elacis z.B. die Gattung und Art Elaeis guineensis [Ölpalme], Papaveraceae wie die Gattung Papaver z.B. die Gattungen und Arten Papaver Orientale, Papaver rhoeas, Papaver dubium
[Mohn], Pedaliaceae wie die Gattung Sesamum z.B. die Gattung und Art Sesamum indicum [Sesam], Piperaceae wie die Gattungen Piper, Artanthe, Peperomia, Steffen- sia z.B. die Gattungen und Arten Piper aduncum, Piper amalago, Piper angustifolium, Piper auritum, Piper betet, Piper cubeba, Piper longum, Piper nigrum, Piper retrofrac- tum, Artanthe adunca, Artanthe elongata, Peperomia elongata, Piper elongatum, Steffensia elongata. [Cayennepfeffer], Poaceae wie die Gattungen Hordeum, Seeale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea (Mais), Triticum z.B. die Gattungen und Arten Hordeum vulgäre, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeum hexastichon., Hordeum hexastichum, Hordeum irreguläre, Hordeum sativum, Hordeum secalinum [Gerste], Seeale cereale [Roggen], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida [Hafer], Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgäre, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum, Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense, Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum, Sorghum vulgäre, Holcus halepen- sis, Sorghum miliaceum, Panicum militaceum [Hirse], Oryza sativa, Oryza latifolia [Reis], Zea mays [Mais] Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum oder Triticum vulgäre [Weizen], Porphyri- diaceae wie die Gattungen Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodeila, Rhodosorus, Vanhoeffenia z.B. die Gattung und Art Porphyridium cruentum, Proteaceae wie die Gattung Macadamia z.B. die Gattung und Art Macadamia intergri- folia [Macadamia], Prasinophyceae wie die Gattungen Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus z.B. die Gattungen und Arten Nephroselmis olivacea, Prasinococcus capsulatus, Scherffelia dubia, Tetraselmis chui, Tetraselmis suecica, Mantoniella squamata, Ostreococcus tauri, Rubiaceae wie die Gattung Coffea z.B. die Gattungen und Arten Cofea spp., Coffea arabica, Coffea canephora oder Coffea liberica [Kaffee], Scrophulariaceae wie die Gattung Verbascum z.B. die Gattungen und Arten Verbascum blattaria, Verbascum chaixii, Verbascum densiflorum, Verbascum lagurus, Verbascum longifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum, Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum oder Verbascum thapsus [Königskerze], Solanaceae wie die Gattungen Capsicum, Nicotiana, Solanum, Lycopersicon z.B. die Gattungen und Arten Capsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens [Pfeffer], Capsicum annuum [Paprika], Nicotiana tabacum, Nicotiana alata, Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotiana obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana rustica, Nicotiana sylvestris [Tabak], Solanum tuberosum [Kartoffel], Solanum melongena [Aubergine] Lycopersicon esculentum, Lycopersicon lycopersicum., Lycopersicon pyriforme,
Solanum integrifolium oder Solanum lycopersicum [Tomate], Sterculiaceae wie die Gattung Theobroma z.B. die Gattung und Art Theobroma cacao [Kakao] oder Thea- ceae wie die Gattung Camellia z.B. die Gattung und Art Camellia sinensis [Tee].Particular preference is given to the following plants selected from the group: Adelotheciaceae such as the genera Physcomitrella eg the genus and species Physcomitrella patens, Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium eg the genus and species Pistacia vera [pistachio], Mangifer indica [ Mango] or Anacardium occidentale [Cashew], Asteraceae such as the genera Calendula, Carthus, Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta, Tagetes, Valeriana eg the genus and species Calendula officinalis [Gardening Marigold], Carthamus tinctorius [ Safflower], Centaurea cyanus [cornflower], Cichorium intybus [chicory], Cynara scolymus [Artichoke], Helianthus annus [sunflower], Lactuca sativa, Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa, Lactuca scariola L. var. integrata, Lactuca scariola L. var. integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta [lettuce], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [marigold], Apiaceae such as the genus Daucus eg the genus and species Daucus carota [carrot], Betulaceae such as the genus Corylus eg the genera and species Corylus avellana or Corylus colurna [hazel], Boraginaceae such as the genus Borago eg the genus and species Borago officinalis [borage], Brassicaceae such as the genera Brassica, Camelina, Melanosinapis, Sinapis, Arabadopsis eg the genera and species Brassica napus, Brassica rapa ssp. [Canola], Sinapis arvensis Brassica juncea, Brassica juncea var. Juncea, Brassica juncea var. Crispifolia, Brassica juncea var. Foliosa, Brassica nigra, Brassica sinapioides, Camelina sativa, Melanosinapis communis [mustard], Brassica oleracea [fodder beet] or Arabica dopsis thaliana, Bromeliaceae such as the genera Anana, Bromelia (pineapple) eg the genera and species Anana comosus, pineapple pineapple or Bromelia comosa [pineapple], Caricaceae such as the genus Carica such as the genus and Art Carica papaya [papaya], Cannabaceae as the genus Cannabis such as the genus and species Cannabis sative [hemp], Convolvulaceae as the genera Ipomea, Convolvulus eg the genera and species Ipomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus [sweet potato, Batate], Chenopodiaceae such as the genus Beta such as the genera and species Beta vulgaris, Beta vulgaris var. Altissima, Beta vulgaris var. Vulgaris, Be ta maritima, Beta vulgaris var. perennis, Beta vulgaris var. conditiva or Beta vulgaris var. esculenta [sugar beet], Crypthecodiniaceae such as the genus Crypthecodinium eg the genus and species Cryptecodinium cohnii, Cucurbitaceae such as the genus Cucubita eg the genera and species Cucurbita maxima, Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [pumpkin], Cymbellaceae such as the genera Amphora, Cymbella, Okedenia, Phaeodactylum, Reimeria eg the genus and species Phaeodactylum tricornutum, Ditrichaceae such as the genera Ditrichaceae, Astomiopsis, Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium, Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon, Skottsbergia eg the genera and species Ceratodon antarcticus, Ceratodon columbiae, Ceratodon heterophyllus, Ceratodon purpurascens, Ceratodon purpureus, Ceratodon purpureus ssp. convolutus, Ceratodon purpureus ssp. stenocarpus, Ceratodon purpureus var. rotundifolius, Ceratodon ratodon, Ceratodon stenocarpus, Chrysoblastella chilensis, Ditrichum ambiguum, Ditrichum brevisetum, Ditrichum crispatissimum, Ditrichum difficile, Ditrichum falcifolium, Ditrichum flexicaule, Ditrichum giganteum, Ditrichum heteromallum, Ditrichum linear, Ditrichum linear, Ditrichum montanum, Ditrichum montanum, Ditrichum pallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillum var. Tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichum tortile, Distichium capillaceum, Distichium hagenii, Distichium inclinatum, Distichium macounii, Eccremidium floridanum, Eccremidium whiteleggei, Lophidion strictus, Pleuridium acuminatum , Pleuridium alternifolium, Pleuridium holdridgei, Pleuridium mexicanum, Pleuridium ravenelii, Pleuridium subulatum, Saelania glaucescens, Trichodon borealis, Trichodon cylindricus or Trichodon cylindricus var. Oblongus, Elaeagnaceae as the genus Elaeagnus eg the genus and Art Olea europaea [Olive], Ericaceae as the genus Kalmia eg the genera and species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistus chamaerhodendros or Kalmia lucida [Berglorbeer], Euphorbiaceae such as the genera Manihot, Janipha, Jatropha Ricinus eg the genera and species Manihot utilissima, Janipha manihot ,, Jatropha manihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta [Manihot] or Ricinus communis [Castor], Fabaceae as the genera Pisum, Albizia , Cathormion, Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine, Dolichos, Phaseolus, Soy eg the genera and species Pisum sativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albizia julibrissin, Albizia lebbeck, Acacia berteriana , Acacia littoralis, Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuillea berteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobium fragrans, Pithecolobium berterianum, Pseudalbizzia berteriana, Acacia julibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck, Acacia macrophyl Ia, Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa [Seidenbaum], Medicago sativa, Medicago falcata, Medicago varia [Alfalfa] Glycine max Dolichos soya, Glycine gracilis, Glycine hispida, Phaseolus max, Soy hispida or Soy max [Soy bean], Funariaceae such as the genera Aphanorrhegma, Entosthodon, Funaria, Physcomitrella, Physcomitrium eg the genera and species Aphanorrhegoma serrateum, Entosthodon attenuatus, Entosthodon bolanderi, Entosthodon bonplandii, Entosthodon californicus, Entosthodon drummondii, Entosthodon jamesonii, Entosthodon leibergii, Entosthodon neoscoticus, Entosthodon rubrisetus, Entosthodon spathulifolia, Entosthodon tucsoni, Funaria americana, Funaria bolanderi, Funaria calcarea, Funaria californica, Funaria calvescens, Funaria convoluta, Funaria flavicans, Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var. Arctica, Funaria hygrometrica var. Calvescens, Funaria hygrometrica var. Convoluta, Funaria hygrometrica var. Muralis, Funaria hygrometrica var. Utahensis, Funaria microstoma, Funaria microstoma var. Obtusifolia, Funaria muhlenbergii, Funaria orcuttii, Funaria plano convexa, Funaria polaris, Funaria ravenelii, Funaria rubriseta, Funaria serrata, Funaria sonorae, Funaria sublimbatus, Funaria tucsoni, Physcomitrella californica, Physcomitrella patens, Physcomitrella readeri, Physcomitrium spp., Physcomitrium californicum, Physcomitrium collenchymatum, Physcomitrium coloradense, Physcomitrum cupuliferum, Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitrium flexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum, Physcomitrium immersum, Physcomitrium kellermanii, Physcomitrium megalocarpum, Physcomitrium pyriforme, Physcomitrium pyriforme var. serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitrium subsphaericum, Physcomitrium washingtoniense, Geraniaceae such as the genera Pelargonium, Cocos, Oleum eg the genera and species Cocos nucifera, Pelargonium grossularioides or Oleum cocois [coconut], Gramineae as the genus Saccharum eg the genus and species Saccharum officinarum, Juglandaceae as the genera Juglans, Wallia eg the genera and species Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra [Walnut], Lauraceae Like the genera Persea, Laurus eg the genera and species Laurus nobilis [Laurel], Persea americana, Persea gratissima or Persea persea [Avocado], Leguminosae the genus Arachis eg the genus and species Arachis hypogaea [peanut], Linaceae wi the genera Linum usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var lewisii, Linum pratense or Linum trigynum [flax], Lythrarieae as the genus Punica eg the genus and species Punica granatum [pomegranate], Malvaceae as the genus Gossypium eg the genera and species Gossypium hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum or Gossypium thurberi [cotton], Marchantiaceae as the genus Marchantia eg the genera and species Marchantia berteroana, Marchantia foliacea, Marchantia macropora, Musaceae as the genus Musa eg the genera and species Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [Banana], Onagraceae such as the genera Camissonia, Oenothera eg the genera and species Oenothera biennis or Camissonia brevipes [evening primrose], Palmae as the genus Elacis eg the genus and species Elaeis guineensis [oil palm], Papaveraceae as the genus Papaver eg the genera and Types Papaver Orientale, Papaver rhoeas, Papaver dubium [Poppy], Pedaliaceae such as the genus Sesamum eg the genus and species Sesamum indicum [sesame], Piperaceae such as the genera Piper, Artanthe, Peperomia, Steffenia eg the genera and species Piper aduncum, Piper amalago, Piper angustifolium, Piper auritum, Piper betet, Piper cubeba, Piper longum, Piper nigrum, Piper retrofracture, Artanthe adunca, Artanthe elongata, Peperomia elongata, Piper elongatum, Steffensia elongata. [Cayenne pepper], Poaceae such as the genera Hordeum, Seeale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea (maize), Triticum eg the genera and species Hordeum vulgare, Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeum hexastichonum, Hordeum hexastichum, Hordeum irregular, Hordeum sativum, Hordeum secalinum [barley], Seeale cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida [oats], Sorghum bicolor , Sorghum halepense, Sorghum saccharatum, Sorghum vulgaris, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum, Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense, Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, sorghum subglabrescens, sorghum verticilliflorum, sorghum vulgaris, holcus halepensis, sorghum miliaceum, panicum militaceum [millet], oryza sativa, oryza latifolia [Rice], Zea mays [maize] Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare [wheat], Porphyridaceae such as the genera Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodeila, Rhodosorus Vanhoeffenia eg the genus and species Porphyridium cruentum, Proteaceae such as the genus Macadamia eg the genus and species Macadamia intergrifolia [Macadamia], Prasinophyceae such as the genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella, Ostreococcus eg the genera and species Nephroselmis olivacea , Prasinococcus capsulatus, Scherffelia dubia, Tetraselmis chui, Tetraselmis suecica, Mantoniella squamata, Ostreococcus tauri, Rubiaceae such as the genus Coffea eg the genera and species Cofea spp., Coffea arabica, Coffea canephora or Coffea liberica [coffee], Scrophulariaceae such as the genus Verbascum eg the genera and species Verbascum Blattaria, Verbascum chaixii, Verbascum densiflorum, Verbascum Lagurus, Verbascum longifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum, Verbascum phlomoides, Verbascum phenicum, Verbascum pulverulentum or Verbascum thapsus [Mullein], Solanaceae such as the genera Capsicum, Nicotiana, Solanum, Lycopersicon eg the genera and species Capsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens [pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata, Nicotiana attenuata, Nicotiana glauca, Nicotiana slowdorffii, Nicotiana obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana rustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato], Solanum melongena [eggplant] Lycopersicon esculentum, Lycopersicon lycopersicum., Lycopersicon pyriforme, Solanum integrifolium or Solanum lycopersicum [tomato], Sterculiaceae such as the genus Theobroma eg the genus and species Theobroma cacao [cacao] or Theaceae such as the genus Camellia eg the genus and species Camellia sinensis [tea].
Multizelluläre Mikroorganismen, die als transgene nicht-humane Organismen eingesetzt werden können, sind vorzugsweise Protisten oder Diatomeen ausgewählt aus der Gruppe der Familien Dinophyceae, Turaniellidae oder Oxytrichidae wie die Gattungen und Arten: Crypthecodinium cohnii, Phaeodactylum tricornutum, Stylonychia mytilus, Stylonychia pustulata, Stylonychia putrina, Stylonychia notophora, Stylonychia sp., Colpidium campylum oder Colpidium sp.Multicellular microorganisms that can be used as transgenic non-human organisms are preferably protists or diatoms selected from the group of the family Dinophyceae, Turaniellidae or Oxytrichidae such as the genera and species: Crypthecodinium cohnii, Phaeodactylum tricornutum, Stylonychia mytilus, Stylonychia pustulata, Stylonychia putrina , Stylonychia notophora, Stylonychia sp., Colpidium campylum or Colpidium sp.
Die Erfindung betrifft ein Verfahren zur Herstellung eines Stoffes, der die Struktur aufweist, die in der folgenden allgemeinen Formel I gezeigt wirdThe invention relates to a process for producing a substance having the structure shown in the following general formula I.
wobei die Variablen und Substiuenten die folgenden sindwhere the variables and substituents are the following
R1 = Hydroxyl, Coenzym A (Thioester), Lysophosphatidylcholin, Lysophospha- tidylethanolamin, Lysophosphatidylglycerol, Lysodiphosphatidylglycerol,R 1 = hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol,
Lysophosphatidylserin, Lysophosphatidylinositol, Sphingo- Base oder ein Radikal der Formel IlLysophosphatidylserine, lysophosphatidylinositol, sphingolase or a radical of formula II
R2 = Wasserstoff, Llysophosphatidylcholin, Lysophosphatidylethanolamin, Lysophosphatidylglycerol, Llysodiphosphatidylglycerol, Lysophosphatidylserin, Lysophosphatidylinositol oder gesättigtes oder ungesättigtes C2-C24- Alkylcarbonyl,R 2 = hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol or saturated or unsaturated C 2 -C 24 -alkylcarbonyl,
R3 = Wasserstoff, gesättigtes oder ungesättigtes C2-C24-Alkylcarbonyl, oder R2 und R3 sind unabhängig voneinander ein Radikal der Formel Ia:
R 3 = hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl, or R 2 and R 3 are independently of one another a radical of the formula Ia:
n = 2, 3, 4, 5, 6, 7 or 9, m = 2, 3, 4, 5 oder 6 und p = 0 oder 3;n = 2, 3, 4, 5, 6, 7 or 9, m = 2, 3, 4, 5 or 6 and p = 0 or 3;
und wobei das Verfahren das Kultivieren von (i) einer erfindungsgemäßen Wirtszelle oder (ii) eines erfindungsgemäßen transgenen, nicht-humanen unter Bedingungen umfasst, die die Biosynthese des Stoffes erlauben. Vorzugsweise wird dabei der zuvor genannte Stoff in einer Menge von mindestens 1 Gew.-% bezogen auf den Gesamtgehalt der Lipide in der Wirtszelle oder dem transgenen Organismus bereitgestellt.and wherein the method comprises culturing (i) a host cell of the invention or (ii) a transgenic non-human of the invention under conditions permitting biosynthesis of the substance. Preferably, the aforementioned substance is provided in an amount of at least 1% by weight based on the total content of the lipids in the host cell or the transgenic organism.
R1 bedeutet in der allgemeinen Formel I Hydroxyl-, CoenzymA-(Thioester), Lyso- Phosphatidylcholin-, Lyso-Phosphatidylethanolamin-, Lyso-Phosphatidylglycerol-, Lyso-Diphosphatidylglycerol-, Lyso-Phosphatidylserin-, Lyso-Phosphatidylinositol-, Sphingobase-, oder einen Rest der allgemeinen Formel IlR 1 in general formula I denotes hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lyso-phosphatidylethanolamine, lyso-phosphatidylglycerol, lyso-diphosphatidylglycerol, lyso-phosphatidylserine, lyso-phosphatidylinositol, sphingobase, or a radical of the general formula II
Die oben genannten Reste von R1 sind immer in Form ihrer Thioester an die Verbindungen der allgemeinen Formel I gebunden. R2 bedeutet in der allgemeinen Formel Il Wasserstoff-, Lyso-Phosphatidylcholin-, Lyso- Phosphatidylethanolamin-, Lyso-Phosphatidylglycerol-, Lyso-Diphosphatidylglycerol-, Lyso-Phosphatidylserin-, Lyso-Phosphatidylinositol- oder gesättigtes oder ungesättigtes C2-C24-Alkylcarbonyl.The abovementioned radicals of R 1 are always bonded in the form of their thioesters to the compounds of general formula I. R 2 in the general formula II denotes hydrogen, lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, lyso-phosphatidylglycerol, lyso-diphosphatidylglycerol, lyso-phosphatidylserine, lyso-phosphatidylinositol or saturated or unsaturated C 2 -C 24 - alkylcarbonyl.
Als Alkylreste seien substituiert oder unsubstituiert, gesättigt oder ungesättigte C2-C24- Alkylcarbonyl-Ketten wie Ethylcarbonyl-, n-Propylcarbonyl-, n-Butylcarbonyl-, n- Pentylcarbonyl-, n-Hexylcarbonyl-,n-Heptylcarbonyl-, n-Octylcarbonyl-, n- Nonylcarbonyl-, n-Decylcarbonyl-, n-Undecylcarbonyl-, n-Dodecylcarbonyl-, n- Tridecylcarbonyl-, n-Tetradecylcarbonyl-, n-Pentadecylcarbonyl-, n- Hexadecylcarbonyl-, n-Heptadecylcarbonyl-, n-Octadecylcarbonyl-, n- Nonadecylcarbonyl-, n-Eicosylcarbonyl-, n-Docosanylcarbonyl- oder n- Tetracosanylcarbonyl- genannt, die ein oder mehrere Doppelbindungen enthalten. Gesättigte oder ungesättigte Cio-C22-Alkylcarbonylreste wie n-Decylcarbonyl-, n-
Undecylcarbonyl-, n-Dodecylcarbonyl-, n-Tridecylcarbonyl-, n-Tetradecylcarbonyl-, n- Pentadecylcarbonyl-, n-Hexadecylcarbonyl-, n-Heptadecylcarbonyl-, n- Octadecylcarbonyl-, n-Nonadecylcarbonyl-, n-Eicosylcarbonyl-, n-Docosanylcarbonyl- oder n-Tetracosanylcarbonyl-., die ein oder mehrere Doppelbindungen enthalten, sind bevorzugt. Besonders bevorzugt sind gesättigte und/oder ungesättigte Ci0-C22- Alkylcarbonylreste wie Cio-Alkylcarbonyl-, Cn-Alkylcarbonyl-, Ci2-Alkylcarbonyl-, Ci3- Alkylcarbonyl-, Ci4-Alkylcarbonyl-, Ci6-Alkylcarbonyl-, Ciβ-Alkylcarbonyl-, C20- Alkylcarbonyl- oder C22-Alkylcarbonylreste, die ein oder mehrere Doppelbindungen enthalten. Ganz besonders bevorzugt sind gesättigte oder ungesättigte Ci6-C22- Alkylcarbonylreste wie Ci6-Alkylcarbonyl-, Ciβ-Alkylcarbonyl-, C20-Alkylcarbonyl- oder C22-Alkylcarbonylreste, die ein oder mehrere Doppelbindungen enthalten. Diese vorteilhaften Reste können zwei, drei, vier, fünf oder sechs Doppelbindungen enthalten. Die besonders vorteilhaften Reste mit 20 oder 22 Kohlenstoffatomen in der Fettsäurekette enthalten bis zu sechs Doppelbindungen, vorteilhaft drei, vier, fünf oder sechs Doppelbindungen, besonders bevorzugt fünf oder sechs Doppelbindungen. Alle genannten Reste leiten sich von den entsprechenden Fettsäuren ab.Suitable alkyl radicals are substituted or unsubstituted, saturated or unsaturated C 2 -C 24 -alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl, n- Octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl, n- Octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl called containing one or more double bonds. Saturated or unsaturated C 1 -C 22 -alkylcarbonyl radicals such as n-decylcarbonyl, n- Undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n- Docosanylcarbonyl or n-tetracosanylcarbonyl- containing one or more double bonds are preferred. Especially preferred are saturated and / or unsaturated C 0 -C 22 - radicals such as Cio-alkylcarbonyl, Cn-alkylcarbonyl, Ci2-alkylcarbonyl, Ci 3 - alkylcarbonyl, Ci 4 -alkylcarbonyl, C 6 alkylcarbonyl, Ciβ-alkylcarbonyl, C 20 - alkylcarbonyl or C 22 -alkylcarbonyl radicals which contain one or more double bonds. Very particular preference is given to saturated or unsaturated C 1 -C 22 -alkylcarbonyl radicals such as C 1 -C 6 -alkylcarbonyl, C 1-6 -alkylcarbonyl, C 20 -alkylcarbonyl or C 22 -alkylcarbonyl radicals which contain one or more double bonds. These advantageous radicals may contain two, three, four, five or six double bonds. The particularly advantageous radicals having 20 or 22 carbon atoms in the fatty acid chain contain up to six double bonds, advantageously three, four, five or six double bonds, more preferably five or six double bonds. All these radicals are derived from the corresponding fatty acids.
R3 bedeutet in der allgemeinen Formel Il Wasserstoff-, gesättigtes oder ungesättigtes C2-C24-Al kylcarbonyl.R 3 in the general formula II is hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl.
Als Alkylreste seien substituiert oder unsubstituiert, gesättigt oder ungesättigte C2-C24- Alkylcarbonyl-Ketten wie Ethylcarbonyl-, n-Propylcarbonyl-, n-Butylcarbonyl-, n- Pentylcarbonyl-, n-Hexylcarbonyl-, n-Heptylcarbonyl-, n-Octylcarbonyl-, n- Nonylcarbonyl-, n-Decylcarbonyl-, n-Undecylcarbonyl-, n-Dodecylcarbonyl-, n- Tridecylcarbonyl-, n-Tetradecylcarbonyl-, n-Pentadecylcarbonyl-, n-Suitable alkyl radicals are substituted or unsubstituted, saturated or unsaturated C 2 -C 24 -alkylcarbonyl chains such as ethylcarbonyl, n-propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl, n-hexylcarbonyl, n-heptylcarbonyl, n- Octylcarbonyl, n-nonylcarbonyl, n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-
Hexadecylcarbonyl-, n-Heptadecylcarbonyl-, n-Octadecylcarbonyl-, n- Nonadecylcarbonyl-, n-Eicosylcarbonyl-, n-Docosanylcarbonyl- oder n- Tetracosanylcarbonyl- genannt, die ein oder mehrere Doppelbindungen enthalten. Gesättigte oder ungesättigte Ci0-C22-Alkylcarbonylreste wie n-Decylcarbonyl-, n- Undecylcarbonyl-, n-Dodecylcarbonyl-, n-Tridecylcarbonyl-, n-Tetradecylcarbonyl-, n- Pentadecylcarbonyl-, n-Hexadecylcarbonyl-, n-Heptadecylcarbonyl-, n- Octadecylcarbonyl-, n-Nonadecylcarbonyl-, n-Eicosylcarbonyl-, n-Docosanylcarbonyl- oder n-Tetracosanylcarbonyl-, die ein oder mehrere Doppelbindungen enthalten, sind bevorzugt. Besonders bevorzugt sind gesättigte und/oder ungesättigte Ci0-C22- Alkylcarbonylreste wie Ciθ-Alkylcarbonyl-, Cn-Alkylcarbonyl-, Ci2-Alkylcarbonyl-, Ci3- Alkylcarbonyl-, Ci4-Alkylcarbonyl-, Ci6-Alkylcarbonyl-, Ciβ-Alkylcarbonyl-, C20- Alkylcarbonyl- oder C22-Alkylcarbonylreste, die ein oder mehrere Doppelbindungen enthalten. Ganz besonders bevorzugt sind gesättigte oder ungesättigte Ci6-C22- Alkylcarbonylreste wie Ci6-Alkylcarbonyl-, Ciβ-Alkylcarbonyl-, C20-Alkylcarbonyl- oder C22-Alkylcarbonylreste, die ein oder mehrere Doppelbindungen enthalten. Diese
vorteilhaften Reste können zwei, drei, vier, fünf oder sechs Doppelbindungen enthalten. Die besonders vorteilhaften Reste mit 20 oder 22 Kohlenstoffatomen in der Fettsäurekette enthalten bis zu sechs Doppelbindungen, vorteilhaft drei, vier, fünf oder sechs Doppelbindungen, besonders bevorzugt fünf oder sechs Doppelbindungen. Alle genannten Reste leiten sich von den entsprechenden Fettsäuren ab.Hexadecylcarbonyl, n-heptadecylcarbonyl, n-octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl, which contain one or more double bonds. Saturated or unsaturated C 1 -C 22 -alkylcarbonyl radicals such as n-decylcarbonyl, n-undecylcarbonyl, n-dodecylcarbonyl, n-tridecylcarbonyl, n-tetradecylcarbonyl, n-pentadecylcarbonyl, n-hexadecylcarbonyl, n-heptadecylcarbonyl , n-octadecylcarbonyl, n-nonadecylcarbonyl, n-eicosylcarbonyl, n-docosanylcarbonyl or n-tetracosanylcarbonyl containing one or more double bonds are preferred. Radicals such as Ci θ alkylcarbonyl, Cn-alkylcarbonyl, Ci2-alkylcarbonyl, Ci - 3 - Particularly preferred are saturated and / or unsaturated C 0 -C 22 are alkylcarbonyl, Ci 4 -alkylcarbonyl, C 6 alkylcarbonyl , Ciβ-alkylcarbonyl, C 20 - alkylcarbonyl or C 22 alkylcarbonyl radicals which contain one or more double bonds. Very particular preference is given to saturated or unsaturated C 1 -C 22 -alkylcarbonyl radicals such as C 1 -C 6 -alkylcarbonyl, C 1-6 -alkylcarbonyl, C 20 -alkylcarbonyl or C 22 -alkylcarbonyl radicals which contain one or more double bonds. These Advantageous radicals may contain two, three, four, five or six double bonds. The particularly advantageous radicals having 20 or 22 carbon atoms in the fatty acid chain contain up to six double bonds, advantageously three, four, five or six double bonds, more preferably five or six double bonds. All these radicals are derived from the corresponding fatty acids.
Die oben genannten Reste von R1, R2 and R3 können mit Hydroxyl- und/oder Epo- xygruppen substituierte sein und/oder können Dreifachbindungen enthalten.The abovementioned radicals of R 1 , R 2 and R 3 may be substituted by hydroxyl and / or epoxy groups and / or may contain triple bonds.
Vorteilhaft enthalten die im erfindungsgemäßen Verfahren hergestellten mehrfach ungesättigten Fettsäuren mindestens zwei vorteilhaft drei, vier, fünf oder sechs Doppelbindungen. Besonders vorteilhaft enthalten die Fettsäuren vier fünf oder sechs Doppelbindungen. Im Verfahren hergestellte Fettsäuren haben vorteilhaft 18-, 20- oder 22-C-Atome in der Fettsäurekette, bevorzugt enthalten die Fettsäuren 20 oder 22 Kohlenstoffatome in der Fettsäurekette. Vorteilhaft werden gesättigte Fettsäuren mit den im Verfahren verwendeten Nukleinsäuren wenig oder gar nicht umgesetzt. Unter wenig ist zu verstehen, das im Vergleich zu mehrfach ungesättigten Fettsäuren die gesättigten Fettsäuren mit weniger als 5 % der Aktivität, vorteilhaft weniger als 3 %, besonders vorteilhaft mit weniger als 2 %, ganz besonders bevorzugt mit weniger als 1 ; 0,5; 0,25 oder 0,125 % umgesetzt werden. Diese hergestellten Fettsäuren können als einziges Produkt im Verfahren hergestellt werden oder in einem Fettsäuregemisch vorliegen.Advantageously, the polyunsaturated fatty acids prepared in the process according to the invention contain at least two, advantageously three, four, five or six double bonds. Particularly advantageously, the fatty acids contain four five or six double bonds. Fatty acids produced in the process advantageously have 18, 20 or 22 carbon atoms in the fatty acid chain, preferably the fatty acids contain 20 or 22 carbon atoms in the fatty acid chain. Advantageously, saturated fatty acids are little or not reacted with the nucleic acids used in the process. Little is understood to mean that compared to polyunsaturated fatty acids, the saturated fatty acids have less than 5% of the activity, advantageously less than 3%, more preferably less than 2%, most preferably less than 1; 0.5; 0.25 or 0.125% are implemented. These produced fatty acids can be produced as the only product in the process or present in a fatty acid mixture.
Vorteilhaft bedeuten die Substituenten R2 oder R3 in den allgemeinen Formeln I und Il unabhängig voneinander gesättigtes oder ungesättigtes Ci8-C22-Alkylcarbonyl-, besonders vorteilhaft bedeuten sie unabhängig voneinander ungesättigtes Ci8-, C2o- oder C22-Alkylcarbonyl- mit mindestens zwei Doppelbindungen.Advantageously, the substituents R 2 or R 3 in the general formulas I and II independently of one another denote saturated or unsaturated C 8 -C 22 -alkylcarbonyl, particularly advantageously independently they denote unsaturated C 8 -, C 2 o- or C 22 -alkylcarbonyl - with at least two double bonds.
Die im Verfahren hergestellten mehrfach ungesättigten Fettsäuren sind vorteilhaft in Membranlipiden und/oder Triacylglyceriden gebunden, können aber auch als freie Fettsäuren oder aber gebunden in Form anderer Fettsäureester in den Organismen vorkommen. Dabei können sie als "Reinprodukte" oder aber vorteilhaft in Form von Mischungen verschiedener Fettsäuren oder Mischungen unterschiedlicher Glyceride vorliegen. Die in den Triacylglyceriden gebundenen verschieden Fettsäuren lassen sich dabei von kurzkettigen Fettsäuren mit 4 bis 6 C-Atomen, mittelkettigen Fettsäuren mit 8 bis 12 C-Atomen oder langkettigen Fettsäuren mit 14 bis 24 C-Atomen ableiten, bevorzugt sind die langkettigen Fettsäuren besonders bevorzugt sind die langkettigen Fettsäuren LCPUFAs von Ci8-, C2o- und/oder C22-Fettsäuren.
Im erfindungsgemäßen Verfahren werden vorteilhaft Fettsäureester mit mehrfach ungesättigten Ci8-, C2o- und/oder C22-Fettsäuremolekülen mit mindestens zwei Doppelbindungen im Fettsäureester, vorteilhaft mit mindestens drei, vier, fünf oder sechs Doppelbindungen im Fettsäureester, besonders vorteilhaft von mindestens fünf oder sechs Doppelbindungen im Fettsäureester hergestellt und führen vorteilhaft zur Synthese von Linolsäure (=LA, C18:2Δ9'12), γ-Linolensäure (= GLA, C18:3Δ6'9'12), Stearidonsäure (= SDA, C18:4 Δ6'9'12 15)' Dihomo-γ-Linolensäure (= DGLA, 20:3 Δ8'11'14), ω-3-Eicosatetraensäure (= ETA, C20:4 Δ5A11'14), Arachidonsäure (ARA, C20:4 Δ5A11'14), Eicosapentaensäure (EPA, C20:5Δ5'8'11'14'17), ω-6-Docosapentaensäure (C22:5Δ4'7'10'13'16), ω-6-Docosatetraensäure (C22:4Δ'7'10'13'16), ω-3-Docosapentaensäure (= DPA, C22:5Δ7'10'13'16'19), Docosahexaensäure (= DHA, C22:6Δ4'7'10'13'16'19) oder deren Mischungen, bevorzugt ARA, EPA und/oder DHA. Ganz besonders bevorzugt werden, ω-3-Fettsäuren wie EPA und/oder DHA hergestellt.The polyunsaturated fatty acids produced in the process are advantageously bound in membrane lipids and / or triacylglycerides, but may also be present as free fatty acids or bound in the form of other fatty acid esters in the organisms. They may be present as "pure products" or advantageously in the form of mixtures of different fatty acids or mixtures of different glycerides. The different fatty acids bound in the triacylglycerides can thereby be derived from short-chain fatty acids having 4 to 6 C atoms, medium-chain fatty acids having 8 to 12 C atoms or long-chain fatty acids having 14 to 24 C atoms, preferably the long-chain fatty acids are particularly preferred the long-chain fatty acids LCPUFAs of Ci 8 , C 2 o and / or C 22 fatty acids. In the inventive process fatty acid ester are advantageously with polyunsaturated C 8 -, C 2 O and / or C 22 -fatty acid molecules having at least two double bonds in the fatty acid ester, advantageously at least three, four, five or six double bonds in the fatty acid ester, particularly advantageously by at least five or six double bonds in the fatty acid ester and lead advantageously to the synthesis of linoleic acid (= LA, C18: 2 Δ9 '12 ), γ-linolenic acid (= GLA, C18: 3 Δ6 ' 9 '12 ), stearidonic acid (= SDA, C18: 4 Δ6 ' 9 ' 12 15) 'Dihomo-γ-linolenic acid (= DGLA, 20: 3 Δ8 ' 11 '14 ), ω-3-eicosatetraenoic acid (= ETA, C20: 4 Δ5A11 ' 14 ), arachidonic acid (ARA, C20: 4 Δ5A11 '14 ), eicosapentaenoic acid (EPA, C20: 5 Δ5 ' 8 ' 11 ' 14 '17 ), ω-6-docosapentaenoic acid (C22: 5 Δ4 ' 7 ' 10 ' 13 '16 ), ω-6-docosatetraenoic acid ( C22: 4 Δ ' 7 ' 10 ' 13 ' 16 ), ω-3-docosapentaenoic acid (= DPA, C22: 5 Δ7 ' 10 ' 13 ' 16 ' 19 ), docosahexaenoic acid (= DHA, C 22: 6 Δ4 ' 7 ' 10 ' 13 ' 16 ' 19 ) or mixtures thereof, preferably ARA, EPA and / or DHA. Very particular preference is given to producing ω-3 fatty acids such as EPA and / or DHA.
Die Fettsäureester mit mehrfach ungesättigten Ci8-, C20- und/oder C22-Fettsäure- molekülen können aus den Organismen, die für die Herstellung der Fettsäureester verwendet wurden, in Form eines Öls oder Lipids beispielsweise in Form von Verbindungen wie Sphingolipide, Phosphoglyceride, Lipide, Glycolipide wie Glycosphingo- lipide, Phospholipide wie Phosphatidylethanolamin, Phosphatidylcholin, Phosphatidyl- serin, Phosphatidylglycerol, Phosphatidylinositol oder Diphosphatidylglycerol, Monoa- cylglyceride, Diacylglyceride, Triacylglyceride oder sonstige Fettsäureester wie die AcetylCoenzymA-Ester, die die mehrfach ungesättigten Fettsäuren mit mindestens zwei, drei, vier, fünf oder sechs bevorzugt fünf oder sechs Doppelbindungen enthalten, isoliert werden, vorteilhaft werden sie in der Form ihrer Diacylglyceride, Triacylglyceride und/oder in Form des Phosphatidylcholin isoliert, besonders bevorzugt in der Form der Triacylglyceride. Neben diesen Estern sind die mehrfach ungesättigten Fettsäuren auch als freie Fettsäuren oder gebunden in anderen Verbindungen in den Organismen vorteilhaft den Pflanzen enthalten. In der Regel liegen die verschiedenen vorgenannten Verbindungen (Fettsäureester und frei Fettsäuren) in den Organismen in einer unge- fähren Verteilung von 80 bis 90 Gew.-% Triglyceride, 2 bis 5 Gew.-% Diglyceride, 5 bis 10 Gew.-% Monoglyceride, 1 bis 5 Gew.-% freie Fettsäuren, 2 bis 8 Gew.-% Phospholipide vor, wobei sich die Summe der verschiedenen Verbindungen zu 100 Gew.-% ergänzt.The fatty acid ester with polyunsaturated C 8 -, C 2 0 and / or C 22 -Fettsäure- molecules can be isolated from the organisms which have been used for the production of the fatty acid ester, in the form of an oil or lipid, for example in the form of compounds such as sphingolipids, Phosphoglycerides, lipids, glycolipids such as glycosphingolipids, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl-coenzymeA esters which contain at least two polyunsaturated fatty acids Preferably, they are isolated in the form of their diacylglycerides, triacylglycerides and / or in the form of the phosphatidylcholine, more preferably in the form of the triacylglycerides. In addition to these esters, the polyunsaturated fatty acids are also included as free fatty acids or bound in other compounds in the organisms beneficial to the plants. In general, the various abovementioned compounds (fatty acid esters and free fatty acids) in the organisms have an approximate distribution of 80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10% by weight of monoglycerides , 1 to 5 wt .-% of free fatty acids, 2 to 8 wt .-% phospholipids ago, wherein the sum of the various compounds to 100 wt .-% complements.
Im erfindungsgemäßen Verfahren werden die hergestellten LCPUFAs mit einem Gehalt von mindestens 3 Gew.-%, vorteilhaft von mindestens 5 Gew.-%, bevorzugt von mindestens 8 Gew.-%, besonders bevorzugt von mindestens 10 Gew.-%, ganz besonders bevorzugt von mindestens 15 Gew.-% bezogen auf die gesamten Fettsäuren in den transgenen Organismen vorteilhaft in einer transgenen Pflanze hergestellt. Dabei werden vorteilhaft Ci8- und/oder C2o-Fettsäuren, die in den Wirtsorganismen
vorhanden sind, zu mindestens 10 %, vorteilhaft zu mindestens 20 %, besonders vorteilhaft zu mindestens 30 %, ganz besonders vorteilhaft zu mindestens 40 % in die entsprechenden Produkte wie DPA oder DHA, um nur zwei beispielhaft zu nennen, umgesetzt. Vorteilhaft werden die Fettsäuren in gebundener Form hergestellt. Mit Hilfe der im erfindungsgemäßen Verfahren verwendeten Nukleinsäuren lassen sich diese ungesättigten Fettsäuren an sn1-, sn2- und/oder sn3-Position der vorteilhaft hergestellten Triglyceride bringen. Da im erfindungsgemäßen Verfahren von den Ausgangsverbindungen Linolsäure (C18:2) bzw. Linolensäure (C18:3) mehrere Reaktionsschritte durchlaufen werden, fallen die Endprodukte des Verfahrens wie beispielsweise Arachidonsäure (ARA), Eicosapentaensäure (EPA), ω-6-Docosapentaensäure oder DHA nicht als absolute Reinprodukte an, es sind immer auch geringe Spuren der Vorstufen im Endprodukt enthalten. Sind in dem Ausgangsorganismus bzw. in der Ausgangspflanze beispielsweise sowohl Linolsäure als auch Linolensäure vorhanden, so liegen die Endprodukte wie ARA, EPA oder DHA als Mischungen vor. Die Vorstufen sollten vorteilhaft nicht mehr als 20 Gew.-%, bevorzugt nicht mehr als 15 Gew.-%, besonders bevorzugt nicht als 10 Gew.-%, ganz besonders bevorzugt nicht mehr als 5 Gew.-% bezogen auf die Menge des jeweilige Endproduktes betragen. Vorteilhaft werden in einer transgenen Pflanze als Endprodukte nur ARA, EPA oder nur DHA im erfindungsgemäßen Verfahren gebunden oder als freie Säuren hergestellt. Werden die Verbindungen ARA, EPA und DHA gleichzeitig hergestellt, werden sie vorteilhaft in einem Verhältnis von mindesten 1 :1 :2 (EPA:ARA:DHA), vorteilhaft von mindestens 1 :1 :3, bevorzugt von 1 :1 :4, besonders bevorzugt von 1 :1 :5 hergestellt.In the process according to the invention, the LCPUFAs produced are present in a content of at least 3% by weight, advantageously of at least 5% by weight, preferably of at least 8% by weight, more preferably of at least 10% by weight, very particularly preferably at least 15 wt .-% based on the total fatty acids in the transgenic organisms advantageously produced in a transgenic plant. In this case, advantageously Ci 8 - and / or C 2 o fatty acids in the host organisms are present, at least 10%, advantageously at least 20%, more preferably at least 30%, most preferably at least 40% in the corresponding products such as DPA or DHA, to name only two exemplified implemented. Advantageously, the fatty acids are prepared in bound form. With the aid of the nucleic acids used in the method according to the invention, these unsaturated fatty acids can be brought to the sn1, sn2 and / or sn3 position of the advantageously prepared triglycerides. Since the starting compounds linoleic acid (C18: 2) or linolenic acid (C18: 3) undergo several reaction steps in the process according to the invention, the end products of the process, such as, for example, arachidonic acid (ARA), eicosapentaenoic acid (EPA), ω-6-docosapentaenoic acid or DHA, are precipitated not as absolute pure products, there are always also small traces of precursors in the final product included. If both linoleic acid and linolenic acid are present in the starting organism or in the starting plant, for example, the end products such as ARA, EPA or DHA are present as mixtures. The precursors should advantageously not more than 20 wt .-%, preferably not more than 15 wt .-%, more preferably not more than 10 wt .-%, most preferably not more than 5 wt .-% based on the amount of the respective Final product amount. Advantageously, only ARA, EPA or only DHA are bound in the process according to the invention or produced as free acids in a transgenic plant as end products. If the compounds ARA, EPA and DHA are prepared simultaneously, they are advantageously used in a ratio of at least 1: 1: 2 (EPA: ARA: DHA), preferably of at least 1: 1: 3, preferably of 1: 1: 4 preferably prepared from 1: 1: 5.
Fettsäureester bzw. Fettsäuregemische, die nach dem erfindungsgemäßen Verfahren hergestellt wurden, enthalten vorteilhaft 6 bis 15 % Palmitinsäure, 1 bis 6 % Stearinsäure; 7 - 85 % Ölsäure; 0,5 bis 8 % Vaccensäure, 0,1 bis 1 % Arachinsäure, 7 bis 25 % gesättigte Fettsäuren, 8 bis 85 % einfach ungesättigte Fettsäuren und 60 bis 85 % mehrfach ungesättigte Fettsäuren jeweils bezogen auf 100 % und auf den Gesamtfettsäuregehalt der Organismen. Als vorteilhafte mehrfach ungesättigte Fettsäure sind in den Fettsäureester bzw. Fettsäuregemische bevorzugt mindestens 0,1 ; 0,2; 0,3; 0,4; 0,5; 0,6; 0,7; 0,8; 0,9 oder 1 % bezogen auf den Gesamtfettsäuregehalt an Arachidonsäure enthalten. Weiterhin enthalten die Fettsäureester bzw. Fettsäuregemische, die nach dem erfindungsgemäßen Verfahren hergestellt wurden, vorteilhaft Fettsäuren ausgewählt aus der Gruppe der Fettsäuren Erucasäure (13- Docosaensäure), Sterculinsäure (9,10-Methylene octadec-9-enonsäure), Malvalinsäure (8,9-Methylen Heptadec-8-enonsäure), Chaulmoogrinsäure (Cyclopenten- dodecansäure), Furan-Fettsäure (9,12-Epoxy-octadeca-9,1 1-dienonsäure), Vernonsäu- re (9,10-Epoxyoctadec-12-enonsäure), Tarinsäure (6-Octadecynonsäure),6- Nonadecynonsäure, Santalbinsäure (t11-Octadecen-9-ynoic acid), 6,9- Octadecenynonsäure, Pyrulinsäure (t10-Heptadecen-8-ynonsäure), Crepenyninsäure
(9-Octadecen-12-ynonsäure), 13,14-Dihydrooropheinsäure, Octadecen-13-ene-9,1 1- diynonsäure, Petroselensäure (cis-6-Octadecenonsäure), 9c,12t-Octadecadiensäure, Calendulasäure (δti Ot^c-Octadecatriensäure), Catalpinsäure (9t1 1t13c- Octadecatriensäure), Eleosterinsäure (θci itiSt-Octadecatriensäure), Jacarinsäure (8c10t12c-Octadecatriensäure), Punicinsäure (9c1 1t13c-Octadecatriensäure), Parina- rinsäure (9c1 1t13t15c-Octadecatetraensäure), Pinolensäure (all-cis-5,9,12-Octa- decatriensäure), Laballensäure (5,6-Octadecadienallensäure), Ricinolsäure (12- Hydroxyölsäure) und/oder Coriolinsäure (IS-Hydroxy-θci it-Octadecadienonsäure). Die vorgenannten Fettsäuren kommen in den nach dem erfindungsgemäßen Verfahren hergestellten Fettsäureester bzw. Fettsäuregemischen in der Regel vorteilhaft nur in Spuren vor, das heißt sie kommen bezogen auf die Gesamtfettsäuren zu weniger als 30 %, bevorzugt zu weniger als 25 %, 24 %, 23 %, 22 % oder 21 %, besonders bevorzugt zu weniger als 20 %, 15 %, 10 %, 9 %, 8 %, 7%, 6 % oder 5%, ganz besonders bevorzugt zu weniger als 4 %, 3 %, 2 % oder 1 % vor. Vorteilhaft enthalten die nach dem erfindungsgemäßen Verfahren hergestellten Fettsäureester bzw. Fettsäuregemische weniger als 0,1 % bezogen auf die Gesamtfettsäuren oder keine Buttersäure, kein Cholesterin, keine Clupanodonsäure (= Docosapentaensäure, C22:5Δ4'8'12'15'21) sowie keine Nisinsäure (Tetracosahexaensäure, C23:6Δ3'8'12'15'18'21). Durch die erfindungsgemäßen Nukleinsäuresequenzen bzw. im erfindungsgemäßen Verfahren verwendeten Nukleinsäuresequenzen kann eine Steigerung der Ausbeute an mehrfach ungesättigten Fettsäuren von mindestens 50 %, vorteilhaft von mindestens 80 %, besonders vorteilhaft von mindestens 100 %, ganz besonders vorteilhaft von mindestens 150 % gegenüber den nicht transgenen Ausgangsorganismus beispielsweise einer Hefe, einer Alge, einem Pilz oder einer Pflanze wie Arabidopsis oder Lein beim Vergleich in der GC-Analyse siehe Beispiele erreicht werden.Fatty acid esters or fatty acid mixtures which have been prepared by the process according to the invention advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid; 7 - 85% oleic acid; 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachidic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids in each case based on 100% and on the total fatty acid content of the organisms. As advantageous polyunsaturated fatty acid in the fatty acid esters or fatty acid mixtures are preferably at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1% based on the total fatty acid content of arachidonic acid. Furthermore, the fatty acid esters or fatty acid mixtures prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8,9 -Methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentenodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,1-dienanoic acid), vernonoic acid (9,10-epoxyoctadec-12-enoic acid) , Taric acid (6-octadecynoic acid), 6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynonic acid), crepenynic acid (9-octadecen-12-ynonic acid), 13,14-dihydrooropheic acid, octadecene-13-ene-9,1-diynoic acid, petroselenoic acid (cis-6-octadecenoic acid), 9c, 12t-octadecadienoic acid, calendulic acid (δti Ot ^ c Octadecatrienoic acid), catalpinic acid (9t1 1t13c octadecatrienoic acid), ele- losteric acid (cis-octadecatrienoic acid), jacric acid (8c10t12c-octadecatrienoic acid), punicic acid (9c1 1t13c-octadecatrienoic acid), parinaric acid (9c1 1t13t15c-octadecatetraenoic acid), pinolenic acid (all-cis -5,9,12-octadecatrienoic acid), labellaric acid (5,6-octadecadienenoic acid), ricinoleic acid (12-hydroxyoleic acid) and / or coriolinic acid (IS-hydroxy-θci it-octadecadienoic acid). The abovementioned fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they are less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, more preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1% ago. Advantageously, the fatty acid esters or mixtures of fatty acids prepared by the process according to the invention contain less than 0.1% based on the total fatty acids or no butyric acid, no cholesterol, no clupanodonic acid (= docosapentaenoic acid, C22: 5 Δ4 ' 8 ' 12 ' 15 ' 21 ) and none Nisic acid (tetracosahexaenoic acid, C23: 6 Δ3 ' 8 ' 12 ' 15 ' 18 '21 ). The nucleic acid sequences according to the invention or the nucleic acid sequences used in the method according to the invention can increase the yield of polyunsaturated fatty acids by at least 50%, advantageously by at least 80%, particularly advantageously by at least 100%, very particularly advantageously by at least 150% compared to the non-transgenic starting organism For example, a yeast, an alga, a fungus, or a plant such as Arabidopsis or flax can be obtained by comparison in GC analysis, see Examples.
Auch chemisch reine mehrfach ungesättigte Fettsäuren oder Fettsäurezusammensetzungen sind nach den vorbeschriebenen Verfahren darstellbar. Dazu werden die Fettsäuren oder die Fettsäurezusammensetzungen aus dem Organismus wie den Mikroorganismen oder den Pflanzen oder dem Kulturmedium, in dem oder auf dem die Organismen angezogen wurden, oder aus dem Organismus und dem Kulturmedium in bekannter Weise beispielsweise über Extraktion, Destillation, Kristallisation, Chromatographie oder Kombinationen dieser Methoden isoliert. Diese chemisch reinen Fettsäuren oder Fettsäurezusammensetzungen sind für Anwendungen im Bereich der Lebensmittelindustrie, der Kosmetikindustrie und besonders der Pharmaindustrie vorteilhaft.Also, chemically pure polyunsaturated fatty acids or fatty acid compositions can be prepared by the methods described above. For this purpose, the fatty acids or fatty acid compositions from the organism such as the microorganisms or plants or the culture medium in which or on which the organisms were grown, or from the organism and the culture medium in a known manner, for example via extraction, distillation, crystallization, chromatography or Isolated combinations of these methods. These chemically pure fatty acids or fatty acid compositions are advantageous for applications in the food industry, the cosmetics industry and especially the pharmaceutical industry.
Im Prinzip können alle Gene des Fettsäure- oder Lipidstoffwechsels vorteilhaft inIn principle, all genes of fatty acid or lipid metabolism can be advantageous in
Kombination mit der(den) erfinderischen Polynucleotiden (im Sinne dieser Anmeldung soll der Plural den Singular und umgekehrt beinhalten) im Verfahren zur Herstellung
mehrfach ungesättigter Fettsäuren verwendet werden vorteilhaft werden Gene des Fettsäure- oder Lipidstoffwechsels ausgewählt aus der Gruppe Acyl-CoA- Dehydrogenase(n), Acyl-ACP[= acyl carrier protein]-Desaturase(n), Acyl-ACP- Thioesterase(n), Fettsäure-Acyl-Transferase(n), Acyl-CoA:Lysophospholipid- Acyltransferasen, Fettsäure-Synthase(n), Fettsäure-Hydroxylase(n), Acetyl-Coenzym A-Carboxylase(n), Acyl-Coenzym A-Oxidase(n), Fettsäure-Desaturase(n), Fettsäure- Acetylenasen, Lipoxygenasen, Triacylglycerol-Lipasen, Allenoxid-Synthasen, Hydro- peroxid-Lyasen oder Fettsäure-Elongase(n) verwendet. Besonders bevorzugt werden Gene ausgewählt aus der Gruppe der Δ-4-Desatu rasen, Δ-5-Desatu rasen, Δ-6- Desaturasen, Δ-9-Desatu rasen, Δ-12-Desaturasen, Δ-6-Elongasen oder Δ-5- Elongasen in Kombination mit den erfindungsgemäßen Polynucleotiden verwendet, wobei einzelne Gene oder mehrere Gene in Kombination verwendet werden können.Combination with the inventive polynucleotide (in the context of this application, the plural shall include the singular and vice versa) in the process of preparation genes of the fatty acid or lipid metabolism selected from the group of acyl-CoA dehydrogenase (s), acyl-ACP [= acyl carrier protein] desaturase (s), acyl-ACP thioesterase (s), Fatty acid acyl transferase (s), acyl CoA: lysophospholipid acyltransferases, fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A oxidase (s) , Fatty acid desaturase (s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allene oxide synthases, hydroperoxide lyases or fatty acid elongase (s). Particular preference is given to genes selected from the group of Δ-4-desaturases, Δ-5-desaturases, Δ-6-desaturases, Δ-9-desaturases, Δ-12-desaturases, Δ-6-elongases or Δ- 5- elongases used in combination with the polynucleotides of the invention, wherein single genes or multiple genes can be used in combination.
Vorteilhaft setzen die im erfindungsgemäßen Verfahren verwendeten Desaturasen ihre jeweiligen Substrate in Form der CoA-Fettsäureester um. Dies führt, wenn vorher ein Elongationsschritt stattgefunden hat, vorteilhaft zu einer erhöhten Produktausbeute. Die jeweiligen Desaturierungsprodukte werden dadurch in höheren Mengen synthetisiert, da der Elongationsschritt in der Regel an den CoA-Fettsäureestern erfolgt, während der Desaturierungsschritt überwiegend an den Phospholipiden oder an den Triglyceriden erfolgt. Eine Ausstauschreaktion, die eine weitere möglicherweise limitierende Enzymreaktion erforderlich machen würde, zwischen den CoA- Fettsäureestern und den Phospholipiden oder Triglyceriden ist somit nicht erforderlich.Advantageously, the desaturases used in the process of the invention convert their respective substrates in the form of the CoA fatty acid esters. This leads, if previously a Elongationsschritt has taken place, advantageously to an increased product yield. The respective desaturation products are thereby synthesized in higher amounts, since the elongation step usually takes place on the CoA fatty acid esters, while the desaturation step takes place predominantly on the phospholipids or on the triglycerides. An exchange reaction that would require another possibly limiting enzyme reaction between the CoA fatty acid esters and the phospholipids or triglycerides is thus not required.
Durch die enzymatische Aktivität der im erfindungsgemäßen Verfahren verwendeten Polypeptide können unterschiedlichste mehrfach ungesättigte Fettsäuren im erfindungsgemäßen Verfahren hergestellt werden. Je nach Auswahl der für das erfindungsgemäße Verfahren verwendeten Organismen wie den bevorzugte Pflanze lassen sich Mischungen der verschiedenen mehrfach ungesättigten Fettsäuren oder einzelne mehrfach ungesättigte Fettsäuren wie EPA oder ARA in freier oder gebundener Form herstellen. Je nachdem welche Fettsäurezusammensetzung in der Ausgangspflanze vorherrscht (C18:2- oder C18:3-Fettsäuren) entstehen so Fettsäuren, die sich von C18:2-Fettsäuren ableiten, wie GLA, DGLA oder ARA oder, die sich von C18:3- Fettsäuren ableiten, wie SDA, ETA oder EPA. Liegt in der für das Verfahren verwendeten Pflanze als ungesättigte Fettsäure nur Linolsäure (= LA, C18:2A9'12) vor, so können als Produkte des Verfahrens nur GLA, DGLA und ARA entstehen, die als freie Fettsäuren oder gebunden vorliegen können. Ist in der im Verfahren verwendeten Pflanze als ungesättigte Fettsäure nur α-Linolensäure (= ALA, C18:3A9'12'15) beispielsweise wie in Lein, so können als Produkte des Verfahrens nur SDA, ETA, EPA und/oder DHA entstehen, die wie oben beschrieben als freie Fettsäuren oder gebun- den vorliegen können. Durch Modifikation der Aktivität der an der Synthese beteiligten
Enzyme Δ-5-Desaturase, Δ-6-Desaturase, Δ-4-Desaturase, Δ-12-Desaturase, Δ-5- Elongase und/oder Δ-6-Elongase lassen sich gezielt in den vorgenannten Organismen vorteilhaft in den vorgenannten Pflanzen nur einzelne Produkte herstellten. Durch die Aktivität der Δ-6-Desaturase und Δ-6-Elongase entstehen beispielsweise GLA und DGLA bzw. SDA und ETA, je nach Ausgangspflanze und ungesättigter Fettsäure. Bevorzugt entstehen DGLA bzw. ETA oder deren Mischungen. Werden die Δ-5- Desaturase, die Δ-5-Elongase und die Δ-4-Desaturase zusätzlich in die Organismen vorteilhaft in die Pflanze eingebracht, so entstehen zusätzlich ARA, EPA und/oder DHA. Vorteilhaft werden nur ARA, EPA oder DHA oder deren Mischungen syntheti- siert, abhängig von den im Organismus bzw. in der Pflanze vorliegenden Fettsäuren, die als Ausgangssubstanz für die Synthese dient. Da es sich um Biosyntheseketten handelt, liegen die jeweiligen Endprodukte nicht als Reinsubstanzen in den Organismen vor. Es sind immer auch geringe Mengen der Vorläuferverbindungen im Endprodukt enthalten. Diese geringen Mengen betragen weniger als 20 Gew.-%, vorteilhaft weniger als 15 Gew.-%, besonders vorteilhaft weniger als 10 Gew.-%, ganz besonders vorteilhaft weniger als 5, 4, 3, 2 oder 1 Gew.-% bezogen auf das Endprodukt DGLA, ETA oder deren Mischungen bzw. ARA, EPA, DHA oder deren Mischungen vorteilhaft EPA oder DHA oder deren Mischungen.The enzymatic activity of the polypeptides used in the process according to the invention makes it possible to prepare a wide variety of polyunsaturated fatty acids in the process according to the invention. Depending on the selection of the organisms used for the process according to the invention, such as the preferred plant, it is possible to prepare mixtures of the various polyunsaturated fatty acids or individual polyunsaturated fatty acids such as EPA or ARA in free or bound form. Depending on which fatty acid composition prevails in the starting plant (C18: 2 or C18: 3 fatty acids), fatty acids derived from C18: 2 fatty acids, such as GLA, DGLA or ARA or those derived from C18: 3 fatty acids derive, such as SDA, ETA or EPA. If only linoleic acid (= LA, C18: 2 A9 '12 ) is present as the unsaturated fatty acid in the plant used for the process, only GLA, DGLA and ARA can arise as products of the process which may be present as free fatty acids or bound. Is in the process used in the process as unsaturated fatty acid only α-linolenic acid (= ALA, C18: 3 A9 ' 12 ' 15 ), for example, as in flax, so can only arise as products of the method SDA, ETA, EPA and / or DHA, which may be present as free fatty acids or bound as described above. By modifying the activity of those involved in the synthesis Enzymes Δ-5-desaturase, Δ-6-desaturase, Δ-4-desaturase, Δ-12-desaturase, Δ-5 elongase and / or Δ-6 elongase can be targeted in the abovementioned organisms advantageously in the abovementioned plants produced only individual products. Due to the activity of Δ-6-desaturase and Δ-6 elongase, for example, GLA and DGLA or SDA and ETA are formed, depending on the starting plant and unsaturated fatty acid. Preference is given to DGLA or ETA or mixtures thereof. If the Δ-5-desaturase, Δ5-elongase and Δ4-desaturase are additionally introduced into the organisms in the plant in an advantageous manner, ARA, EPA and / or DHA are additionally produced. Advantageously, only ARA, EPA or DHA or mixtures thereof are synthesized, depending on the fatty acids present in the organism or in the plant, which serves as the starting substance for the synthesis. Since these are biosynthetic chains, the respective end products are not present as pure substances in the organisms. There are always small amounts of precursor compounds in the final product. These small amounts are less than 20 wt .-%, advantageously less than 15 wt .-%, more preferably less than 10 wt .-%, most preferably less than 5, 4, 3, 2 or 1 wt .-% based to the end product DGLA, ETA or mixtures thereof or ARA, EPA, DHA or mixtures thereof advantageously EPA or DHA or mixtures thereof.
Neben der Produktion der Ausgangsfettsäuren für die im erfindungsgemäßen Verfahren verwendeten Polypeptide direkt im Organismus können die Fettsäuren auch von außen gefüttert werden. Aus kostengründen ist die Produktion im Organismus bevorzugt. Bevorzugte Substrate sind die Linolsäure (C18:2A9'12), die γ-Linolensäure (C18:3Δ6'9'12), die Eicosadiensäure (C20:2Δ11'14), die Dihomo-γ-linolensäure (C20:3Δ8'11'14), die Arachidonsäure (C20:4Δ5A11'14), die Docosatetraensäure (C22:4Δ7'10'13'16) und die Docosapentaensäure (C22:5Δ4'7'10'13'15).In addition to the production of the starting fatty acids for the polypeptides used in the process according to the invention directly in the organism, the fatty acids can also be fed from the outside. For cost reasons, production in the organism is preferred. Preferred substrates are linoleic acid (C18: 2 A9 '12 ), γ-linolenic acid (C18: 3 Δ6 ' 9 '12 ), eicosadienoic acid (C20: 2 Δ11 ' 14 ), dihomo-γ-linolenic acid (C20: 3 Δ8 ' 11 ' 14 ), the arachidonic acid (C20: 4 Δ5A11 '14 ), the docosatetraenoic acid (C22: 4 Δ7 ' 10 ' 13 ' 16 ) and the docosapentaenoic acid (C22: 5 Δ4 ' 7 ' 10 ' 13 ' 15 ).
Zur Steigerung der Ausbeute im beschriebenen Verfahren zur Herstellung von Ölen und/oder Triglyceriden mit einem vorteilhaft erhöhten Gehalt an mehrfach ungesättig- ten Fettsäuren ist es vorteilhaft die Menge an Ausgangsprodukt für die Fettsäuresynthese zu steigern, dies kann beispielsweise durch das Einbringen einer Nukleinsäure in den Organismus, die für ein Polypeptid mit Δ-12-Desaturase codiert, erreicht werden. Dies ist besonders vorteilhaft in Öl-produzierenden Organismen wie der Familie der Brassicaceae wie der Gattung Brassica z.B. Raps; der Familie der Elaeagnaceae wie die Gattung Elaeagnus z.B. die Gattung und Art Olea europaea oder der Familie Fabaceae wie der Gattung Glycine z.B. die Gattung und Art Glycine max, die einen hohen Ölsäuregehalt aufweisen. Da diese Organismen nur einen geringen Gehalt an Linolsäure aufweisen (Mikoklajczak et al., Journal of the American OiI Chemical Society, 38, 1961 , 678 - 681 ) ist die Verwendung der genannten Δ-12-Desaturasen zur Herstellung des Ausgangsprodukts Linolsäure vorteilhaft.
Vorteilhaft werden im erfindungsgemäßen Verfahren die vorgenannten Nukleinsäure- sequenzen oder deren Derivat oder Homologe, die für Polypeptide codieren, die noch die enzymatische Aktivität der durch Nukleinsäuresequenzen codierten Proteine besitzen. Diese Sequenzen werden einzeln oder in Kombination mit den erfindungsgemäßen Polynucleotiden in Expressionskonstrukte cloniert und zum Einbringen und zur Expression in Organismen verwendet. Diese Expressionskonstrukte ermöglichen durch ihre Konstruktion eine vorteilhafte optimale Synthese der im erfindungsgemäßen Verfahren produzierten mehrfach ungesättigten Fettsäuren.To increase the yield in the process described for the preparation of oils and / or triglycerides with an advantageously increased content of polyunsaturated fatty acids, it is advantageous to increase the amount of starting material for the fatty acid synthesis, this can for example by introducing a nucleic acid into the organism , which codes for a polypeptide with Δ-12-desaturase can be achieved. This is particularly advantageous in oil-producing organisms such as the family Brassicaceae such as the genus Brassica eg rape; the family of Elaeagnaceae as the genus Elaeagnus eg the genus and species Olea europaea or family Fabaceae as the genus Glycine eg the genus and species Glycine max, which have a high oleic acid content. Since these organisms have only a low content of linoleic acid (Mikoklajczak et al., Journal of the American Oil Chemical Society, 38, 1961, 678-681), the use of said Δ-12-desaturases for the preparation of the starting product linoleic acid is advantageous. Advantageous in the process according to the invention are the abovementioned nucleic acid sequences or their derivative or homologs which code for polypeptides which still possess the enzymatic activity of the proteins encoded by nucleic acid sequences. These sequences are cloned individually or in combination with the polynucleotides of the invention in expression constructs and used for introduction and expression in organisms. By their construction, these expression constructs enable a favorable optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.
Bei einer bevorzugten Ausführungsform umfasst das Verfahren ferner den Schritt des Gewinnens einer Zelle oder eines ganzen Organismus, der die im Verfahren verwendeten Nukleinsäuresequenzen enthält, wobei die Zelle und/oder der Organismus mit einem erfindungsgemäßen Polynucleotiden einem Genkonstrukt oder einem Vektor wie nachfolgend beschrieben, allein oder in Kombination mit weiteren Nukleinsäuresequenzen, die für Proteine des Fettsäure- oder Lipidsstoffwechsels codieren, transformiert wird. Bei einer weiteren bevorzugten Ausführungsform umfasst dieses Verfahren ferner den Schritt des Gewinnens der Öle, Lipide oder freien Fettsäuren aus dem Organismus oder aus der Kultur. Bei der Kultur kann es sich beispielsweise um eine Fermentationskultur beispielsweise im Falle der Kultivierung von Mikroorganismen wie z.B. Mortierella, Thalassiosira, Mantoniella, Ostreococcus, Saccharomyces oder Thraustochytrium oder um eine Treibhaus oder Feldkultur einer Pflanze handeln. Die so hergestellte Zelle oder der so hergestellte Organismus ist vorteilhaft eine Zelle eines Öl-produzierenden Organismus wie einer Ölfruchtpflanze wie beispielsweise Erdnuss, Raps, Canola, Lein, Hanf, Erdnuss, Soja, Safflower, Hanf, Sonnenblumen oder Borretsch.In a preferred embodiment, the method further comprises the step of obtaining a cell or whole organism containing the nucleic acid sequences used in the method, wherein the cell and / or organism with a polynucleotide of the invention is a gene construct or a vector as described below, alone or in combination with other nucleic acid sequences coding for proteins of the fatty acid or lipid metabolism. In a further preferred embodiment, this method further comprises the step of recovering the oils, lipids or free fatty acids from the organism or from the culture. The culture may be, for example, a fermentation culture, for example, in the case of culturing microorganisms such as e.g. Mortierella, Thalassiosira, Mantoniella, Ostreococcus, Saccharomyces or Thraustochytrium or to act as a greenhouse or field crop of a plant. The cell or organism thus produced is advantageously a cell of an oil-producing organism such as an oil crop such as peanut, canola, canola, flax, hemp, peanut, soybean, safflower, hemp, sunflower or borage.
Unter Anzucht ist beispielsweise die Kultivierung im Falle von Pflanzenzellen, -gewebe oder -organe auf oder in einem Nährmedium oder der ganzen Pflanze auf bzw. in einem Substrat beispielsweise in Hydrokultur, Blumentopferde oder auf einem Ackerboden zu verstehen.Cultivation is, for example, culturing in the case of plant cells, tissue or organs on or in a nutrient medium or the whole plant on or in a substrate, for example in hydroponics, potting soil or on arable land.
Als Organismen bzw. Wirtszellen für das erfindungsgemäße Verfahren sind solche geeignet, die in der Lage sind Fettsäuren speziell ungesättigte Fettsäuren zu syntheti- sieren bzw. für die Expression rekombinanter Gene geeignet sind. Beispielhaft seien Pflanzen wie Arabidopsis, Asteraceae wie Calendula oder Kulturpflanzen wie Soja, Erdnuss, Rizinus, Sonnenblume, Mais, Baumwolle, Flachs, Raps, Kokosnuss, Ölpal- me, FärberSaflor (Carthamus tinctorius) oder Kakaobohne, Mikroorganismen wie Pilze beispielsweise die Gattung Mortierella, Thraustochytrium, Saprolegnia, Phytophtora oder Pythium, Bakterien wie die Gattung Escherichia oder Shewanella, Hefen wie die
Gattung Saccharomyces, Cyanobakterien, Ciliaten, Algen wie Mantoniella oder Ostreococcus oder Protozoen wie Dinoflagellaten wie Thalassiosira oder Crypthecodi- nium genannt. Bevorzugt werden Organismen, die natürlicherweise Öle in größeren Mengen synthetisieren können wie Pilze wie Mortierella alpina, Pythium insidiosum, Phytophtora infestans oder Pflanzen wie Soja, Raps, Kokosnuss, Ölpalme, Färber- Saflor, Flachs, Hanf, Rizinus, Calendula, Erdnuss, Kakaobohne oder Sonnenblume oder Hefen wie Saccharomyces cerevisiae, besonders bevorzugt werden Soja, Flachs, Raps, FärberSaflor, Sonnenblume, Calendula, Mortierella oder Saccharomyces cerevisiae. Prinzipiell sind als Wirtsorganismen neben den vorgenannten transgenen Organismen auch transgene Tiere vorteilhaft nicht-humane Tiere geeignet beispielsweise C. elegans. Weitere geeignete Wirtszellen und Organismen wurden bereits zuvor ausführlich beschrieben.Suitable organisms or host cells for the process according to the invention are those which are able to synthesize fatty acids, especially unsaturated fatty acids, or are suitable for the expression of recombinant genes. Examples include plants such as Arabidopsis, Asteraceae such as Calendula or crops such as soybean, peanut, castor, sunflower, corn, cotton, flax, rape, coconut, oil palm, dyer safflower (Carthamus tinctorius) or cocoa bean, microorganisms such as fungi, for example, the genus Mortierella, Thraustochytrium, Saprolegnia, Phytophthora or Pythium, bacteria such as the genus Escherichia or Shewanella, yeasts like the Genus Saccharomyces, cyanobacteria, ciliates, algae such as Mantoniella or Ostreococcus or protozoa such as dinoflagellates such as Thalassiosira or Crypthecodi- nium called. Preference is given to organisms which can naturally synthesize oils in large quantities, such as fungi such as Mortierella alpina, Pythium insidiosum, Phytophtora infestans or plants such as soybean, rapeseed, coconut, oil palm, dyer safflower, flax, hemp, castor, calendula, peanut, cocoa bean or Sunflower or yeasts such as Saccharomyces cerevisiae, with particular preference being given to soybean, flax, rapeseed, dyer's safflower, sunflower, calendula, mortierella or Saccharomyces cerevisiae. In principle, as host organisms, in addition to the abovementioned transgenic organisms, transgenic animals are also advantageously suitable for non-human animals, for example C. elegans. Other suitable host cells and organisms have previously been described in detail.
Transgene Pflanzen, die die im erfindungsgemäßen Verfahren synthetisierten mehr- fach ungesättigten Fettsäuren enthalten, können vorteilhaft direkt vermarktet werden ohne dass die synthetisierten Öle, Lipide oder Fettsäuren isoliert werden müssen. Unter Pflanzen im erfindungsgemäßen Verfahren sind ganze Pflanzen sowie alle Pflanzenteile, Pflanzenorgane oder Pflanzenteile wie Blatt, Stiel, Samen, Wurzel, Knollen, Antheren, Fasern, Wurzelhaare, Stängel, Embryos, KaIIi, Kotelydonen, Petiolen, Erntematerial, pflanzliches Gewebe, reproduktives Gewebe, Zellkulturen, die sich von der transgenen Pflanze abgeleiten und/oder dazu verwendet werden können, die transgene Pflanze hervorzubringen. Der Samen umfasst dabei alle Samenteile wie die Samenhüllen, Epidermis- und Samenzellen, Endosperm oder Embyrogewebe. Die im erfindungsgemäßen Verfahren hergestellten Verbindungen können aber auch aus den Organismen vorteilhaft Pflanzen in Form ihrer Öle, Fett, Lipide und/oder freien Fettsäuren isoliert werden. Durch dieses Verfahren hergestellte mehrfach ungesättigte Fettsäuren lassen sich durch Ernten der Organismen entweder aus der Kultur, in der sie wachsen, oder vom Feld ernten. Dies kann über Pressen oder Extraktion der Pflanzenteile bevorzugt der Pflanzensamen erfolgen. Dabei können die Öle, Fette, Lipide und/oder freien Fettsäuren durch so genanntes kalt schlagen oder kalt pressen ohne Zuführung von Wärme durch Pressen gewonnen werden. Damit sich die Pflanzenteile speziell die Samen leichter aufschließen lassen, werden sie vorher zerkleinert, gedämpft oder geröstet. Die so vorbehandelten Samen können anschließend gepresst werden oder mit Lösungsmittel wie warmes Hexan extrahiert werden. Anschließend wird das Lösungsmittel wieder entfernt. Im Falle von Mikroorganismen werden diese nach Ernte beispielsweise direkt ohne weitere Arbeitsschritte extrahiert oder aber nach Aufschluss über verschiedene dem Fachmann bekannte Methoden extrahiert. Auf diese Weise können mehr als 96 % der im Verfahren hergestellten Verbindungen isoliert werden. Anschließend werden die so erhaltenen Produkte weiter bearbeitet, das heißt raffiniert. Dabei werden zunächst beispielsweise die Pflanzenschleime und
Trübstoffe entfernt. Die sogenannte Entschleimung kann enzymatisch oder beispielsweise chemisch/physikalisch durch Zugabe von Säure wie Phosphorsäure erfolgen. Anschließend werden die freien Fettsäuren durch Behandlung mit einer Base beispielsweise Natronlauge entfernt. Das erhaltene Produkt wird zur Entfernung der im Produkt verbliebenen Lauge mit Wasser gründlich gewaschen und getrocknet. Um die noch im Produkt enthaltenen Farbstoffe zu entfernen werden die Produkte einer Bleichung mit beispielsweise Bleicherde oder Aktivkohle unterzogen. Zum Schluss wird das Produkt noch beispielsweise mit Wasserdampf noch desodoriert.Transgenic plants which contain the polyunsaturated fatty acids synthesized in the process according to the invention can advantageously be marketed directly without the synthesized oils, lipids or fatty acids having to be isolated. Plants in the process according to the invention include whole plants and all plant parts, plant organs or plant parts such as leaves, stems, seeds, roots, tubers, anthers, fibers, root hairs, stems, embryos, callosis, kotelydons, petioles, crop material, plant tissue, reproductive tissue, Cell cultures that can be derived from the transgenic plant and / or used to produce the transgenic plant. The seed includes all seed parts such as the seed shells, epidermis and sperm cells, endosperm or embryonic tissue. However, the compounds prepared in the process according to the invention can also be isolated from the organisms advantageously plants in the form of their oils, fat, lipids and / or free fatty acids. Polyunsaturated fatty acids produced by this process can be harvested by harvesting the organisms either from the culture in which they grow or from the field. This can be done by pressing or extraction of the plant parts, preferably the plant seeds. The oils, fats, lipids and / or free fatty acids can be obtained by so-called cold pressing or cold pressing without supplying heat by pressing. In order for the plant parts, especially the seeds, to be easier to digest, they are first crushed, steamed or roasted. The pretreated seeds can then be pressed or extracted with solvents such as warm hexane. Subsequently, the solvent is removed again. In the case of microorganisms, these are harvested after harvesting, for example, directly without further working steps, or else extracted after digestion by various methods known to the person skilled in the art. In this way, more than 96% of the compounds prepared in the process can be isolated. Subsequently, the products thus obtained are further processed, that is refined. First, for example, the mucus and Turbidity removed. The so-called degumming can be carried out enzymatically or, for example, chemically / physically by adding acid, such as phosphoric acid. Subsequently, the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution. The product obtained is thoroughly washed with water to remove the lye remaining in the product and dried. In order to remove the dyes still contained in the product, the products are subjected to bleaching with, for example, bleaching earth or activated carbon. Finally, the product is still deodorized, for example, with steam.
Vorzugsweise sind die durch dieses Verfahren produzierten PUFAs bzw. LCPUFAs Ci8-, C2O- oder C22-Fettsäuremoleküle vorteilhaft C2o- oder C22-Fettsäuremoleküle mit mindestens zwei Doppelbindungen im Fettsäuremolekül, vorzugsweise drei, vier, fünf oder sechs Doppelbindungen. Diese Ci8-, C2o- oder C22-Fettsäuremoleküle lassen sich aus dem Organismus in Form eines Öls, Lipids oder einer freien Fettsäure isolieren. Geeignete Organismen sind beispielsweise die vorstehend erwähnten. Bevorzugte Organismen sind transgene Pflanzen.Preferably, the PUFAs or LCPUFAs produced by this process are C 8 -, C 2 O- or C 22 -fatty acid molecules and advantageously C 2 o- or C 22 -fatty acid molecules having at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds. These Ci 8 -, C 2 o- or C 22 -Fettsäuremoleküle can be isolated from the organism in the form of an oil, lipid or a free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.
Eine Ausführungsform der Erfindung sind deshalb Öle, Lipide oder Fettsäuren oder Fraktionen davon, die durch das oben beschriebene Verfahren hergestellt worden sind, besonders bevorzugt Öl, Lipid oder eine Fettsäurezusammensetzung, die PUFAs umfassen und von transgenen Pflanzen herrühren.One embodiment of the invention is therefore oils, lipids or fatty acids or fractions thereof which have been prepared by the method described above, more preferably oil, lipid or fatty acid composition comprising PUFAs derived from transgenic plants.
Diese Öle, Lipide oder Fettsäuren enthalten wie oben beschrieben vorteilhaft 6 bis 15 % Palmitinsäure, 1 bis 6 % Stearinsäure; 7 - 85 % Ölsäure; 0,5 bis 8 % Vaccensäure, 0,1 bis 1 % Arachinsäure, 7 bis 25 % gesättigte Fettsäuren, 8 bis 85 % einfach ungesättigte Fettsäuren und 60 bis 85 % mehrfach ungesättigte Fettsäuren jeweils bezogen auf 100 % und auf den Gesamtfettsäuregehalt der Organismen. Als vorteilhafte mehrfach ungesättigte Fettsäure sind in den Fettsäureester bzw. Fettsäuregemische bevorzugt mindestens 0,1 ; 0,2; 0,3; 0,4; 0,5; 0,6; 0,7; 0,8; 0,9 oder 1 % bezogen auf den Gesamtfettsäuregehalt an Arachidonsäure enthalten. Weiterhin enthalten die Fettsäureester bzw. Fettsäuregemische, die nach dem erfindungsgemäßen Verfahren hergestellt wurden, vorteilhaft Fettsäuren ausgewählt aus der Gruppe der Fettsäuren Erucasäure (13-Docosaensäure), Sterculinsäure (9,10-Methylene octadec-9- enonsäure), Malvalinsäure (8,9-Methylen Heptadec-8-enonsäure), Chaulmoogrinsäure (Cyclopenten-dodecansäure), Furan-Fettsäure (9,12-Epoxy-octadeca-9,11- dienonsäure), Vernonsäure (9,10-Epoxyoctadec-12-enonsäure), Tarinsäure (6- Octadecynonsäure),6-Nonadecynonsäure, Santalbinsäure (t11-Octadecen-9-ynoic acid), 6,9-Octadecenynonsäure, Pyrulinsäure (t10-Heptadecen-8-ynonsäure), Crepe- nyninsäure (9-Octadecen-12-ynonsäure), 13,14-Dihydrooropheinsäure, Octadecen-13- ene-9,1 1-diynonsäure, Petroselensäure (cis-6-Octadecenonsäure), 9c,12t-Octa- decadiensäure, Calendulasäure (8t10t12c-Octadecatriensäure), Catalpinsäure
(9t1 itiSc-Octadecatriensäure), Eleosterinsäure (9c1 1t13t-Octadecatriensäure), Jacarinsäure (8c10t12c-Octadecatriensäure), Punicinsäure (9c11t13c-Octadecatrien- säure),Parinarinsäure (9c1 1t13t15c-Octadecatetraensäure), Pinolensäure (all-cis- 5,9,12-Octadecatriensäure), Laballensäure (5,6-Octadecadienallensäure), Ricinolsäure (12-Hydroxyölsäure) und/oder Coriolinsäure (13-Hydroxy-9c,11t-Octadecadienon- säure). Die vorgenannten Fettsäuren kommen in den nach dem erfindungsgemäßen Verfahren hergestellten Fettsäureester bzw. Fettsäuregemischen in der Regel vorteilhaft nur in Spuren vor, das heißt sie kommen bezogen auf die Gesamtfettsäuren zu weniger als 30 %, bevorzugt zu weniger als 25 %, 24 %, 23 %, 22 % oder 21 %, besonders bevorzugt zu weniger als 20 %, 15 %, 10 %, 9 %, 8 %, 7%, 6 % oder 5%, ganz besonders bevorzugt zu weniger als 4 %, 3 %, 2 % oder 1 % vor. Vorteilhaft enthalten die nach dem erfindungsgemäßen Verfahren hergestellten Fettsäureester bzw. Fettsäuregemische weniger als 0,1 % bezogen auf die Gesamtfettsäuren oder keine Butterbuttersäure, kein Cholesterin, keine Clupanodonsäure (= Docosapentaen- säure, C22:5A4'8'12'15'21) sowie keine Nisinsäure (Tetracosahexaensäure,These oils, lipids or fatty acids advantageously contain 6 to 15% palmitic acid, 1 to 6% stearic acid as described above; 7 - 85% oleic acid; 0.5 to 8% of vaccenic acid, 0.1 to 1% of arachidic acid, 7 to 25% of saturated fatty acids, 8 to 85% of monounsaturated fatty acids and 60 to 85% of polyunsaturated fatty acids in each case based on 100% and on the total fatty acid content of the organisms. As advantageous polyunsaturated fatty acid in the fatty acid esters or fatty acid mixtures are preferably at least 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1% based on the total fatty acid content of arachidonic acid. Furthermore, the fatty acid esters or fatty acid mixtures prepared by the process according to the invention advantageously contain fatty acids selected from the group of the fatty acids erucic acid (13-docosaic acid), sterculic acid (9,10-methylene octadec-9-enoic acid), malvalic acid (8,9 -Methylene heptadec-8-enoic acid), chaulmoogric acid (cyclopentenodecanoic acid), furan fatty acid (9,12-epoxy-octadeca-9,11-dienoic acid), vernonic acid (9,10-epoxyoctadec-12-enoic acid), tartric acid ( 6- octadecynoic acid), 6-nonadecynoic acid, santalbic acid (t11-octadecen-9-ynoic acid), 6,9-octadecenynoic acid, pyrulic acid (t10-heptadecen-8-ynonic acid), crepenyninic acid (9-octadecen-12-ynonic acid) , 13,14-dihydrooropheic acid, octadecene-13-ene-9,1-diynoic acid, petroselenoic acid (cis-6-octadecenoic acid), 9c, 12t-octadecadienoic acid, calendulic acid (8t10t12c octadecatrienoic acid), catalpinic acid (9t1 itiSc-octadecatrienoic acid), elecetic acid (9c1 1t13t octadecatrienoic acid), jacaric acid (8c10t12c octadecatrienoic acid), punicic acid (9c11t13c octadecatrienoic acid), parinaric acid (9c1 1t13t15c octadecatetraenoic acid), pinolenic acid (all-cis 5, 9, 12) Octadecatrienoic acid), labellaric acid (5,6-octadecadienenoic acid), ricinoleic acid (12-hydroxyoleic acid) and / or coriolinic acid (13-hydroxy-9c, 11t-octadecadienoic acid). The abovementioned fatty acids are generally advantageously present only in traces in the fatty acid esters or fatty acid mixtures prepared by the process according to the invention, that is to say they are less than 30%, preferably less than 25%, 24%, 23%, based on the total fatty acids. , 22% or 21%, more preferably less than 20%, 15%, 10%, 9%, 8%, 7%, 6% or 5%, most preferably less than 4%, 3%, 2% or 1% ago. Advantageously, the fatty acid esters or mixtures of fatty acids prepared by the process according to the invention contain less than 0.1% based on the total fatty acids or no butter butyric acid, no cholesterol, no clupanodonic acid (= docosapentaenoic acid, C22: 5 A4 ' 8 ' 12 ' 15 ' 21 ) and no nisic acid (tetracosahexaenoic acid,
C23"6A3 ' 8 ' 12 ' 15 ' 18 ' 2"KC23 " 6 A3 ' 8 ' 12 ' 15 ' 18 ' 2 " K
Bevorzugt enthalten die erfindungsgemäßen Öle, Lipide oder Fettsäuren mindestens 0,5%, 1%, 2%, 3%, 4% oder 5%, vorteilhaft mindestens 6%, 7%, 8%, 9% oder 10%, besonders vorteilhaft mindestens 11 %, 12%, 13%, 14% oder 15% ARA oder mindestens 0,5%, 1%, 2%, 3%, 4% oder 5%, vorteilhaft mindestens 6%, oder 7%, besonders vorteilhaft mindestens 8%, 9% oder 10% EPA und/oder DHA bezogen auf den Gesamtfettsäuregehalt des Produktionsorganismus vorteilhaft einer Pflanze, besonders vorteilhaft einer Ölfruchtpflanze wie Soja, Raps, Kokosnuss, Ölpalme, Färber- safflor, Flachs, Hanf, Rizinus, Calendula, Erdnuss, Kakaobohne, Sonnenblume oder den oben genannten weiteren ein- oder zweikeimblättrigen Ölfruchtpflanzen.The oils, lipids or fatty acids according to the invention preferably contain at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, 7%, 8%, 9% or 10%, particularly advantageously at least 11%, 12%, 13%, 14% or 15% ARA or at least 0.5%, 1%, 2%, 3%, 4% or 5%, advantageously at least 6%, or 7%, more preferably at least 8 %, 9% or 10% EPA and / or DHA based on the total fatty acid content of the production organism advantageously a plant, particularly advantageous an oil crop such as soy, rapeseed, coconut, oil palm, safflower, flax, hemp, castor, calendula, peanut, cocoa bean , Sunflower or the above-mentioned other monocotyledonous or dicotyledonous oil crops.
Eine weitere erfindungsgemäße Ausführungsform ist die Verwendung des Öls, Lipids, der Fettsäuren und/oder der Fettsäurezusammensetzung in Futtermitteln, Nahrungs- mittein, Kosmetika oder Pharmazeutika. Die erfindungsgemäßen Öle, Lipide, Fettsäuren oder Fettsäuregemische können in der dem Fachmann bekannten Weise zur Abmischung mit anderen Ölen, Lipiden, Fettsäuren oder Fettsäuregemischen tierischen Ursprungs wie z.B. Fischölen verwendet werden. Auch diese Öle, Lipide, Fettsäuren oder Fettsäuregemische, die aus pflanzlichen und tierischen Bestandteilen bestehen, können zur Herstellung von Futtermitteln, Nahrungsmitteln, Kosmetika oder Pharmazeutika verwendet werden.Another embodiment of the invention is the use of the oil, lipid, fatty acids and / or fatty acid composition in feed, food, cosmetics or pharmaceuticals. The oils, lipids, fatty acids or fatty acid mixtures according to the invention may be mixed with other oils, lipids, fatty acids or fatty acid mixtures of animal origin, such as those described in the art, for example. Fish oils are used. These oils, lipids, fatty acids or fatty acid mixtures, which consist of vegetable and animal components, can be used for the production of feed, food, cosmetics or pharmaceuticals.
Unter dem Begriff "Öl", "Lipid" oder "Fett" wird ein Fettsäuregemisch verstanden, das ungesättigte, gesättigte, vorzugsweise veresterte Fettsäure(n) enthält. Bevorzugt ist, dass das Öl, Lipid oder Fett einen hohen Anteil an mehrfach ungesättigten freien oder
vorteilhaft veresterten Fettsäure(n), insbesondere Linolsäure, γ-Linolensäure, Dihomo- γ-Linolensäure, Arachidonsäure, α-Linolensäure, Stearidonsäure, Eicosatetraensäure, Eicosapentaensäure, Docosapentaensäure oder Docosahexaensäure hat. Vorzugsweise ist der Anteil an ungesättigten veresterten Fettsäuren ungefähr 30 %, mehr bevorzugt ist ein Anteil von 50 %, noch mehr bevorzugt ist ein Anteil von 60 %, 70 %, 80 % oder mehr. Zur Bestimmung kann z.B. der Anteil an Fettsäure nach Überführung der Fettsäuren in die Methylestern durch Umesterung gaschromatographisch bestimmt werden. Das Öl, Lipid oder Fett kann verschiedene andere gesättigte oder ungesättigte Fettsäuren, z.B. Calendulasäure, Palmitin-, Palmitolein-, Stearin-, Ölsäure etc., enthalten. Insbesondere kann je nach Ausgangsorganismus der Anteil der verschiedenen Fettsäuren in dem Öl oder Fett schwanken.The term "oil", "lipid" or "fat" is understood as meaning a fatty acid mixture which contains unsaturated, saturated, preferably esterified fatty acid (s). It is preferred that the oil, lipid or fat contain a high proportion of polyunsaturated free or advantageously esterified fatty acid (s), in particular linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid. Preferably, the proportion of unsaturated esterified fatty acids is about 30%, more preferred is a proportion of 50%, even more preferred is a proportion of 60%, 70%, 80% or more. For the determination, for example, the proportion of fatty acid after conversion of the fatty acids into the methyl esters can be determined by transesterification by gas chromatography. The oil, lipid or fat may contain various other saturated or unsaturated fatty acids, eg calendulic acid, palmitic, palmitoleic, stearic, oleic acid, etc. In particular, depending on the starting organism, the proportion of the various fatty acids in the oil or fat may vary.
Bei den im Verfahren hergestellten mehrfach ungesättigte Fettsäuren mit vorteilhaft mindestens zwei Doppelbindungen enthalten, handelt es sich wie oben beschrieben beispielsweise um Sphingolipide, Phosphoglyceride, Lipide, Glycolipide, Phospholipi- de, Monoacylglycerin, Diacylglycerin, Triacylglycerin oder sonstige Fettsäureester.The polyunsaturated fatty acids having advantageously at least two double bonds which are produced in the process are, as described above, for example sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.
Aus den so im erfindungsgemäßen Verfahren hergestellten mehrfach ungesättigte Fettsäuren mit vorteilhaft mindestens fünf oder sechs Doppelbindungen lassen sich die enthaltenden mehrfach ungesättigten Fettsäuren beispielsweise über eine Alkalibehandlung beispielsweise wässrige KOH oder NaOH oder saure Hydrolyse vorteilhaft in Gegenwart eines Alkohols wie Methanol oder Ethanol oder über eine enzymatische Abspaltung freisetzen und isolieren über beispielsweise Phasentrennung und anschließender Ansäuerung über z.B. H2SO4. Die Freisetzung der Fettsäuren kann auch direkt ohne die vorhergehend beschriebene Aufarbeitung erfolgen.From the polyunsaturated fatty acids thus produced in the process according to the invention advantageously having at least five or six double bonds, the polyunsaturated fatty acids containing, for example, an alkali treatment such as aqueous KOH or NaOH or acid hydrolysis advantageously in the presence of an alcohol such as methanol or ethanol or via an enzymatic cleavage liberate and isolate via, for example, phase separation and subsequent acidification via, for example, H 2 SO 4 . The release of the fatty acids can also be carried out directly without the workup described above.
Die im Verfahren verwendeten Nukleinsäuren können nach Einbringung in einem Organismus vorteilhaft einer Pflanzenzelle bzw. Pflanze entweder auf einem separaten Plasmid liegen oder vorteilhaft in das Genom der Wirtszelle integriert sein. Bei Inte- gration in das Genom kann die Integration zufallsgemäß sein oder durch derartige Rekombination erfolgen, dass das native Gen durch die eingebrachte Kopie ersetzt wird, wodurch die Produktion der gewünschten Verbindung durch die Zelle moduliert wird, oder durch Verwendung eines Gens in „trans", so dass das Gen mit einer funktionellen Expressionseinheit, welche mindestens eine die Expression eines Gens gewährleistende Sequenz und mindestens eine die Polyadenylierung eines funktionell transkribierten Gens gewährleistende Sequenz enthält, funktionell verbunden ist. Vorteilhaft werden die Nukleinsäuren über Multiexpressionskassetten oder Konstrukte zur multiparallelen Expression in die Organismen vorteilhaft zur multiparallelen samenspezifischen Expression von Genen in die Pflanzen gebracht.
Moose und Algen sind die einzigen bekannten Pflanzensysteme, die erhebliche Mengen an mehrfach ungesättigten Fettsäuren, wie Arachidonsäure (ARA) und/oder Eicosapentaensäure (EPA) und/oder Docosahexaensäure (DHA) herstellen. Moose enthalten PUFAs in Membranlipiden während Algen, algenverwandte Organismen und einige Pilze auch nennenswerte Mengen an PUFAs in der Triacylglycerolfraktion akkumulieren. Daher eignen sich Nukleinsäuremoleküle, die aus solchen Stämmen isoliert werden, die PUFAs auch in der Triacylglycerolfraktion akkumulieren, besonders vorteilhaft für das erfindungsgemäße Verfahren und damit zur Modifikation des Lipid- und PUFA-Produktionssystems in einem Wirt, insbesondere Pflanzen, wie Ölfrucht- pflanzen, beispielsweise Raps, Canola, Lein, Hanf, Soja, Sonnenblumen, Borretsch. Sie sind deshalb vorteilhaft im erfindungsgemäßen Verfahren verwendbar.After incorporation into an organism, the nucleic acids used in the method can advantageously be either a plant cell or plant, either on a separate plasmid or advantageously integrated into the genome of the host cell. Upon integration into the genome, integration may be at random or by such recombination as replacing the native gene with the incorporated copy, thereby modulating production of the desired compound by the cell, or by using a gene in "trans". in such a way that the gene is functionally linked to a functional expression unit which contains at least one sequence ensuring the expression of a gene and at least one sequence ensuring the polyadenylation of a functionally transcribed gene Advantageously, the nucleic acids are introduced into the organisms via multi-expression cassettes or constructs for multiparallel expression advantageous for multiparallel seed-specific expression of genes brought into the plants. Moose and algae are the only known plant systems that produce significant amounts of polyunsaturated fatty acids, such as arachidonic acid (ARA) and / or eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA). Moose contain PUFAs in membrane lipids, while algae, algae-related organisms and some fungi also accumulate significant levels of PUFAs in the triacylglycerol fraction. Therefore, nucleic acid molecules which are isolated from strains which also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the process according to the invention and thus for modification of the lipid and PUFA production system in a host, in particular plants, such as oilseed plants, for example Rapeseed, canola, flax, hemp, soy, sunflower, borage. They are therefore advantageous for use in the process according to the invention.
Als Substrate der im erfindungsgemäßen Polypeptide oder des Polypeptids des Fettsäure- oder Lipidstoffwechsels ausgewählt aus der Gruppe Acyl-CoA- Dehydrogenase(n), Acyl-ACP[= acyl carrier protein]-Desaturase(n), Acyl-ACP- Thioesterase(n), Fettsäure-Acyl-Transferase(n), Acyl-CoA:Lysophospholipid- Acyltransferase(n), Fettsäure-Synthase(n), Fettsäure-Hydroxylase(n), Acetyl- Coenzym A-Carboxylase(n), Acyl-Coenzym A-Oxidase(n), Fettsäure-Desaturase(n), Fettsäure-Acetylenase(n), Lipoxygenase(n), Triacylglycerol-Lipase(n), Allenoxid- Synthase(n), Hydroperoxid-Lyase(n) oder Fettsäure-Elongase(n) eignen sich bevorzugt Ci6-, Ci8- oder C2o-Fettsäuren. Bevorzugt werden die im Verfahren als Substrate umgesetzten Fettsäuren in Form ihrer Acyl-CoA-Ester und/oder ihrer Phospholipid- Ester umgesetzt.As substrates of the polypeptides according to the invention or of the polypeptide of the fatty acid or lipid metabolism selected from the group acyl-CoA-dehydrogenase (s), acyl-ACP [acyl carrier protein] desaturase (s), acyl-ACP thioesterase (s) , Fatty acid acyltransferase (s), acyl-CoA: lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A carboxylase (s), acyl coenzyme A- Oxidase (s), fatty acid desaturase (s), fatty acid acetylenase (s), lipoxygenase (s), triacylglycerol lipase (s), allene oxide synthase (s), hydroperoxide lyase (s) or fatty acid elongase (n ) are preferably Ci6, Ci8 or C 2 o fatty acids. The fatty acids reacted as substrates in the process are preferably reacted in the form of their acyl-CoA esters and / or their phospholipid esters.
Zur Herstellung der erfindungsgemäßen langkettigen PUFAs müssen die mehrfach ungesättigten Ciβ-Fettsäuren zunächst durch die enzymatische Aktivität einer Desatu- rase zunächst desaturiert und anschließend über eine Elongase um mindestens zwei Kohlenstoffatome verlängert werden. Nach einer Elongationsrunde führt diese Enzymaktivität zu C2o-Fettsäuren, und nach zwei Elongationsrunden zu C22-Fettsäuren. Die Aktivität der erfindungsgemäßen Verfahren verwendeten Desaturasen und Elongasen führt vorzugsweise zu Ciβ-, C20- und/oder C22-Fettsäuren vorteilhaft mit mindestens zwei Doppelbindungen im Fettsäuremolekül, vorzugsweise mit drei, vier, fünf oder sechs Doppelbindungen, besonders bevorzugt zu C2o- und/oder C22-Fettsäuren mit mindestens zwei Doppelbindungen im Fettsäuremolekül, vorzugsweise mit drei, vier, fünf oder sechs Doppelbindungen, ganz besonders bevorzugt mit fünf oder sechs Doppelbindungen im Molekül. Nachdem eine erste Desaturierung und die Verlängerung stattfand, können weitere Desaturierungs- und Elongierungsschritte wie z.B. eine solche Desaturierung in Δ-5- und Δ-4-Position erfolgen. Besonders bevorzugt als Produkte des erfindungsgemäßen Verfahrens sind Dihomo-γ-linolensäure, Arachidon- säure, Eicosapentaensäure, Docosapetaensäure und/oder Docosahesaensäure. Die
C2o-Fettsäuren mit mindestens zwei Doppelbindungen in der Fettsäure können durch die erfindungsgemäße enzymatische Aktivität in Form der freien Fettsäure oder in Form der Ester, wie Phospholipide, Glycolipide, Sphingolipide, Phosphoglyceride, Monoacylglycerin, Diacylglycerin oder Triacylglycerin, verlängert werden.To prepare the long-chain PUFAs according to the invention, the polyunsaturated C 16 -fatty acids must first be desaturated by the enzymatic activity of a desaturase and then be extended by at least two carbon atoms via an elongase. After one round of elongation this enzyme activity leads to C 2 o-fatty acids, and after two rounds of elongation to C 22 -fatty acids. Desaturases and elongases used in the activity of the process according to the invention preferably leads to Ciβ-, C 2 0 and / or C 22 fatty acids advantageously having at least two double bonds in the fatty acid molecule, preferably with three, four, five or six double bonds, especially preferably C 2 o- and / or C 22 -fatty acids having at least two double bonds in the fatty acid molecule, preferably having three, four, five or six double bonds, very particularly preferably having five or six double bonds in the molecule. After a first desaturation and extension, further desaturation and elongation steps such as desaturation at Δ-5 and Δ-4 positions may occur. Particularly preferred products of the process according to the invention are dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and / or docosaheic acid. The C 2 o fatty acids having at least two double bonds in the fatty acid can be extended by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.
Der bevorzugte Biosyntheseort von Fettsäuren, Ölen, Lipiden oder Fette in den vorteilhaft verwendeten Pflanzen ist beispielsweise im allgemeinen der Samen oder Zellschichten des Samens, so dass eine samenspezifische Expression der im Verfahren verwendeten Nukleinsäuren sinnvoll ist. Es ist jedoch naheliegend, dass die Biosynthese von Fettsäuren, Ölen oder Lipiden nicht auf das Samengewebe beschränkt sein muss, sondern auch in allen übrigen Teilen der Pflanze - beispielsweise in Epidermiszellen oder in den Knollen - gewebespezifisch erfolgen kann.The preferred biosynthesis site of fatty acids, oils, lipids or fats in the advantageously used plants is, for example, generally the seeds or cell layers of the seed, so that a seed-specific expression of the nucleic acids used in the method is useful. However, it is obvious that the biosynthesis of fatty acids, oils or lipids need not be limited to the seed tissue, but may also be tissue-specific in all other parts of the plant - for example in epidermal cells or in the tubers.
Werden im erfindungsgemäßen Verfahren als Organismen Mikroorganismus wie Hefen wie Saccharomyces oder Schizosaccharomyces, Pilze wie Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor oder Thraustochytrium Algen wie Isochrysis, Mantoniella, Ostreococcus, Phaeodactylum oder Crypthecodiniu verwendet, so werden diese Organismen vorteilhaft fermentativ angezogen.If microorganisms such as yeasts such as Saccharomyces or Schizosaccharomyces, fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium algae such as Isochrysis, Mantoniella, Ostreococcus, Phaeodactylum or Crypthecodiniu are used in the method according to the invention as organisms, these organisms are advantageously attracted to fermentation.
Durch die Verwendung der erfindungsgemäßen Nukleinsäuren, die für eine Δ-5- Elongase codieren, können im Verfahren die hergestellten mehrfach ungesättigten Fettsäuren mindestens um 5 %, bevorzugt mindestens um 10 %, besonders bevorzugt mindestens um 20 %, ganz besonders bevorzugt um mindestens 50 % gegenüber dem Wildtyp der Organismen, die die Nukleinsäuren nicht rekombinant enthalten, erhöht werden.By using the nucleic acids according to the invention which code for a Δ-5 elongase, the polyunsaturated fatty acids prepared in the process can be at least 5%, preferably at least 10%, more preferably at least 20%, very particularly preferably at least 50%. be increased compared to the wild type of organisms that do not contain the nucleic acids recombinantly.
Durch das erfindungsgemäße Verfahren können die hergestellten mehrfach ungesättigten Fettsäuren in den im Verfahren verwendeten Organismen prinzipiell auf zwei Arten erhöht werden. Es kann vorteilhaft der Pool an freien mehrfach ungesättigten Fettsäuren und/oder der Anteil der über das Verfahren hergestellten veresterten mehrfach ungesättigten Fettsäuren erhöht werden. Vorteilhaft wird durch das erfindungsgemäße Verfahren der Pool an veresterten mehrfach ungesättigten Fettsäuren in den transgenen Organismen erhöht.By the method according to the invention, the polyunsaturated fatty acids produced in the organisms used in the process can in principle be increased in two ways. Advantageously, the pool of free polyunsaturated fatty acids and / or the proportion of esterified polyunsaturated fatty acids produced by the process can be increased. Advantageously, the process according to the invention increases the pool of esterified polyunsaturated fatty acids in the transgenic organisms.
Werden im erfindungsgemäßen Verfahren als Organismen Mikroorganismen verwendet, so werden sie je nach Wirtsorganismus in dem Fachmann bekannter Weise angezogen bzw. gezüchtet. Mikroorganismen werden in der Regel in einem flüssigen Medium, das eine Kohlenstoffquelle meist in Form von Zuckern, eine Stickstoffquelle meist in Form von organischen Stickstoffquellen wie Hefeextrakt oder Salzen wie Ammoniumsulfat, Spurenelemente wie Eisen-, Mangan-, Magnesiumsalze und
gegebenenfalls Vitamine enthält, bei Temperaturen zwischen O0C und 10O0C, bevorzugt zwischen 100C bis 60°C unter Sauerstoffbegasung angezogen. Dabei kann der pH der Nährflüssigkeit auf einen festen Wert gehalten werden, das heißt während der Anzucht reguliert werden oder nicht. Die Anzucht kann batch weise, semi batch weise oder kontinuierlich erfolgen. Nährstoffe können zu Beginn der Fermentation vorgelegt oder semikontinuierlich oder kontinuierlich nachgefüttert werden. Die hergestellten mehrfach ungesättigten Fettsäuren können nach dem Fachmann bekannten Verfahren wie oben beschrieben aus den Organismen isoliert werden. Beispielsweise über Extraktion, Destillation, Kristallisation, ggf. Salzfällung und/oder Chromatographie. Die Organismen können dazu vorher noch vorteilhaft aufgeschlossen werden.If microorganisms are used as organisms in the process according to the invention, they are grown or grown, depending on the host organism, in a manner known to the person skilled in the art. Microorganisms are usually in a liquid medium containing a carbon source usually in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally contains vitamins, at temperatures between 0 0 C and 10O 0 C, preferably between 10 0 C to 60 ° C attracted under oxygen fumigation. In this case, the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation or not. The cultivation can be batchwise, semi-batch wise or continuous. Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously. The polyunsaturated fatty acids prepared can be isolated from the organisms by methods known to those skilled in the art as described above. For example, extraction, distillation, crystallization, optionally salt precipitation and / or chromatography. The organisms can be opened up for this purpose yet advantageous.
Das erfindungsgemäße Verfahren wird, wenn es sich bei den Wirtsorganismen um Mikroorganismen handelt, vorteilhaft bei einer Temperatur zwischen 00C bis 95° , bevorzugt zwischen 100C bis 85°C, besonders bevorzugt zwischen 15°C bis 75°C, ganz besonders bevorzugt zwischen 15°C bis 45°C durchgeführt.The inventive method, when it is in the host organisms are microorganisms, advantageously carried out at a temperature between 0 0 C to 95 °, preferably between 10 0 C to 85 ° C, more preferably between 15 ° C to 75 ° C, most preferably carried out between 15 ° C to 45 ° C.
Der pH Wert wird dabei vorteilhaft zwischen pH 4 und 12, bevorzugt zwischen pH 6 und 9, besonders bevorzugt zwischen pH 7 und 8 gehalten.The pH is advantageously maintained between pH 4 and 12, preferably between pH 6 and 9, more preferably between pH 7 and 8.
Das erfindungsgemäße Verfahren kann batchweise, semi-batchweise oder kontinuierlich betrieben werden. Eine Zusammenfassung über bekannte Kultivierungsmethoden ist im Lehrbuch von Chmiel (Bioprozeßtechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991 )) oder im Lehrbuch von Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)) zu finden.The process according to the invention can be operated batchwise, semi-batchwise or continuously. A summary of known cultivation methods is in the textbook by Chmiel (Bioprozesstechnik 1. Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) Find.
Das zu verwendende Kulturmedium hat in geeigneter Weise den Ansprüchen der jeweiligen Stämme zu genügen. Beschreibungen von Kulturmedien verschiedener Mikroorganismen sind im Handbuch "Manual of Methods für General Bacteriology" der American Society für Bacteriology (Washington D. C, USA, 1981 ) enthalten.The culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington D.C, USA, 1981).
Diese erfindungsgemäß einsetzbaren Medien umfassen wie oben beschrieben gewöhnlich eine oder mehrere Kohlenstoffquellen, Stickstoffquellen, anorganische Salze, Vitamine und/oder Spurenelemente.As described above, these media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
Bevorzugte Kohlenstoffquellen sind Zucker, wie Mono-, Di- oder Polysaccharide. Sehr gute Kohlenstoffquellen sind beispielsweise Glucose, Fructose, Mannose, Galactose, Ribose, Sorbose, Ribulose, Lactose, Maltose, Saccharose, Raffinose, Stärke oder Cellulose. Man kann Zucker auch über komplexe Verbindungen, wie Melassen, oder andere Nebenprodukte der Zucker-Raffinierung zu den Medien geben. Es kann auch
vorteilhaft sein, Gemische verschiedener Kohlenstoffquellen zuzugeben. Andere mögliche Kohlenstoffquellen sind Öle und Fette wie z.B. Sojaöl, Sonnenblumenöl, Erdnussöl und/oder Kokosfett, Fettsäuren wie z.B. Palmitinsäure, Stearinsäure und/oder Linolsäure, Alkohole und/oder Polyalkohole wie z. B. Glycerin, Methanol und/oder Ethanol und/oder organische Säuren wie z.B. Essigsäure und/oder Milchsäure.Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It can also be advantageous to add mixtures of different carbon sources. Other possible sources of carbon are oils and fats such as soybean oil, sunflower oil, peanut oil and / or coconut oil, fatty acids such as palmitic acid, stearic acid and / or linoleic acid, alcohols and / or polyalcohols such. As glycerol, methanol and / or ethanol and / or organic acids such as acetic acid and / or lactic acid.
Stickstoffquellen sind gewöhnlich organische oder anorganische Stickstoffverbindungen oder Materialien, die diese Verbindungen enthalten. Beispielhafte Stickstoffquel- len umfassen Ammoniak in flüssiger- oder gas- Form oder Ammoniumsalze, wie Ammoniumsulfat, Ammoniumchlorid, Ammoniumphosphat, Ammoniumcarbonat oder Ammoniumnitrat, Nitrate, Harnstoff, Aminosäuren oder komplexe Stickstoffquellen, wie Maisquellwasser, Sojamehl, Sojaprotein, Hefeextrakt, Fleischextrakt und andere. Die Stickstoffquellen können einzeln oder als Mischung verwendet werden.Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds. Exemplary nitrogen sources include ammonia in liquid or gas form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soy protein, yeast extract, meat extract and others. The nitrogen sources can be used singly or as a mixture.
Anorganische Salzverbindungen, die in den Medien enthalten sein können, umfassen die Chlorid-, Phosphor- oder Sulfatsalze von Calcium, Magnesium, Natrium, Kobalt, Molybdän, Kalium, Mangan, Zink, Kupfer und Eisen.Inorganic salt compounds which may be included in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
Als Schwefelquelle für die Herstellung von schwefelhaltigen Feinchemikalien, insbesondere von Methionin, können anorganische schwefelhaltige Verbindungen wie beispielsweise Sulfate, Sulfite, Dithionite, Tetrathionate, Thiosulfate, Sulfide aber auch organische Schwefelverbindungen, wie Mercaptane und Thiole, verwendet werden.As the sulfur source for the production of sulfur-containing fine chemicals, in particular methionine, inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic sulfur compounds, such as mercaptans and thiols can be used.
Als Phosphorquelle können Phosphorsäure, Kaliumdihydrogenphosphat oder Dikali- umhydrogenphosphat oder die entsprechenden Natriumhaitigen Salze verwendet werden.Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
Chelatbildner können zum Medium gegeben werden, um die Metallionen in Lösung zu halten. Besonders geeignete Chelatbildner umfassen Dihydroxyphenole, wie Catechol oder Protocatechuat, oder organische Säuren, wie Zitronensäure.Chelating agents can be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
Die erfindungsgemäß zur Kultivierung von Mikroorganismen eingesetzten Fermentationsmedien enthalten üblicherweise auch andere Wachstumsfaktoren, wie Vitamine oder Wachstumsförderer, zu denen beispielsweise Biotin, Riboflavin, Thiamin, Folsäure, Nikotinsäure, Panthothenat und Pyridoxin gehören. Wachstumsfaktoren und Salze stammen häufig von komplexen Medienkomponenten, wie Hefeextrakt, Melassen, Maisquellwasser und dergleichen. Dem Kulturmedium können überdies geeignete Vorstufen zugesetzt werden. Die genaue Zusammensetzung der Medien- Verbindungen hängt stark vom jeweiligen Experiment ab und wird für jeden spezifi-
sehen Fall individuell entschieden. Information über die Medienoptimierung ist erhältlich aus dem Lehrbuch "Applied Microbiol. Physiology, A Practical Approach" (Hrsg. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) S. 53-73, ISBN 0 19 963577 3). Wachstumsmedien lassen sich auch von kommerziellen Anbietern beziehen, wie Standard 1 (Merck) oder BHI (Brain heart infusion, DIFCO) und dergleichen.The fermentation media used according to the invention for the cultivation of microorganisms usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are often derived from complex media components, such as yeast extract, molasses, corn steep liquor, and the like. In addition, suitable precursors can be added to the culture medium. The exact composition of the media compounds will depend heavily on the experiment and will be specific to each specific see case individually decided. Information about the media optimization is available from the textbook "Applied Microbiol Physiology, A Practical Approach" (Ed PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media may also be obtained from commercial suppliers such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
Sämtliche Medienkomponenten werden, entweder durch Hitze (20 min bei 1 ,5 bar und 1210C) oder durch Sterilfiltration, sterilisiert. Die Komponenten können entweder zusammen oder nötigenfalls getrennt sterilisiert werden. Sämtliche Medien- komponenten können zu Beginn der Anzucht zugegen sein oder wahlfrei kontinuierlich oder Chargenweise hinzu gegeben werden.All media components are sterilized either by heat (20 min at 1, 5 bar and 121 0 C) or by sterile filtration. The components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or added randomly or batchwise, as desired.
Die Temperatur der Kultur liegt normalerweise zwischen 15°C und 45°C, vorzugsweise bei 25°C bis 400C und kann während des Experimentes konstant gehalten oder verändert werden. Der pH-Wert des Mediums sollte im Bereich von 5 bis 8,5, vorzugsweise um 7,0 liegen. Der pH-Wert für die Anzucht lässt sich während der Anzucht durch Zugabe von basischen Verbindungen wie Natriumhydroxid, Kaliumhydroxid, Ammoniak bzw. Ammoniakwasser oder sauren Verbindungen wie Phosphorsäure oder Schwefelsäure kontrollieren. Zur Kontrolle der Schaumentwicklung können Anti- schaummittel wie z. B. Fettsäurepolyglykolester, eingesetzt werden. Zur Aufrechterhaltung der Stabilität von Plasmiden können dem Medium geeignete selektiv wirkende Stoffe, wie z. B. Antibiotika, hinzugefügt werden. Um aerobe Bedingungen aufrechtzuerhalten, werden Sauerstoff oder Sauerstoffhaltige Gasmischungen, wie z.B. Umgebungsluft, in die Kultur eingetragen. Die Temperatur der Kultur liegt normalerwei- se bei 200C bis 45°C und vorzugsweise bei 25°C bis 400C. Die Kultur wird solange fortgesetzt, bis sich ein Maximum des gewünschten Produktes gebildet hat. Dieses Ziel wird normalerweise innerhalb von 10 Stunden bis 160 Stunden erreicht.The temperature of the culture is usually between 15 ° C and 45 ° C, preferably at 25 ° C to 40 0 C and can be kept constant or changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid. To control the foaming anti-foaming agents such. As fatty acid polyglycol, are used. To maintain the stability of plasmids, the medium can be selected selectively acting substances such. As antibiotics, are added. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as ambient air, are introduced into the culture. The temperature of the culture is nor- mally at 20 0 C to 45 ° C and preferably 25 ° C to 40 0 C. The culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.
Die so erhaltenen, insbesondere mehrfach ungesättigte Fettsäuren enthaltenden, Fermentationsbrühen haben üblicherweise eine Trockenmasse von 7,5 bis 25 Gew.-%.The fermentation broths thus obtained, in particular containing polyunsaturated fatty acids, usually have a dry matter content of 7.5 to 25% by weight.
Die Fermentationsbrühe kann anschließend weiterverarbeitet werden. Je nach Anforderung kann die Biomasse ganz oder teilweise durch Separationsmethoden, wie z. B. Zentrifugation, Filtration, Dekantieren oder einer Kombination dieser Methoden aus der Fermentationsbrühe entfernt oder vollständig in ihr belassen werden. Vorteilhaft wird die Biomasse nach Abtrennung aufgearbeitet.The fermentation broth can then be further processed. Depending on the requirement, the biomass can be wholly or partly by separation methods, such. As centrifugation, filtration, decantation or a combination of these methods are removed from the fermentation broth or completely left in it. Advantageously, the biomass is worked up after separation.
Die Fermentationsbrühe kann aber auch ohne Zellabtrennung mit bekannten Methoden, wie z. B. mit Hilfe eines Rotationsverdampfers, Dünnschichtverdampfers, Fallfilmverdampfers, durch Umkehrosmose, oder durch Nanofiltration, eingedickt
beziehungsweise aufkonzentriert werden. Diese aufkonzentrierte Fermentationsbrühe kann schließlich zur Gewinnung der darin enthaltenen Fettsäuren aufgearbeitet werden.The fermentation broth can also without cell separation with known methods such. B. with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or be concentrated. This concentrated fermentation broth may eventually be worked up to recover the fatty acids contained therein.
Die Polynucleotide bzw. Polypeptide der vorliegenden Erfindung, die am Stoffwechsel von Lipiden und Fettsäuren, PUFA-Cofaktoren und Enzymen oder am Transport lipophiler Verbindungen über Membranen beteiligt sind, werden im erfindungsgemäßen Verfahren zur Modulation der Produktion von PUFAs in transgenen Organismen vorteilhaft in Pflanzen, wie Mais, Weizen, Roggen, Hafer, Triticale, Reis, Gerste, Sojabohne, Erdnuss, Baumwolle, Linum Arten wie Öl- oder Faserlein, Brassica-Arten, wie Raps, Canola und Rübsen, Pfeffer, Sonnenblume, Borretsch, Nachtkerze und Tagetes, Solanacaen-Pflanzen, wie Kartoffel, Tabak, Aubergine und Tomate, Vicia- Arten, Erbse, Maniok, Alfalfa, Buschpflanzen (Kaffee, Kakao, Tee), Salix-Arten, Bäume (Ölpalme, Kokosnuss) und ausdauernden Gräsern und Futterfeldfrüchten, entweder direkt (z.B. wenn die Überexpression oder Optimierung eines Fettsäurebiosynthese- Proteins einen direkten Einfluss auf die Ausbeute, Produktion und/oder Effizienz der Produktion der Fettsäure aus modifizierten Organismen hat) verwendet und/oder können eine indirekt Auswirkung haben, die dennoch zu einer Steigerung der Ausbeute, Produktion und/oder Effizienz der Produktion der PUFAs oder einer Abnahme unerwünschter Verbindungen führt (z.B. wenn die Modulation des Stoffwechsels von Lipiden und Fettsäuren, Cofaktoren und Enzymen zu Veränderungen der Ausbeute, Produktion und/oder Effizienz der Produktion oder der Zusammensetzung der gewünschten Verbindungen innerhalb der Zellen führt, was wiederum die Produktion einer oder mehrerer Fettsäuren beeinflussen kann).The polynucleotides or polypeptides of the present invention, which are involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds via membranes, are advantageously used in plants according to the invention for modulating the production of PUFAs in transgenic organisms Corn, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, linum species such as oil or fiberine, Brassica species such as oilseed rape, canola and turnip rape, pepper, sunflower, borage, evening primrose and Tagetes, Solanacaen - plants, such as potato, tobacco, aubergine and tomato, Vicia species, pea, cassava, alfalfa, bush plants (coffee, cocoa, tea), Salix species, trees (oil palm, coconut) and perennial grasses and forage crops, either directly ( For example, if overexpression or optimization of a fatty acid biosynthesis protein has a direct impact on the yield, production, and / or production efficiency of the F acidic acid from modified organisms) and / or may have an indirect effect which nevertheless results in an increase in the yield, production and / or efficiency of the production of the PUFAs or a decrease in undesired compounds (e.g. if the modulation of the metabolism of lipids and fatty acids, cofactors and enzymes results in changes in the yield, production and / or efficiency of production or composition of the desired compounds within the cells, which in turn may affect the production of one or more fatty acids).
Die Kombination verschiedener Vorläufermoleküle und Biosyntheseenzyme führt zur Herstellung verschiedener Fettsäuremoleküle, was eine entscheidende Auswirkung auf die Zusammensetzung der Lipide hat. Da mehrfach ungesättigte Fettsäuren (= PUFAs) nicht nur einfach in Triacylglycerin sondern auch in Membranlipide eingebaut werden.The combination of different precursor molecules and biosynthetic enzymes leads to the production of various fatty acid molecules, which has a decisive effect on the composition of the lipids. Since polyunsaturated fatty acids (= PUFAs) are not only simply incorporated in triacylglycerol but also in membrane lipids.
Besonders zur Herstellung von PUFAs, beispielsweise Stearidonsäure, Eicosapen- taensäure und Docosahexaensäure eignen sich Brasicaceae, Boraginaceen, Primula- ceen, oder Linaceen. Besonders vorteilhaft eignet sich Lein (Linum usitatissimum) zur Herstellung von PUFAS mit dem erfindungsgemäßen Nukleinsäuresequenzen vorteilhaft, wie beschrieben, in Kombination mit weiteren Desaturasen und Elongasen.Particularly suitable for the production of PUFAs, for example stearidonic acid, eicosapentaenoic acid and docosahexaenoic acid, are Brasicaceae, boraginaceous plants, primulas or linaceae. Particularly advantageous is Lein (Linum usitatissimum) for the production of PUFAS with the nucleic acid sequences of the invention advantageously, as described, in combination with other desaturases and elongases.
Die Lipidsynthese lässt sich in zwei Abschnitte unterteilen: die Synthese von Fettsäuren und ihre Bindung an sn-Glycerin-3-Phosphat sowie die Addition oder Modifikation einer polaren Kopfgruppe. Übliche Lipide, die in Membranen verwendet werden, umfassen Phospholipide, Glycolipide, Sphingolipide und Phosphoglyceride. Die
OOThe lipid synthesis can be divided into two sections: the synthesis of fatty acids and their attachment to sn-glycerol-3-phosphate and the addition or modification of a polar head group. Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and phosphoglycerides. The OO
Fettsäuresynthese beginnt mit der Umwandlung von Acetyl-CoA in Malonyl-CoA durch die Acetyl-CoA-Carboxylase oder in Acetyl-ACP durch die Acetyltransacylase. Nach einer Kondensationsreaktion bilden diese beiden Produktmoleküle zusammen Aceto- acetyl-ACP, das über eine Reihe von Kondensations-, Reduktions- und Dehydratisie- rungsreaktionen umgewandelt wird, so dass ein gesättigtes Fettsäuremolekül mit der gewünschten Kettenlänge erhalten wird. Die Produktion der ungesättigten Fettsäuren aus diesen Molekülen wird durch spezifische Desaturasen katalysiert, und zwar entweder aerob mittels molekularem Sauerstoff oder anaerob (bezüglich der Fettsäuresynthese in Mikroorganismen siehe F. C. Neidhardt et al. (1996) E. coli und Salmonella. ASM Press: Washington, D. C, S. 612-636 und darin enthaltene Literaturstellen; Lengeier et al. (Hrsgb.) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, und die enthaltene Literaturstellen, sowie Magnuson, K., et al. (1993) Microbiolo- gical Reviews 57:522-542 und die enthaltenen Literaturstellen). Die so hergestellten an Phospholipide gebundenen Fettsäuren müssen anschließend wieder für die weitere Elongationen aus den Phospholipiden in den FettsäureCoA-Ester-Pool überführt werden. Dies ermöglichen Acyl-CoA^ysophospholipid-Acyltransferasen. Weiterhin können diese Enzyme die elongierten Fettsäuren wieder von den CoA-Estern auf die Phospholipide übertragen. Diese Reaktionsabfolge kann gegebenenfalls mehrfach durchlaufen werden.Fatty acid synthesis begins with the conversion of acetyl-CoA into malonyl-CoA by the acetyl-CoA carboxylase or into acetyl-ACP by the acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydration reactions, so that a saturated fatty acid molecule with the desired chain length is obtained. The production of unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically by molecular oxygen or anaerobically (for fatty acid synthesis in microorganisms see FC Neidhardt et al., (1996) E. coli and Salmonella ASM Press: Washington, D Lenienier et al., Eds., (1999) Biology of Procaryotes, Thieme: Stuttgart, New York, and the references contained therein, and Magnuson, K., et al. (1985) C, pp. 612-636 and references therein; 1993) Microbiological Reviews 57: 522-542 and the references contained therein). The fatty acids thus bound to phospholipids must then be converted again for the further elongations from the phospholipids into the fatty acid CoA ester pool. This is facilitated by acyl-CoA3sophospholipid acyltransferases. Furthermore, these enzymes can transfer the elongated fatty acids again from the CoA esters to the phospholipids. This reaction sequence can optionally be run through several times.
Vorläufer für die PUFA-Biosynthese sind beispielsweise Ölsäure, Linol- und Linolensäure. Diese Ciβ-Kohlenstoff-Fettsäuren müssen auf C2o und C22 verlängert werden, damit Fettsäuren vom Eicosa- und Docosa-Kettentyp erhalten werden. Mithilfe der im Verfahren verwendeten Desaturasen wie der Δ-12-, Δ-4-, Δ-5- und Δ-6-Desatu rasen und/oder der Δ-5-, Δ-6-Elongasen können Arachidonsäure, Eicosapentaensäure, Docosapentaensäure oder Docosahexaensäure vorteilhaft Eicosapentaensäure und/oder Docosahexaensäure hergestellt werden und anschließend für verschiedene Zwecke bei Nahrungsmittel-, Futter-, Kosmetik- oder pharmazeutischen Anwendungen verwendet werden. Mit den genannten Enzymen können C2o- und/oder C22-Fettsäuren mit mindestens zwei vorteilhaft mindestens drei, vier, fünf oder sechs Doppelbindungen im Fettsäuremolekül, vorzugsweise C20- oder C22-Fettsäuren mit vorteilhaft vier, fünf oder sechs Doppelbindungen im Fettsäuremolekül hergestellt werden. Die Desaturie- rung kann vor oder nach Elongation der entsprechenden Fettsäure erfolgen. Daher führen die Produkte der Desaturaseaktivitäten und der möglichen weiteren Desaturie- rung und Elongation zu bevorzugten PUFAs mit höherem Desaturierungsgrad, einschließlich einer weiteren Elongation von C20 zu C22-Fettsäuren,zu Fettsäuren wie v- Linolensäure, Dihomo-γ-linolensäure, Arachidonsäure, Stearidonsäure, Eicosatetraen- säure oder Eicosapentaensäure. Substrate der verwendeten Desaturasen und Elongasen im erfindungsgemäßen Verfahren sind C16-, C18- oder C2o-Fettsäuren wie zum Beispiel Linolsäure, γ-Linolensäure, α-Linolensäure, Dihomo-γ-linolensäure,
Eicosatetraensäure oder Stearidonsäure. Bevorzugte Substrate sind Linolsäure, v- Linolensäure und/oder α-Linolensäure, Dihomo-γ-linolensäure bzw. Arachidonsäure, Eicosatetraensäure oder Eicosapentaensäure. Die synthetisierten C2o- oder C22- Fettsäuren mit mindestens zwei, drei, vier, fünf oder sechs Doppelbindungen in der Fettsäure fallen im erfindungsgemäßen Verfahren in Form der freien Fettsäure oder in Form ihrer Ester beispielsweise in Form ihrer Glyceride an.Precursors for the PUFA biosynthesis are, for example, oleic acid, linoleic acid and linolenic acid. These Ciβ-carbon fatty acids must be extended to C 2 o and C 22 in order to obtain fatty acids of the eicosa- and docosa-chain type. By means of the desaturases used in the process, such as the Δ-12, Δ-4, Δ-5 and Δ-6 desaturases and / or the Δ-5, Δ-6 elongases, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or Docosahexaensäure advantageously eicosapentaenoic acid and / or docosahexaenoic acid are prepared and then used for various purposes in food, feed, cosmetic or pharmaceutical applications. C 2 - and / or C 22 -fatty acids having at least two, preferably at least three, four, five or six double bonds in the fatty acid molecule, preferably C 2 0 or C 22 -fatty acids with advantageously four, five or six double bonds, can be used with the abovementioned enzymes be prepared in the fatty acid molecule. The desaturation can take place before or after elongation of the corresponding fatty acid. Thus, the products of desaturase activities and possible further desaturation and elongation result in preferred PUFAs having a higher degree of desaturation, including a further elongation of C 20 to C 22 fatty acids, to fatty acids such as α-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, Stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. Substrates of the desaturases and elongases used in the process of this invention are C 1 6-, C linolenic dihomo-γ-1 8- or C2o fatty acids such as linoleic acid, γ-linolenic acid, α-linolenic acid, Eicosatetraenoic acid or stearidonic acid. Preferred substrates are linoleic acid, v-linolenic acid and / or α-linolenic acid, dihomo-γ-linolenic acid or arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. The synthesized C 2 o- or C 22 -fatty acids with at least two, three, four, five or six double bonds in the fatty acid are obtained in the process according to the invention in the form of the free fatty acid or in the form of their esters, for example in the form of their glycerides.
Unter dem Begriff "Glycerid" wird ein mit ein, zwei oder drei Carbonsäureresten verestertes Glycerin verstanden (Mono-, Di- oder Triglycerid). Unter "Glycerid" wird auch ein Gemisch an verschiedenen Glyceriden verstanden. Das Glycerid oder das Glyceridgemisch kann weitere Zusätze, z.B. freie Fettsäuren, Antioxidantien, Proteine, Kohlenhydrate, Vitamine und/oder andere Substanzen enthalten.The term "glyceride" is understood to mean a glycerol esterified with one, two or three carboxylic acid residues (mono-, di- or triglyceride). By "glyceride" is also meant a mixture of different glycerides. The glyceride or glyceride mixture may contain other additives, e.g. contain free fatty acids, antioxidants, proteins, carbohydrates, vitamins and / or other substances.
Unter einem "Glycerid" im Sinne des erfindungsgemäßen Verfahrens werden ferner vom Glycerin abgeleitete Derivate verstanden. Dazu zählen neben den oben beschriebenen Fettsäureglyceriden auch Glycerophospholipide und Glyceroglycolipide. Bevorzugt seien hier die Glycerophospholipide wie Lecithin (Phosphatidylcholin), Cardiolipin, Phosphatidylglycerin, Phosphatidylserin und Alkylacylglycerophospholipide beispielhaft genannt.A "glyceride" in the sense of the method according to the invention is also understood to mean derivatives derived from glycerol. In addition to the fatty acid glycerides described above, these also include glycerophospholipids and glyceroglycolipids. The glycerophospholipids, such as lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and alkylacylglycerophospholipids, may be mentioned by way of example here.
Ferner müssen Fettsäuren anschließend an verschiedene Modifikationsorte transportiert und in das Triacylglycerin-Speicherlipid eingebaut werden. Ein weiterer wichtiger Schritt bei der Lipidsynthese ist der Transfer von Fettsäuren auf die polaren Kopfgruppen, beispielsweise durch Glycerin-Fettsäure-Acyltransferase (siehe Frentzen, 1998, Lipid, 100(4-5):161-166).Furthermore, fatty acids must then be transported to various modification sites and incorporated into the triacylglycerol storage lipid. Another important step in lipid synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol-fatty acid acyltransferase (see Frentzen, 1998, Lipid, 100 (4-5): 161-166).
Veröffentlichungen über die Pflanzen-Fettsäurebiosynthese, Desaturierung, den Lipidstoffwechsel und Membrantransport von fetthaltigen Verbindungen, die Betaoxida- tion, Fettsäuremodifikation und Cofaktoren, Triacylglycerin-Speicherung und - Assemblierung einschließlich der Literaturstellen darin siehe in den folgenden Artikeln: Kinney, 1997, Genetic Engeneering, Hrsgb.: JK Setlow, 19:149-166; Ohlrogge und Browse, 1995, Plant Cell 7:957-970; Shanklin und Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:61 1-641 ; Voelker, 1996, Genetic Engeneering, Hrsgb.: JK Setlow, 18:11 1-13; Gerhardt, 1992, Prog. Lipid R. 31 :397-417; Gühnemann-Schäfer & Kindl, 1995, Biochim. Biophys Acta 1256:181-186; Kunau et al., 1995, Prog. Lipid Res. 34:267-342; Stymne et al., 1993, in: Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants, Hrsgb.: Murata und Somerville, Rockville, American Society of Plant Physiologists, 150-158, Murphy & Ross 1998, Plant Journal. 13(1 ):1- 16.
Die im Verfahren hergestellten PUFAs, umfassen eine Gruppe von Molekülen, die höhere Tiere nicht mehr synthetisieren können und somit aufnehmen müssen oder die höhere Tiere nicht mehr ausreichend selbst herstellen können und somit zusätzlich aufnehmen müssen, obwohl sie leicht von anderen Organismen, wie Bakterien, synthetisiert werden, beispielsweise können Katzen Arachidonsäure nicht mehr synthetisieren.For publications on plant fatty acid biosynthesis, desaturation, lipid metabolism and membrane transport of fatty compounds, beta oxidation, fatty acid modification and cofactors, triacylglycerol storage and assembly, including references therein, see the following articles: Kinney, 1997, Genetic Engineering, eds .: JK Setlow, 19: 149-166; Ohlrogge and Browse, 1995, Plant Cell 7: 957-970; Shanklin and Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 61-641; Voelker, 1996, Genetic Engineering, eds. JK Setlow, 18:11 1-13; Gerhardt, 1992, Prog. Lipid R. 31: 397-417; Gühnemann-Schäfer & Kindl, 1995, Biochim. Biophys Acta 1256: 181-186; Kunau et al., 1995, Prog. Lipid Res. 34: 267-342; Stymne et al., 1993, in: Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants, eds .: Murata and Somerville, Rockville, American Society of Plant Physiologists, 150-158, Murphy & Ross 1998, Plant Journal. 13 (1): 1-16. The PUFAs produced in the process comprise a group of molecules that are no longer able to synthesize, and therefore need to take up, higher animals, or that can no longer sufficiently produce higher animals themselves, and thus have to additionally take up, even though they are readily synthesized by other organisms, such as bacteria For example, cats can no longer synthesize arachidonic acid.
Unter Phospholipiden im Sinne der Erfindung sind zu verstehen Phosphatidylcholin, Phosphatidylethanolamin, Phosphatidylserin, Phosphatidylglycerin und/oder Phospha- tidylinositol vorteilhafterweise Phosphatidylcholin. Die Begriffe Produktion oder Produktivität sind im Fachgebiet bekannt und beinhalten die Konzentration des Fermentationsproduktes (Verbindungen der Formel I), das in einer bestimmten Zeitspanne und einem bestimmten Fermentationsvolumen gebildet wird (z.B. kg Produkt pro Stunde pro Liter). Es umfasst auch die Produktivität innerhalb einer Pflanzenzelle oder einer Pflanze, das heißt den Gehalt an den gewünschten im Verfahren hergestellten Fettsäuren bezogen auf den Gehalt an allen Fettsäuren in dieser Zelle oder Pflanze. Der Begriff Effizienz der Produktion umfasst die Zeit, die zur Erzielung einer bestimmten Produktionsmenge nötig ist (z.B. wie lange die Zelle zur Aufrichtung einer bestimmten Durchsatzrate einer Feinchemikalie benötigt). Der Begriff Ausbeute oder Produkt/Kohlenstoff-Ausbeute ist im Fachgebiet bekannt und umfasst die Effizienz der Umwandlung der Kohlenstoffquelle in das Produkt (d.h. die Feinchemikalie). Dies wird gewöhnlich beispielsweise ausgedrückt als kg Produkt pro kg Kohlenstoffquelle. Durch Erhöhen der Ausbeute oder Produktion der Verbindung wird die Menge der gewonnenen Moleküle oder der geeigneten gewonnenen Moleküle dieser Verbindung in einer bestimmten Kulturmenge über einen festgelegten Zeitraum erhöht. Die Begriffe Biosynthese oder Biosyntheseweg sind im Fachgebiet bekannt und umfassen die Synthese einer Verbindung, vorzugsweise einer organischen Verbindung, durch eine Zelle aus Zwischenverbindungen, beispielsweise in einem Mehrschritt- und stark regulierten Prozess. Die Begriffe Abbau oder Abbauweg sind im Fachgebiet bekannt und umfassen die Spaltung einer Verbindung, vorzugsweise einer organischen Verbindung, durch eine Zelle in Abbauprodukte (allgemeiner gesagt, kleinere oder weniger komplexe Moleküle) beispielsweise in einem Mehrschritt- und stark regulierten Prozess. Der Begriff Stoffwechsel ist im Fachgebiet bekannt und umfasst die Gesamtheit der biochemischen Reaktionen, die in einem Organismus stattfinden. Der Stoffwechsel einer bestimmten Verbindung (z.B. der Stoffwechsel einer Fettsäure) umfasst dann die Gesamtheit der Biosynthese-, Modifikations- und Abbauwege dieser Verbindung in der Zelle, die diese Verbindung betreffen.In the context of the invention, phospholipids are to be understood as meaning phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and / or phosphatidylinositol, advantageously phosphatidylcholine. The terms production or productivity are known in the art and include the concentration of the fermentation product (compounds of formula I) formed in a given period of time and fermentation volume (e.g., kg of product per hour per liter). It also includes productivity within a plant cell or plant, that is, the content of the desired fatty acids produced in the process based on the content of all fatty acids in that cell or plant. The term efficiency of production includes the time required to achieve a certain amount of production (e.g., how long the cell takes to establish a given throughput rate of a fine chemical). The term yield or product / carbon yield is known in the art and includes the efficiency of converting the carbon source into the product (i.e., the fine chemical). This is usually expressed, for example, as kg of product per kg of carbon source. By increasing the yield or production of the compound, the amount of the recovered molecules or molecules of this compound obtained in a given amount of culture is increased over a fixed period of time. The terms biosynthesis or biosynthetic pathway are known in the art and involve the synthesis of a compound, preferably an organic compound, by a cell from intermediates, for example in a multi-step and highly regulated process. The terms degradation or degradation pathway are well known in the art and involve the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), for example in a multi-step and highly regulated process. The term metabolism is known in the art and includes the entirety of the biochemical reactions that take place in an organism. The metabolism of a particular compound (e.g., the metabolism of a fatty acid) then comprises all of the biosynthetic, modification, and degradation pathways of that compound in the cell that affect that compound.
Durch den Einsatz der erfindungsgemäßen Polynucleotide und optional weiterer Polynucleotide, die für Enzyme des Lipid oder Fettsäurestoffwechsels kodieren, in dem
erfindungsgemäßen Verfahren können verschiedene vorteilhafte Effekte erzielt werden. So kann die Ausbeute, Produktion und/oder Effizienz der Produktion der mehrfach ungesättigten Fettsäuren in einer Pflanze, bevorzugt in einer Ölfruchtpflanze, oder einem Mikroorganismus beeinflusst werden kann. Die Anzahl oder Aktivität der erfindungsgemäßen Polypeptide bzw. Polynucleotide kann erhöht werden, so dass größere Mengen der Genprodukte und damit letztlich größere Mengen der Verbindungen der allgemeinen Formel I hergestellt werden. Auch eine de novo Synthese in einem Organismus, dem die Aktivität und Fähigkeit zur Biosynthese der Verbindungen vor dem Einbringen des/der entsprechenden Gens/Gene fehlte, ist möglich. Entspre- chendes gilt für die Kombination mit weiteren Desaturasen oder Elongasen oder weiteren Enzymen aus dem Fettsäure- und Lipidstoffwechsel. Auch die Verwendung verschiedener divergenter, d.h. auf DNA-Sequenzebene unterschiedlicher Sequenzen kann dabei vorteilhaft sein bzw. die Verwendung von Promotoren zur Genexpression, die eine andere zeitliche Genexpression z.B. abhängig vom Reifegrad eines Samens oder Öl-speichernden Gewebes ermöglicht.By using the polynucleotides according to the invention and optionally further polynucleotides which code for enzymes of the lipid or fatty acid metabolism in which According to the invention, various advantageous effects can be achieved. Thus, the yield, production and / or efficiency of the production of the polyunsaturated fatty acids in a plant, preferably in an oil crop, or a microorganism can be influenced. The number or activity of the polypeptides or polynucleotides according to the invention can be increased, so that larger amounts of the gene products and thus ultimately larger amounts of the compounds of general formula I are produced. Also, a de novo synthesis in an organism lacking the activity and ability to biosynthesize the compounds before introducing the gene (s) of interest is possible. The same applies to the combination with other desaturases or elongases or other enzymes from the fatty acid and lipid metabolism. The use of different divergent, ie different sequences on DNA sequence level may be advantageous or the use of promoters for gene expression, which allows a different time gene expression, for example, depending on the degree of ripeness of a seed or oil-storing tissue.
Durch das Einbringen eines erfindungsgemäßen Polynucleotids in einen Organismus allein oder in Kombination mit anderen Genen in eine Zelle kann nicht nur den Biosynthesefluss zum Endprodukt erhöht, sondern auch die entsprechende Triacylgly- cerin- Zusammensetzung erhöht oder de novo geschaffen werden. Ebenso kann die Anzahl oder Aktivität anderer Gene, die am Import von Nährstoffen, die zur Biosynthese einer oder mehrerer Fettsäuren, Ölen, polaren und/oder neutralen Lipiden nötig sind, erhöht sein, so dass die Konzentration dieser Vorläufer, Cofaktoren oder Zwischenverbindungen innerhalb der Zellen oder innerhalb des Speicherkompartiments erhöht ist, wodurch die Fähigkeit der Zellen zur Produktion von PUFAs weiter gesteigert wird. Durch Optimierung der Aktivität oder Erhöhung der Anzahl einer oder mehrerer erfindungsgemäßer Polynucleotide bzw. Polypeptide, die an der Biosynthese dieser Verbindungen beteiligt sind, oder durch Zerstören der Aktivität einer oder mehrerer Gene, die am Abbau dieser Verbindungen beteiligt sind, kann es möglich sein, die Ausbeute, Produktion und/oder Effizienz der Produktion von Fettsäure- und Lipidmolekülen aus Organismen insbesondere aus Pflanzen zu steigern. Die im Verfahren gewonnenen Fettsäuren eignen sich als Ausgangsmaterial für die chemische Synthese von weiteren Wertprodukten. Sie können beispielsweise in Kombination miteinander oder allein zur Herstellung von Pharmaka, Nahrungsmittel, Tierfutter oder Kosmetika verwendet werden.By introducing a polynucleotide according to the invention into an organism alone or in combination with other genes into a cell, not only can the biosynthesis flux to the end product be increased, but also the corresponding triacylglycerol composition can be increased or created de novo. Likewise, the number or activity of other genes necessary for the import of nutrients necessary for the biosynthesis of one or more fatty acids, oils, polar and / or neutral lipids may be increased, such that the concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment, thereby further increasing the ability of the cells to produce PUFAs. By optimizing the activity or increasing the number of one or more polynucleotides or polypeptides of the invention involved in the biosynthesis of these compounds, or by disrupting the activity of one or more genes involved in the degradation of these compounds, it may be possible to use the To increase the yield, production and / or efficiency of the production of fatty acid and lipid molecules from organisms, in particular from plants. The fatty acids obtained in the process are suitable as starting material for the chemical synthesis of other valuable products. They may be used, for example, in combination with each other or solely for the manufacture of pharmaceuticals, foods, animal feed or cosmetics.
Aus den zuvor gemachten Ausführungen versteht sich, dass die Erfindung auch ein Verfahren zur Herstellung einer Öl-, Lipid- oder Fettsäurezusammensetzung umfas-
send die Schritte des erfindungsgemäßen Verfahrens und den weiteren Schritt des Formulierens des Stoffes als Öl-, Lipid- oder Fettsäurezusammensetzung betrifft.It will be understood from the foregoing that the invention also encompasses a process for preparing an oil, lipid or fatty acid composition. send relates to the steps of the method according to the invention and the further step of formulating the substance as an oil, lipid or fatty acid composition.
In einer bevorzugten Ausführungsform dieses Verfahren wird die Öl-, Lipid- oder Fettsäurezusammensetzung weiter formuliert zu einem Arzneimittel, zu Kosmetik, zu einem Nahrungsmittel, zu einem Futtermittel, vorzugsweise Fischfutter, oder zu einem Nahrungsergänzungsmittel.In a preferred embodiment of this method, the oil, lipid or fatty acid composition is further formulated into a medicament, a cosmetic, a food, a feed, preferably a fish feed, or a dietary supplement.
Schließlich betrifft die Erfindung grundsätzlich die Verwendung des Polynucleotids, des Vektors, der Wirtszelle, des Polypeptids oder des transgenen, nicht-humanen Organsismus der vorliegenden Erfindung für die Herstellung einer Öl-, Lipid- oder Fettsäurezusammensetzung. Diese ist dann bevorzugt als Arzneimittel, Kosmetik, Nahrungsmittel, Futtermittel, vorzugsweise Fischfutter, oder Nahrungsergänzungsmittel einzuset- zen.Finally, the invention generally relates to the use of the polynucleotide, the vector, the host cell, the polypeptide or the transgenic, non-human organism of the present invention for the preparation of an oil, lipid or fatty acid composition. This is then preferably used as pharmaceuticals, cosmetics, foods, animal feed, preferably fish feed, or dietary supplements.
Der Inhalt sämtlicher in dieser Patentanmeldung zitierten Literaturstellen, Patentan- meidungen, Patente und veröffentlichten Patentanmeldungen ist hiermit durch Bezugnahme auf den jeweiligen speziellen Offenbarungsgehalt aufgenommen.The contents of all references cited in this patent application, patent applications, patents and published patent applications are hereby incorporated by reference to the respective specific disclosure content.
Figurencharacters
Figur 1 zeigt einen Sequenzvergleich der Δ5- und Δ6-Elongase Aminosäuresequenzen aus O. lucimarinus, O. tauri und T. pseudonana im ClustalW-Vergleich.FIG. 1 shows a sequence comparison of the Δ5 and Δ6 elongase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
Figur 2 zeigt einen Sequenzvergleich der Δ4-Desaturase Aminosäuresequenzen aus O. lucimarinus, O. tauri und T. pseudonana im ClustalW-Vergleich.FIG. 2 shows a sequence comparison of the Δ4-desaturase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
Figur 3 zeigt einen Sequenzvergleich der Δ5-Desaturase Aminosäuresequenzen aus O. lucimarinus, O. tauri und T. pseudonana im ClustalW-Vergleich.FIG. 3 shows a sequence comparison of the Δ5-desaturase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
Figur 4 zeigt einen Sequenzvergleich der Δ6-Desaturase Aminosäuresequenzen aus O. lucimarinus, O. tauri und T. pseudonana im ClustalW-Vergleich.FIG. 4 shows a sequence comparison of the Δ6-desaturase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison.
Figur 5 zeigt einen Sequenzvergleich der Δ12-Desaturase Aminosäuresequenzen aus O. lucimarinus, O. tauri und T. pseudonana im ClustalW-Vergleich.
Figur 6 zeigt die gaschromatographische Bestimmung der Fettsäuren aus Hefen, die mit dem Plasmid pYES (A, B) bzw. pYES-D5Elo(OI) (C) transformiert wurden. Die Fettsäure 20:4Δ5,8,1 1 ,14 wurde zugefüttert (B, C).FIG. 5 shows a sequence comparison of the Δ12-desaturase amino acid sequences from O. lucimarinus, O. tauri and T. pseudonana in the ClustalW comparison. 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, 1, 14 was fed (B, C).
Figur 7 zeigt die gaschromatographische Bestimmung der Fettsäuren aus Hefen, die mit dem Plasmid pYES (A, B, C) bzw. pYES-DΘEIo(OI) (D, E) transformiert wurden. Die Fettsäuren 18:3Δ6,9,12 bzw. 18:4Δ6,9, 12,15 wurde zugefüttert (B, D) bzw. (C, E).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-DΘEIo (OI) (D, E). The fatty acids 18: 3Δ6,9,12 and 18: 4Δ6,9, 12,15 were fed (B, D) and (C, E), respectively.
Figur 8 zeigt die gaschromatographische Bestimmung der Fettsäuren aus Hefen, die mit dem Plasmid pYES (A, B) bzw. pYES-D5Des(OI_2) (C) transformiert wurden. Die Fettsäuren 20:3Δ5,8,1 1 ,14 wurde zugefüttert in (B) und (C).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 acids 20: 3Δ5, 8, 1, 14 were fed into (B) and (C).
Figur 9 zeigt die gaschromatographische Bestimmung der Fettsäuren aus Hefen, die mit dem Plasmid pYES (A) bzw. pYES-D12Des(OI) (B) transformiert wurden.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).
Figur 10 zeigt die gaschromatographische Bestimmung der Fettsäuren aus Hefe. pYes-d5Des(OI_1 ) in Hefe Stamm InvSc ohne Zugabe von Fettsäuren (A); pYes- d5Des(OI_1 ) in Hefe Stamm InvSc nach Zugabe der Fettsäure 20:3n-6 (B), pYes- d5Des(OI_1 ) in Hefe Stamm InvSc nach Zugabe der Fettsäure 20:4n-3 (C).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 fatty acid 20: 3n-6 (B), pYes- d5Des (OI_1) in yeast strain InvSc after addition of fatty acid 20: 4n-3 (C).
Die Erfindung wird durch die nachstehenden Beispiele weiter veranschaulicht, die nicht als beschränkend aufgefasst werden sollten.The invention is further illustrated by the following examples, which should not be construed as limiting.
BeispieleExamples
Beispiel 1 : Allgemeine KlonierungsverfahrenExample 1: General Cloning Methods
Die Klonierungsverfahren wie z.B. Restriktionsspaltungen, Agarose-Gelelektrophorese, Reinigung von DNA-Fragmenten, Transfer von Nukleinsäuren auf Nitrozellulose und Nylon Membranen, Verknüpfen von DNA-Fragmenten, Transformation von Escherichia coli Zellen, Anzucht von Bakterien und die Sequenzanalyse rekombinanter DNA wurden wie bei Sambrook et al. (1989) (CoId Spring Harbor Laboratory Press: ISBN 0- 87969-309-6) beschrieben durchgeführt.The cloning methods, e.g. Restriction cleavage, 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, culture of bacteria and sequence analysis of recombinant DNA were performed as described in Sambrook et al. (1989) (CoId Spring Harbor Laboratory Press: ISBN 0-87969-309-6).
Beispiel 2: Sequenzanalyse rekombinanter DNA
Die Sequenzierung rekombinanter DNA-Moleküle erfolgte mit einem Laserfluoreszenz- DNA-Sequenzierer der Firma ABI nach der Methode von Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sei. USA74, 5463-5467). Fragmente resultierend aus einer Polymerase Kettenreaktion wurden zur Vermeidung von Polymerasefehlern in zu exprimieren- den Konstrukten sequenziert und überprüft.Example 2 Sequence Analysis of Recombinant DNA The sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad. See, USA74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed.
Beispiel 3: Lipidextraktion aus HefenExample 3 Lipid Extraction from Yeasts
Die Auswirkung der genetischen Modifikation in Pflanzen, Pilzen, Algen, Ciliaten oder auf die Produktion einer gewünschten Verbindung (wie einer Fettsäure) kann bestimmt werden, indem die modifizierten Mikroorganismen oder die modifizierte Pflanze unter geeigneten Bedingungen (wie den vorstehend beschriebenen) gezüchtet werden und das Medium und/oder die zellulären Komponenten auf die erhöhte Produktion des gewünschten Produktes (d.h. von Lipiden oder einer Fettsäure) untersucht wird. Diese Analysetechniken sind dem Fachmann bekannt und umfassen Spektroskopie, Dünnschichtchromatographie, Färbeverfahren verschiedener Art, enzymatische und mikrobiologische Verfahren sowie analytische Chromatographie, wie Hochleistungs- Flüssigkeitschromatographie (siehe beispielsweise Ullman, Encyclopedia of Industrial Chemistry, Bd. A2, S. 89-90 und S. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,The effect of genetic modification in plants, fungi, algae, ciliates or on the production of a desired compound (such as a fatty acid) may be determined by cultivating the modified microorganism or modified plant under suitable conditions (such as those described above), and Medium and / or the cellular components on the increased production of the desired product (ie of lipids or a fatty acid) is examined. These analytical techniques are well known to those skilled in the art and include spectroscopy, thin layer chromatography, staining methods of various types, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.
(1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Bioche- mistry and Molecular Biology, Bd. 17; Rehm et al. (1993) Biotechnology, Bd. 3, Kapitel III: "Product recovery and purification", S. 469-714, VCH: Weinheim; Belter, P.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", pp. 469-714, VCH: Weinheim; Belter, P.A., et al.
(1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J. F., und Cabral, J. M. S. (1992) Recovery processes for biological(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., und Henry, J. D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Bd. B3; Kapitel 1 1 , S. 1-27, VCH: Weinheim; und Dechow, FJ. (1989) Separation and purification techniques in biotechnology, Noyes Publications).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 1 1, pp. 1-27, VCH: Weinheim; and Dechow, FJ. (1989) Separation and purification techniques in biotechnology, Noyes Publications).
Neben den oben erwähnten Verfahren werden Pflanzenlipide aus Pflanzenmaterial wie von Cahoon et al. (1999) Proc. Natl. Acad. Sei. USA 96 (22): 12935-12940, und Browse et al. (1986) Analytic Biochemistry 152:141-145, beschrieben extrahiert. Die qualitative und quantitative Lipid- oder Fettsäureanalyse ist beschrieben bei Christie, William W., Advances in Lipid Methodology, Ayr/Scotland: OiIy Press (OiIy Press Lipid Library; 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland: OiIy Press, 1989, Repr. 1992, IX, 307 S. (OiIy Press Lipid Library; 1 ); "Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) - 16 (1977) u.d.T.: Progress in the Chemistry of Fats and Other Lipids CODEN.
Zusätzlich zur Messung des Endproduktes der Fermentation ist es auch möglich, andere Komponenten der Stoffwechselwege zu analysieren, die zur Produktion der gewünschten Verbindung verwendet werden, wie Zwischen- und Nebenprodukte, um die Gesamteffizienz der Produktion der Verbindung zu bestimmen. Die Analyse- verfahren umfassen Messungen der Nährstoffmengen im Medium (z.B. Zucker, Kohlenwasserstoffe, Stickstoffquellen, Phosphat und andere Ionen), Messungen der Biomassezusammensetzung und des Wachstums, Analyse der Produktion üblicher Metabolite von Biosynthesewegen und Messungen von Gasen, die während der Fermentation erzeugt werden. Standardverfahren für diese Messungen sind in Applied Microbial Physiology; A Practical Approach, P.M. Rhodes und P. F. Stanbury, Hrsgb., IRL Press, S. 103-129; 131-163 und 165-192 (ISBN: 0199635773) und darin angegebenen Literaturstellen beschrieben.In addition to the above-mentioned methods, plant lipids derived from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Be. USA 96 (22): 12935-12940, and Browse et al. (1986) Analytic Biochemistry 152: 141-145. Qualitative and quantitative lipid or fatty acid analysis is described in Christie, William W., Advances in Lipid Methodology, Ayr / Scotland: OiIy Press (Oli Press Lipid Library, 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland: OiIy Press, 1989, Repr. 1992, IX, 307 p. (OiIy Press Lipid Library, 1); "Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) - 16 (1977) udT: Progress in the Chemistry of Fats and Other Lipids CODES. In addition to measuring the end product of the fermentation, it is also possible to analyze other components of the metabolic pathways used to produce the desired compound, such as by-products and by-products, to determine the overall efficiency of production of the compound. Analytical methods include measurements of nutrient levels in the medium (eg, sugars, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of biomass composition and growth, analysis of production of common biosynthetic pathway metabolites, and measurements of gases produced during fermentation. Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, PM Rhodes and PF Stanbury, Eds., IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and references cited therein.
Ein Beispiel ist die Analyse von Fettsäuren (Abkürzungen: FAME, Fettsäuremethyl- ester; GC-MS, Gas-Flüssigkeitschromatographie-Massenspektrometrie; TAG, Tria- cylglycerin; TLC, Dünnschichtchromatographie).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).
Der unzweideutige Nachweis für das Vorliegen von Fettsäureprodukten kann mittels Analyse rekombinanter Organismen nach Standard-Analyseverfahren erhalten werden: GC, GC-MS oder TLC, wie verschiedentlich beschrieben von Christie und den Literaturstellen darin (1997, in: Advances on Lipid Methodology, Vierte Aufl.: Christie, OiIy Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie- Verfahren, Lipide 33:343-353).The unambiguous evidence for the presence of fatty acid products can be obtained by analysis of recombinant organisms by standard analytical methods: GC, GC-MS or TLC as variously described by Christie and the references therein (1997, in: Advances on Lipid Methodology, Fourth Edition. Christie, Oliver Press, Dundee, 119-169, 1998, Gas Chromatography Mass Spectrometry Method, Lipids 33: 343-353).
Das zu analysierende Material kann durch Ultraschallbehandlung, Mahlen in der Glasmühle, flüssigen Stickstoff und Mahlen oder über andere anwendbare Verfahren aufgebrochen werden. Das Material muss nach dem Aufbrechen zentrifugiert werden. Das Sediment wird in Aqua dest. resuspendiert, 10 min bei 1000C erhitzt, auf Eis abgekühlt und erneut zentrifugiert, gefolgt von Extraktion in 0,5 M Schwefelsäure in Methanol mit 2 % Dimethoxypropan für 1 Std. bei 900C, was zu hydrolysierten Öl- und Lipidverbindungen führt, die transmethylierte Lipide ergeben. Diese Fettsäuremethylester werden in Petrolether extrahiert und schließlich einer GC-Analyse unter Verwendung einer Kapillarsäule (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 mikrom, 0,32 mm) bei einem Temperaturgradienten zwischen 170°C und 2400C für 20 min und 5 min bei 2400C unterworfen. Die Identität der erhaltenen Fettsäuremethylester muss unter Verwendung von Standards, die aus kommerziellen Quellen erhältlich sind (d.h. Sigma), definiert werden.
Beispiel 4: Klonierung und Charakterisierung von Elongase-Genen aus Ostreococcus lucimarinusThe material to be analyzed may be broken up by sonication, milling in the glass mill, liquid nitrogen and milling or other applicable methods. The material must be centrifuged after rupture. The sediment is distilled in aqua. re-suspended, heated at 100 ° C. for 10 minutes, cooled on ice and recentrifuged, followed by extraction into 0.5 M sulfuric acid in methanol with 2% dimethoxypropane for 1 hour at 90 ° C., resulting in hydrolyzed oil and lipid compounds, which give transmethylated lipids. These fatty acid methyl ester are extracted in petroleum ether and finally subjected to GC analysis (mikrom Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25, 0.32 mm) using a capillary column with a temperature gradient between 170 ° C and 240 0 C for 20 min and 5 min at 240 0 C subjected. The identity of the resulting fatty acid methyl esters must be defined using standards available from commercial sources (ie Sigma). Example 4 Cloning and Characterization of Elongase Genes from Ostreococcus lucimarinus
Durch Suche nach konservierten Bereichen in den Proteinsequenzen in Elongase- Genen konnten zwei Sequenzen mit entsprechenden Motiven in einer Ostreococcus lucimarinus Sequenzdatenbank identifiziert werden. In einem weiteren Schritt wurden die Gene mittels Sequenzvergleich, Genvorhersage und der Suche nach kodierenden Bereichen charakterisiert. Folgende kodierende Bereiche wurden gefunden:By searching for conserved regions in the protein sequences in elongase genes, two sequences with corresponding motifs could be identified in an Ostreococcus lucimarinus sequence database. In a further step, the genes were characterized by sequence comparison, gene prediction and the search for coding regions. The following coding areas were found:
Tabelle 1 : Kodierende BereicheTable 1: Coding ranges
Figur 1 zeigt die Sequenzähnlichkeiten zu anderen Algen (Ostreococcus tauri, Thalassiosira pseudonana) für die verschiedenen Elongase-Aminosäure Sequenzen im ClustalW Sequenzvergleich. Überraschenderweise unterscheiden sich die Sequenzen von O. lucimarinus in ihrer Aminosäuresequenz deutlich von den anderen Algen.FIG. 1 shows the sequence similarities to other algae (Ostreococcus tauri, Thalassiosira pseudonana) for the different elongase amino acid sequences in the ClustalW sequence comparison. Surprisingly, the sequences of O. lucimarinus differ significantly in their amino acid sequence from the other algae.
Tabelle 2: Sequenzidentitäten einzelner ElongasenTable 2: Sequence identities of individual elongases
Die Klonierung wurde wie folgt durchgeführt:The cloning was carried out as follows:
40 ml einer Ostreococcus lucimarinus Kultur in der stationären Phase wurden abzentri- fugiert und in 100 μl Aqua bidest resuspendiert und bei -200C gelagert. Auf der Basis des PCR-Verfahren wurden die zugehörigen genomischen DNAs amplifiziert. Die entsprechenden Primerpaare wurden so ausgewählt, dass sie die Hefe-Konsensus- Sequenz für hocheffiziente Translation (Kozak, Cell 1986, 44:283-292) neben dem Startcodon trugen. Die Amplifizierung der DNAs wurde jeweils mit 1 μl aufgetauten Zellen, 200 μM dNTPs, 2,5 U Tag-Polymerase und 100 pmol eines jeden Primers in einem Gesamtvolumen von 50 μl durchgeführt. Die Bedingungen für die PCR waren wie folgt: Erste Denaturierung bei 95°C für 5 Minuten, gefolgt von 30 Zyklen bei 94°C für 30 Sekunden, 55°C für 1 Minute und 72°C für 2 Minuten sowie ein letzter Verlängerungsschritt bei 72°C für 10 Minuten.
Zur Charakterisierung der Funktion der Elongasen aus Ostreococcus lucimarinus werden die offenen Leserahmen der jeweiligen DNAs stromabwärts des Galactose- induzierbaren GAL1 -Promotors von pYES2.1A/5-His-TOPO (Invitrogen) kloniert, wobei pOLE1 und pOLE2 erhalten werden.40 ml of a Ostreococcus lucimarinus culture in the stationary phase were fugal abzentri- and resuspended in 100 .mu.l double-distilled water and stored at -20 0 C. Based on the PCR method, the associated genomic DNAs were amplified. The corresponding primer pairs were selected to carry the yeast consensus high-efficiency translation sequence (Kozak, Cell 1986, 44: 283-292) adjacent to the start codon. The amplification of the DNAs was carried out in each case with 1 μl of thawed cells, 200 μM dNTPs, 2.5 U of tag 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 extension step at 72 ° C for 10 minutes. To characterize the function of the elongases from Ostreococcus lucimarinus, the open reading frames of the respective DNAs are cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1A / 5-His-TOPO (Invitrogen) to yield pOLE1 and pOLE2.
Der Saccharomyces cerev/s/ae-Stamm 334 wird durch Elektroporation (1500 V) mit dem Vektor pOLE1 bzw. pOLE2 transformiert. Als Kontrolle wird eine Hefe verwendet, die mit dem leeren Vektor pYES2 transformiert wird. Die Selektion der transformierten Hefen erfolgt auf Komplett-Minimalmedium (CMdum)-Agarplatten mit 2% Glucose, aber ohne Uracil. Nach der Selektion werden je drei Transformanten zur weiteren funktionellen Expression ausgewählt.The Saccharomyces cerev / s / ae strain 334 is transformed by electroporation (1500 V) with the vector pOLE1 or pOLE2, respectively. As a control, a yeast is used, which is transformed with the empty vector pYES2. The selection of the transformed yeasts is carried out on complete minimal medium (CMdum) agar plates with 2% glucose, but without uracil. After selection, three transformants each are selected for further functional expression.
Für die Expresssion der Ol-Elongasen werden zunächst Vorkulturen aus jeweils 5 ml CMdum-Flüssigmedium mit 2% (w/v) Raffinose aber ohne Uracil mit den ausgewählten Transformanten angeimpft und 2 Tage bei 300C, 200 rpm inkubiert. 5 ml CMdum- Flüssigmedium (ohne Uracil) mit 2% Raffinose und 300 μM verschiedener Fettsäuren werden dann mit den Vorkulturen auf eine OD6oo von 0,05 angeimpft. Die Expression wird durch die Zugabe von 2% (w/v) Galactose induziert. Die Kulturen wurden für weitere 96 h bei 200C inkubiert.For the expression of the oil elongases, first precultures each of 5 ml of CMdum liquid medium with 2% (w / v) raffinose but without uracil are inoculated with the selected transformants and incubated for 2 days at 30 ° C., 200 rpm. 5 ml of CMdum liquid medium (without uracil) with 2% raffinose and 300 μM of various fatty acids are then inoculated with the precultures to an OD 6 oo of 0.05. Expression is induced by the addition of 2% (w / v) galactose. The cultures were incubated for a further 96 h at 20 ° C.
Hefen, die mit den Plasmiden pYES2, pOLE1 und pOLE2 transformiert sind, werden folgendermaßen analysiert:Yeasts transformed with plasmids pYES2, pOLE1 and pOLE2 are analyzed as follows:
Die Hefezellen aus den Hauptkulturen werden durch Zentrifugation (100 x g, 5 min, 20°C) geerntet und mit 100 mM NaHCO3, pH 8,0 gewaschen, um restliches Medium und Fettsäuren zu entfernen. Aus den Hefe-Zellsedimenten werden Fettsäuremethylester (FAMEs) durch saure Methanolyse hergestellt. Hierzu werden die Zellsedimente mit 2 ml 1 N methanolischer Schwefelsäure und 2% (v/v) Dimethoxypropan für 1 h bei 800C inkubiert. Die Extraktion der FAMES erfolgt durch zweimalige Extraktion mit Petrolether (PE). Zur Entfernung nicht derivatisierter Fettsäuren werden die organischen Phasen je einmal mit 2 ml 100 mM NaHCO3, pH 8,0 und 2 ml Aqua dest. gewaschen. Anschließend wurden die PE-Phasen mit Na2SO4 getrocknet, unter Argon eingedampft und in 100 μl PE aufgenommen. Die Proben werden auf einer DB-23- Kapillarsäule (30 m, 0,25 mm, 0,25 μm, Agilent) in einem Hewlett-Packard 6850- Gaschromatographen mit Flammenionisationsdetektor getrennt. Die Bedingungen für die GLC-Analyse sind wie folgt: Die Ofentemperatur wurde von 50°C bis 250°C mit einer Rate von 5°C/min und schließlich 10 min bei 250°C(halten) programmiert.
Die Identifikation der Signale erfolgt durch Vergleiche der Retentionszeiten mit entsprechenden Fettsäurestandards (Sigma). Die Methodik ist beschrieben zum Beispiel 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 und Michaelson et al., 1998, FEBS Letters. 439(3):215-218.The yeast cells from the major cultures are harvested by centrifugation (100 xg, 5 min, 20 ° C) and washed with 100 mM NaHCO 3 , pH 8.0 to remove residual medium and fatty acids. From the yeast cell sediments, fatty acid methyl esters (FAMEs) are produced by acid methanolysis. For this purpose, the cell sediments are incubated with 2 ml of 1N methanolic sulfuric acid and 2% (v / v) dimethoxypropane for 1 h at 80 ° C. Extraction of the FAMES is carried out by extracting twice with petroleum ether (PE). To remove non-derivatized fatty acids, the organic phases are distilled once each with 2 ml of 100 mM NaHCO 3 , pH 8.0 and 2 ml of distilled water. washed. Subsequently, the PE phases were dried with Na 2 SO 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 with flame ionization detector. The conditions for the GLC analysis are as follows: The oven temperature was programmed from 50 ° C to 250 ° C at a rate of 5 ° C / min and finally 10 min at 250 ° C (hold). The signals are identified by comparison of 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.
Aktivitäts- und Substratbestimmung von D5Elo(OI):Activity and Substrate Determination of D5Elo (OI):
Zur Ermittlung der Aktivität und Substratspezifität der d5Elo(OI) wurden verschiedene Fettsäuren gefüttert (Tab. 3). Die gefütterten Substrate sind dabei in großen Mengen in allen transgenen Hefen nachzuweisen. Die transgenen Hefen zeigen die Synthese neuer Fettsäuren, den Produkten der d5Elo(OI)-Reaktion. Dies bedeutet, dass das Gen d5Elo(OI) funktional exprimiert wurde.To determine the activity and substrate specificity of d5Elo (OI), various fatty acids were fed (Table 3). The lined substrates are to be detected in large quantities in all transgenic yeasts. The transgenic yeasts show the synthesis of new fatty acids, the products of the d5Elo (OI) reaction. This means that the gene d5Elo (OI) has been functionally expressed.
Figur 6 zeigt die Chromatogramme der einzelnen Experimente. Als Kontrolle wurde in Fig. 6A Hefen transformiert mit pYES ohne Zugabe von Fettsäuren analysiert. In Fig. 1 B wurde zu den Hefen transformiert mit pYES die Fettsäure 20:4Δ5,8,1 1 ,14 gefüttert. Dabei kann die gefütterte Fettsäure in großen Mengen detektiert werden. Dasselbe Experiment ist in Fig. 6C für Hefen transformiert mit dem Plasmid pYES-D5Elo(OI) durchgeführt. Im Unterschied zu Fig. 6B kann in den Hefen mit pYES-D5Elo(OI) eine zusätzliche Fettsäure detektiert werden, die auf die Aktivität der D5Elo(OI) zurückgehen muss. Anhand der Aktivität kann D5Elo(OI) als Δ5-Elongase charakterisiert werden.FIG. 6 shows the chromatograms of the individual experiments. As a control, yeasts transformed with pYES without addition of fatty acids were analyzed in FIG. 6A. In Fig. 1B the yeast was transformed with pYES the fatty acid 20: 4Δ5, 8, 1, 14 fed. In this case, the fed fatty acid can be detected in large quantities. The same experiment is performed in Fig. 6C for yeasts transformed with the plasmid pYES-D5Elo (OI). In contrast to FIG. 6B, an additional fatty acid can be detected in the yeasts with pYES-D5Elo (OI), which must be due to the activity of the D5Elo (OI). Based on the activity, D5Elo (OI) can be characterized as a Δ5 elongase.
Zusammenfassung der Ergebnisse zu D5Elo(OI):Summary of the results for D5Elo (OI):
In den Hefefütterungsexperimenten konnte gezeigt werden, dass das Monierte GenIn the yeast feeding experiments it could be shown that the Monierte Gen
D5Elo(OI) SEQ ID12 funktional exprimiert wurde und Elongase-Aktivität besitzt. Anhand der gefütterten Fettsäure kann D5Elo(OI) als Δ5-Elongase charakterisiert werden, d.h. C20-Fettsäuren mit einer Δ5-Doppelbindung werden spezifisch verlängert.
Aktivitäts- und Substratbestimmung von D6Elo(OI):D5Elo (OI) SEQ ID12 was functionally expressed and possesses elongase activity. Based on the fed fatty acid, D5Elo (OI) can be characterized as a Δ5 elongase, ie C20 fatty acids with a Δ5 double bond are specifically extended. Activity and Substrate Determination of D6Elo (OI):
Zur Ermittlung der Aktivität und Substratspezifität der D6Elo(OI) wurden verschiedene Fettsäuren gefüttert (Tab. 4). Die gefütterten Substrate sind dabei in großen Mengen in allen transgenen Hefen nachzuweisen. Die transgenen Hefen zeigen die Synthese neuer Fettsäuren, den Produkten der D6Elo(OI)-Reaktion. Dies bedeutet, dass das Gen D6Elo(OI) funktional exprimiert wurde.To determine the activity and substrate specificity of D6Elo (OI), various fatty acids were fed (Table 4). The lined substrates are to be detected in large quantities in all transgenic yeasts. The transgenic yeasts show the synthesis of new fatty acids, the products of the D6Elo (OI) reaction. This means that the gene D6Elo (OI) has been functionally expressed.
Tabelle 4: Fütterun / Umsetzun verschiedener Fettsäuren durch D6Elo OITable 4: Feeding / Implementation of Various Fatty Acids by D6Elo OI
Figur 7 zeigt die Chromatogramme der einzelnen Experimente. Als Kontrolle wurde in Fig. 7A Hefen transformiert mit pYES ohne Zugabe von Fettsäuren analysiert. In Fig. 7B und 7C wurden den Hefen transformiert mit pYES die Fettsäure 18:3Δ6,9,12 (B) bzw. 18:4Δ6,9,12,15 (C) zugegeben. Dabei können die gefütterte Fettsäure in großen Mengen detektiert werden. Dasselbe Experiment ist in Fig. 7C und 7D für Hefen transformiert mit dem Plasmid pYES-DΘEIo(OI) durchgeführt. Im Unterschied zu Fig. 7B und 7C kann in den Hefen mit pYES-DΘEIo(OI) eine zusätzliche Fettsäure detektiert werden, die auf die Aktivität der D6Elo(OI) zurückgehen muss. Anhand der Aktivität kann D6Elo(OI) als Δ6-Elongase charakterisiert werden.FIG. 7 shows the chromatograms of the individual experiments. As a control, yeasts transformed with pYES without addition of fatty acids were analyzed in FIG. 7A. In Figs. 7B and 7C, the yeasts transformed with pYES were added with the fatty acid 18: 3Δ6, 9, 12 (B) and 18: 4Δ6, 9, 12, 15 (C), respectively. The fed fatty acid can be detected in large quantities. The same experiment is performed in Figs. 7C and 7D for yeasts transformed with the plasmid pYES-DΘEIo (OI). In contrast to FIGS. 7B and 7C, an additional fatty acid can be detected in the yeasts with pYES-DΘEIo (OI), which must be due to the activity of the D6Elo (OI). Based on the activity D6Elo (OI) can be characterized as Δ6 elongase.
Zusammenfassung der Ergebnisse zu D6Elo(OI):Summary of the results for D6Elo (OI):
In den Hefefütterungsexperimenten konnte gezeigt werden, dass das Monierte GenIn the yeast feeding experiments it could be shown that the Monierte Gen
D6Elo(OI) SEQ ID16 funktional exprimiert wurde und Elongase-Aktivität besitzt.
Anhand der gefütterten Fettsäure kann D6Elo(OI) als Δ6-Elongase charakterisiert werden, d.h. C18-Fettsäuren mit einer Δ6-Doppelbindung werden spezifisch verlängert.D6Elo (OI) SEQ ID16 was functionally expressed and possesses elongase activity. Based on the fed fatty acid, D6Elo (OI) can be characterized as a Δ6 elongase, ie C18 fatty acids with a Δ6 double bond are specifically extended.
Beispiel 5: Klonierung und Charakterisierung von Desaturase-Genen aus Ostreococcus lucimarinusExample 5 Cloning and Characterization of Desaturase Genes from Ostreococcus lucimarinus
Durch Suche nach konservierten Bereichen in den Proteinsequenzen mit Hilfe von konservierten Motiven (His-Boxen, Domergue et al. 2002, Eur. J. Biochem. 269, 4105- 41 13) konnten fünf Sequenzen mit entsprechenden Motiven in einer Ostreococcus lucimarinus Sequenzdatenbank (genomische Sequenzen) identifiziert werden. In einem weiteren Schritt wurden die Gene mittels Sequenzvergleich, Genvorhersage und der Suche nach kodierenden Bereichen charakterisiert. Folgende kodierende Bereiche wurden gefunden::By searching for conserved regions in the protein sequences using conserved motifs (His-Boxes, Domergue et al., 2002, Eur. J. Biochem., 269, 4105-4113), five sequences with corresponding motifs in an Ostreococcus lucimarinus sequence database (genomic Sequences) are identified. In a further step, the genes were characterized by sequence comparison, gene prediction and the search for coding regions. The following coding areas were found ::
Tabelle 5: Kodierende BereicheTable 5: Coding ranges
Zur Charakterisierung der Funktion der Desaturase d6Des(OI) (= Δ-6-Desaturase) aus Ostreococcus lucimarinus wird der offenen Leserahmen der DNA stromabwärts des Galactose-induzierbaren GAL1 -Promotors von pYES2.1A/5-His-TOPO (Invitrogen) kloniert, wobei der entsprechenden pYES2.1-d6Elo(OI) Klon erhalten wird. In entsprechender Art und Weise können weitere Desaturase-Gene aus Ostreococcus kloniert werden.To characterize the function of desaturase d6Des (OI) (= Δ-6-desaturase) from Ostreococcus lucimarinus, the open reading frame of the DNA is cloned downstream of the galactose-inducible GAL1 promoter of pYES2.1A / 5-His-TOPO (Invitrogen), whereby the corresponding pYES2.1-d6Elo (OI) clone is obtained. In a corresponding manner, further desaturase genes from Ostreococcus can be cloned.
Der Saccharomyces cerev/s/ae-Stamm 334 wird durch Elektroporation (1500 V) mit dem Vektor pYES2.1-d6Elo(OI) transformiert. Als Kontrolle wurde eine Hefe verwendet, die mit dem leeren Vektor pYES2 transformiert wird. Die Selektion der transformierten Hefen erfolgt auf Komplett-Minimalmedium (CMdum)-Agarplatten mit 2% Glucose, aber ohne Uracil. Nach der Selektion werden je drei Transformanten zur weiteren funktionellen Expression ausgewählt.
Für die Expresssion der d6Elo(OI) Desaturase werden zunächst Vorkulturen aus jeweils 5 ml CMdum-Flüssigmedium mit 2% (w/v) Raffinose aber ohne Uracil mit den ausgewählten Transformanten angeimpft und 2 Tage bei 300C, 200 rpm inkubiert. 5 ml CMdum-Flüssigmedium (ohne Uracil) mit 2% Raffinose und 300 μM verschiedener Fettsäuren werden dann mit den Vorkulturen auf eine OD6oo von 0,05 angeimpft. Die Expression wird durch die Zugabe von 2% (w/v) Galactose induziert. Die Kulturen werden für weitere 96 h bei 200C inkubiert.The Saccharomyces cerev / s / ae strain 334 is transformed by electroporation (1500 V) with the vector pYES2.1-d6Elo (OI). As a control, a yeast was used, which is transformed with the empty vector pYES2. The selection of the transformed yeasts is carried out on complete minimal medium (CMdum) agar plates with 2% glucose, but without uracil. After selection, three transformants each are selected for further functional expression. For the expression of d6Elo (OI) desaturase first precultures from 5 ml of CMdum liquid medium with 2% (w / v) raffinose but without uracil are inoculated with the selected transformants and incubated for 2 days at 30 0 C, 200 rpm. 5 ml CMdum liquid medium (without uracil) with 2% raffinose and 300 μM different fatty acids are then inoculated with the precultures to an OD 6 oo of 0.05. Expression is induced by the addition of 2% (w / v) galactose. The cultures are incubated for a further 96 h at 20 ° C.
Die Figuren 2 bis 5 zeigen die Sequenzähnlichkeiten zu anderen Algen (Ostreococcus tauri, Thalassiosira pseudonana) für die verschiedenen Desaturase-Aminosäure Sequenzen im ClustalW Sequenzvergleich. Überraschenderweise unterscheiden sich die Sequenzen von O. lucimarinus in ihrer Aminosäuresequenz deutlich von den anderen Algen.Figures 2 to 5 show the sequence similarities to other algae (Ostreococcus tauri, Thalassiosira pseudonana) for the different desaturase amino acid sequences in ClustalW sequence comparison. Surprisingly, the sequences of O. lucimarinus differ significantly in their amino acid sequence from the other algae.
Tabelle 6: Sequenzidentitäten einzelner DesaturasenTable 6: Sequence identities of individual desaturases
Die Gene werden wie folgt characterisiert:The genes are characterized as follows:
Zur Expression der Desaturasen in Hefezellen werden aus den Hauptkulturen durch Zentrifugation (100 x g, 5 min, 20°C) geerntet und mit 100 mM NaHCO3, pH 8,0 gewaschen, um restliches Medium und Fettsäuren zu entfernen. Die Hefe- Zellsedimente werden 4 h mit Chloroform/Methanol (1 :1 ) extrahiert. Die resultierende
organische Phase wird mit 0,45 % NaCI extrahiert, mit Na2SC>4 getrocknet und unter Vakuum evaporiert. Der Lipidextrakt wird durch Dünnschicht-Chromatographie (Horizontal-Tank, Chloroform:Methanol:Essigsäure 65:35:8) weiter in die Lipidklassen Phosphatidylcholin (PC), Phosphatidiylinositol (PI), Phosphatidyserin (PS), Phosphati- dylethanolamine (PE) und Neutral-Lipide (NL) aufgetrennt. Die verschiedenen aufgetrennten Spots auf der Dünnschichtplatte weden abgekratzt. Für die gaschromatografi- sche Analyse wurde Fettsäuremethylester (FAMEs) durch saure Methanolyse hergestellt. Hierzu werden die Zellsedimente mit 2 ml 1 N methanolischer Schwefelsäure und 2% (v/v) Dimethoxypropan für 1 h bei 800C inkubiert. Die Extraktion der FAMES erfolgte durch zweimalige Extraktion mit Petrolether (PE). Zur Entfernung nicht derivatisierter Fettsäuren wird die organische Phase je einmal mit 2 ml 100 mM NaHCC>3, pH 8,0 und 2 ml Aqua dest. gewaschen. Anschließend werden die PE- Phasen mit Na2SO4 getrocknet, unter Argon eingedampft und in 100 μl PE aufgenommen. Die Proben werden auf einer DB-23-Kapillarsäule (30 m, 0,25 mm, 0,25 μm, Agilent) in einem Hewlett-Packard 6850-Gaschromatographen mit Flammenionisationsdetektor getrennt. Die Bedingungen für die GLC-Analyse sind wie folgt: Die Ofentemperatur wird von 500C bis 250°C mit einer Rate von 5°C/min und schließlich 10 min bei 250°C(halten) programmiert.For expression of the desaturases in yeast cells, they are harvested from the main cultures by centrifugation (100 × g, 5 min, 20 ° C.) and washed with 100 mM NaHCO 3 , pH 8.0 to remove residual medium and fatty acids. The yeast cell pellets are extracted with chloroform / methanol (1: 1) for 4 h. The resulting organic phase is extracted with 0.45% NaCl, dried with Na 2 SC> 4 and evaporated under vacuum. The lipid extract is further purified by thin-layer chromatography (horizontal tank, chloroform: methanol: acetic acid 65: 35: 8) into the lipid classes phosphatidylcholine (PC), phosphatidiylinositol (PI), phosphatidyserine (PS), phosphatidylethanolamine (PE) and neutral Lipids (NL) separated. The various split spots on the thin-layer plate are scraped off. For gas chromatographic analysis, fatty acid methyl ester (FAMEs) was prepared by acid methanolysis. For this purpose, the cell sediments are incubated with 2 ml of 1N methanolic sulfuric acid and 2% (v / v) dimethoxypropane for 1 h at 80 ° C. The extraction of the FAMES was carried out by extraction twice with petroleum ether (PE). To remove non-derivatized fatty acids, the organic phase is distilled once each with 2 ml of 100 mM NaHCO 3, pH 8.0 and 2 ml of distilled water. washed. Subsequently, the PE phases are dried with Na 2 SO 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 with flame ionization detector. The conditions for the GLC analysis are as follows: The oven temperature is from 50 0 C to 250 ° C at a rate of 5 ° C / min and finally 10 min at 250 ° C (hold) programmed.
Die Identifikation der Signale erfolgt durch Vergleiche der Retentionszeiten mit entsprechenden Fettsäurestandards (Sigma). Die Methodik ist beschrieben zum Beispiel 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 und Michaelson et al., 1998, FEBS Letters. 439(3):215-218.The signals are identified by comparison of 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.
Aktivitäts- und Substratbestimmung von D5Des_2 (Ol):Activity and Substrate Determination of D5Des_2 (Ol):
Zur Ermittlung der Aktivität und Substratspezifität der D5Des_2 (Ol) SEQ ID 24 wurden verschiedene Fettsäuren gefüttert (Tab. 7). Die gefütterten Substrate sind dabei in großen Mengen in allen transgenen Hefen nachzuweisen. Die transgenen Hefen zeigen die Synthese neuer Fettsäuren, den Produkten der D5Des_2 (Ol)-Reaktion. Dies bedeutet, dass das Gen D5Des_2 (Ol) funktional exprimiert wurde.To determine the activity and substrate specificity of D5Des_2 (Ol) SEQ ID 24, various fatty acids were fed (Table 7). The lined substrates are to be detected in large quantities in all transgenic yeasts. The transgenic yeasts show the synthesis of new fatty acids, the products of the D5Des_2 (Ol) reaction. This means that the gene D5Des_2 (Ol) has been functionally expressed.
Tabelle 7: Fütterun / Umsetzun verschiedener Fettsäuren durch D5Des OI 2).Table 7: Administration / Implementation of Different Fatty Acids by D5Des OI 2).
Figur 8 zeigt die Chromatogramme der einzelnen Experimente. Als Kontrolle wurde in Fig. 8A Hefen, transformiert mit pYES, ohne Zugabe von Fettsäuren analysiert. In Fig. 8B wurden den Hefen transformiert mit pYES die Fettsäure 20:3Δ8, 1 1 ,14 zugegeben. Dabei können die gefütterte Fettsäure in großen Mengen detektiert werden. Dasselbe Experiment ist in Fig. 8C für Hefen transformiert mit dem Plasmid pYES-D5Des(OI_2) durchgeführt worden. Im Unterschied zu Fig. 8B kann in den Hefen mit pYES- D5Des(OI_2) eine zusätzliche Fettsäure detektiert werden, die auf die Aktivität der D5Des(OI_2) zurückgehen muss. Anhand der Aktivität kann D5Des(OI_2) als Δ5- Desaturase charakterisiert werden.FIG. 8 shows the chromatograms of the individual experiments. As a control, yeasts transformed with pYES were analyzed in Fig. 8A without addition of fatty acids. In Fig. 8B, the yeasts transformed with pYES were added the fatty acid 20: 3Δ8, 11, 14. The fed fatty acid can be detected in large quantities. The same experiment was performed in Fig. 8C for yeasts transformed with the plasmid pYES-D5Des (OI_2). In contrast to FIG. 8B, an additional fatty acid can be detected in the yeasts with pYES-D5Des (OI_2), which must be due to the activity of D5Des (OI_2). Based on the activity D5Des (OI_2) can be characterized as Δ5-desaturase.
Zusammenfassung der Ergebnisse zu D5Des_2 (Ol):Summary of the results for D5Des_2 (Ol):
In den Hefefütterungsexperimenten konnte gezeigt werden, dass das Monierte Gen D5Des_2 (Ol) SEQ ID 24 funktional exprimiert wurde und Desaturase-Aktivität besitzt. Anhand der gefütterten Fettsäure kann D5Des_2 (Ol) als Δ5-Desaturase charakterisiert werden, d.h. C20-Fettsäuren mit einer Δ8-Doppelbindung werden spezifisch an der Δ5- Position dehydriert.In the yeast feeding experiments it could be shown that the monten gene D5Des_2 (Ol) SEQ ID 24 was functionally expressed and possesses desaturase activity. From the fed fatty acid, D5Des_2 (Ol) can be characterized as Δ5-desaturase, i. C20 fatty acids with a Δ8 double bond are specifically dehydrogenated at the Δ5 position.
Aktivitäts- und Substratbestimmung von D12Des(OI):Activity and Substrate Determination of D12Des (OI):
Zur Ermittlung der Aktivität und Substratspezifität der D12Des(OI) SEQ ID 18 wurden verschiedene Fettsäuren gefüttert (Tab. 8). Die gefütterten Substrate sind dabei in großen Mengen in allen transgenen Hefen nachzuweisen. Die transgenen Hefen zeigen die Synthese neuer Fettsäuren, den Produkten der D12Des(OI)-Reaktion. Dies bedeutet, dass das Gen D12Des(OI) funktional exprimiert wurde.To determine the activity and substrate specificity of D12Des (OI) SEQ ID 18, various fatty acids were fed (Table 8). The lined substrates are to be detected in large quantities in all transgenic yeasts. The transgenic yeasts show the synthesis of new fatty acids, the products of the D12Des (OI) reaction. This means that the gene D12Des (OI) has been functionally expressed.
Tabelle 8: Fütterung/ Umsetzung verschiedener Fettsäuren durch D12Des(OI).Table 8: Feeding / conversion of various fatty acids by D12Des (OI).
Figur 9 zeigt die Chromatogramme der einzelnen Experimente. Als Kontrolle wurde in Fig. 9A Hefen, transformiert mit pYES, ohne Zugabe von Fettsäuren analysiert. In Fig. 9B wurden den Hefen transformiert mit pYES-D12Des(OI) analysiert. Im Unterschied zu Fig. 9A kann in den Hefen mit pYES-D12Des(OI) eine zusätzliche Fettsäure detektiert werden, die auf die Aktivität der D12Des(OI) zurückgehen muss. Anhand der Aktivität kann D12Des(OI) als Δ12-Desaturase charakterisiert werden.FIG. 9 shows the chromatograms of the individual experiments. As a control, yeasts transformed with pYES were analyzed in Fig. 9A without addition of fatty acids. In Fig. 9B, yeasts transformed with pYES-D12Des (OI) were analyzed. In contrast to FIG. 9A, an additional fatty acid can be detected in the yeasts with pYES-D12Des (OI), which must be due to the activity of the D12Des (OI). Based on the activity D12Des (OI) can be characterized as Δ12-desaturase.
Zusammenfassung der Ergebnisse zu D12Des(OI):Summary of the results for D12Des (OI):
In den Hefefütterungsexperimenten konnte gezeigt werden, dass das Monierte Gen D12Des(OI) SEQ ID 18 funktional exprimiert wurde und Desaturase-Aktivität besitzt. Anhand des Fettsäurespektrums kann D12Des(OI) als Δ12-Desaturase charakterisiert werden, d.h. C18-Fettsäuren mit einer Δ9-Doppelbindung werden spezifisch an der Δ12-Position dehydriert.In the yeast feeding experiments it could be shown that the monten gene D12Des (OI) SEQ ID 18 was functionally expressed and possesses desaturase activity. From the fatty acid spectrum, D12Des (OI) can be characterized as Δ12-desaturase, i. C18 fatty acids with a Δ9 double bond are specifically dehydrogenated at the Δ12 position.
Aktivitäts- und Substratbestimmung von D5Des(OI):Activity and Substrate Determination of D5Des (OI):
Zur Ermittlung der Aktivität und Substratspezifität der D5Des(OI) SEQ ID 26 wurden verschiedene Fettsäuren gefüttert (Tab. 9). Die gefütterten Substrate sind dabei in großen Mengen in allen transgenen Hefen nachzuweisen. Die transgenen Hefen zeigen die Synthese neuer Fettsäuren, den Produkten der D5Des(OI)-Reaktion. Dies bedeutet, dass das Gen D5Des(OI) funktional exprimiert wurde.To determine the activity and substrate specificity of D5Des (OI) SEQ ID 26, various fatty acids were fed (Table 9). The lined substrates are to be detected in large quantities in all transgenic yeasts. The transgenic yeasts show the synthesis of new fatty acids, the products of the D5Des (OI) reaction. This means that the gene D5Des (OI) has been functionally expressed.
Tabelle 9: Umsetzung verschiedener Fettsäuren durch D5Des(OI)Table 9: Reaction of various fatty acids by D5Des (OI)
Figur 10 zeigt die gaschromatographische Analyse von Hefefütterungsexperimenten. Nach Expression von pYes-d5Des(OI_1 ) in Hefe Stamm InvSc ohne Zugabe von Fettsäuren (Fig. 10A) wurde keine Umsetzung der vorhandenen Fettsäuren festgestellt. pYes-d5Des(OI_1 ) Expression in Hefe Stamm InvSc nach Zugabe der Fettsäure 20:3n-6 (B) führt zur spezifischen Umsetzung von 20:3n-6 zu 20:4n-6 (Arachidonsäure) und Expression von pYes-d5Des(OI_1 ) in Hefe Stamm InvSc nach Zugabe der Fettsäure 20:4n-3 (C) zur spezifische Umsetzung von 20:4n-3 zu 20:5n-3 (Eicosapen- taensäure). Die spezifische Inkorporation von d5-Doppelbindungen in die gefütterten Fettsäuren zeigt die d5-Desaturase Aktivität von d5Des(OI). FIG. 10 shows the gas chromatographic analysis of yeast feeding experiments. After expression of pYes-d5Des (OI_1) in yeast strain InvSc without addition of fatty acids (Figure 10A), no conversion of the fatty acids present was found. pYes-d5Des (OI_1) expression in yeast strain InvSc after addition of fatty acid 20: 3n-6 (B) leads to the specific conversion of 20: 3n-6 to 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) for specific conversion of 20: 4n-3 to 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).
Zusammenfassung der Ergebnisse zu D5Des(OI):Summary of the results for D5Des (OI):
In den Hefefütterungsexperimenten konnte gezeigt werden, dass das Monierte GenIn the yeast feeding experiments it could be shown that the Monierte Gen
D5Des(OI) SEQ ID 26 funktional exprimiert wurde und Desaturase-Aktivität besitzt.D5Des (OI) SEQ ID 26 was functionally expressed and has desaturase activity.
Anhand des Fettsäurespektrums kann D5Des(OI) als Δ5-Desaturase charakterisiert werden, d.h. C20-Fettsäuren mit einer Δ8-Doppelbindung werden spezifisch an der Δ5-From the fatty acid spectrum, D5Des (OI) can be characterized as Δ5-desaturase, i. C20 fatty acids with a Δ8 double bond are bound specifically to the Δ5-
Position dehydriert.
Dehydrated position.
Claims
1. Polynucleotid umfassend eine Nukleinsäuresequenz, die ausgewählt ist aus derA polynucleotide comprising a nucleic acid sequence selected from
Gruppe bestehend aus:Group consisting of:
(a) Nukleinsäuresequenz wie in einer der SEQ ID No. 1 , 3, 5, 7, 9, 1 1 , 13, 15 oder gezeigt;(a) Nucleic acid sequence as in one of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15 or 14;
(b) Nukleinsäuresequenz, die ein Polypeptid kodiert, das eine Aminosäu- resequenz wie in einer der SEQ ID No. 2, 4, 6, 8, 10, 12, 14 oder 16 gezeigt aufweist;(b) Nucleic acid sequence which encodes a polypeptide which has an amino acid sequence as described in one of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16;
(c) Nukleinsäuresequenz, die ein Polypeptid kodiert, das mindestens 80% identisch zu einem Polypeptid ist, das von der Nucleinsäurese- quenzen aus (a) oder (b) kodiert wird, wobei das Polypeptid Desatu- rase oder Elongase Aktivität aufweist; und(c) a nucleic acid sequence encoding a polypeptide that is at least 80% identical to a polypeptide encoded by the nucleic acid sequences of (a) or (b), wherein the polypeptide has desaturase or elongase activity; and
(d) Nukleinsäuresequenz für ein Fragment einer Nukleinsäure aus (a), (b) oder (c), wobei das Fragment ein Polypeptid mit einer Desaturase oder Elongase Aktivität kodiert.(d) Nucleic acid sequence for a fragment of a nucleic acid from (a), (b) or (c), wherein the fragment encodes a polypeptide having a desaturase or elongase activity.
2. Polynucleotid nach Anspruch 1 , wobei das Polynucleotid aus RNA oder DNA besteht.2. Polynucleotide according to claim 1, wherein the polynucleotide consists of RNA or DNA.
3. Vektor umfassend das Polynucleotid nach Anspruch 1 oder 2.3. A vector comprising the polynucleotide according to claim 1 or 2.
4. Vektor nach Anspruch 3, wobei der Vektor ein Expressionsvektor ist.The vector of claim 3, wherein the vector is an expression vector.
5. Vektor nach Anspruch 3 oder 4, wobei der Vektor mindestens ein weiteres Polynucleotid umfasst, das ein weiteres Enzym kodiert, das in die Biosynthese von Lipiden oder Fettsäuren eingebunden ist.5. A vector according to claim 3 or 4, wherein the vector comprises at least one further polynucleotide encoding a further enzyme which is involved in the biosynthesis of lipids or fatty acids.
6. Wirtszelle umfassend das Polynucleotid nach Anspruch 1 oder 2 oder den Vektor nach einem der Ansprüche 3 bis 5.A host cell comprising the polynucleotide of claim 1 or 2 or the vector of any one of claims 3 to 5.
7. Wirtszelle nach Anspruch 6, wobei die Wirtszelle zusätzlich mindestens ein weiteres Enzym umfasst, das in die Biosynthese von Lipiden oder Fettsäuren eingebunden ist. 7. The host cell according to claim 6, wherein the host cell additionally comprises at least one further enzyme which is involved in the biosynthesis of lipids or fatty acids.
8. Vektor nach Anspruch 5 oder Wirtszelle nach Anspruch 7 wobei das Enzym ausgewählt ist aus der Gruppe bestehend aus: Acyl-CoA-Dehydrogenase(n), A- cyl-ACP[= acyl carrier protein]-Desaturase(n), Acyl-ACP-Thioesterase(n), Fett- säure-Acyl-Transferase(n), Acyl-CoA:Lysophospholipid-Acyltransferase(n), Fett- säure-Synthase(n), Fettsäure-Hydroxylase(n), Acetyl-Coenzym A- Carboxylase(n), Acyl-Coenzym A-Oxidase(n), Fettsäure-Desaturase(n), Fett- säure-Acetylenase(n), Lipoxygenase(n), Triacylglycerol-Lipase(n), Allenoxid- Synthase(n), Hydroperoxid-Lyase(n), Fettsäure-Elongase(n), Δ4-Desaturase(n), Δ5-Desaturase(n), Δ6-Desaturase(n), Δδ-Desaturase(n), Δ9-Desaturase(n), Δ12- Desaturase(n), Δ5-Elongase(n), Δθ-Elongase(n) und Δ9-Elongase(n).8. A vector according to claim 5 or a host cell according to claim 7, wherein the enzyme is selected from the group consisting of: acyl-CoA-dehydrogenase (s), acyl-ACP [= acyl carrier protein] desaturase (s), acyl- ACP thioesterase (s), fatty acid acyltransferase (s), acyl CoA: lysophospholipid acyltransferase (s), fatty acid synthase (s), fatty acid hydroxylase (s), acetyl coenzyme A- Carboxylase (s), acyl coenzyme A oxidase (s), fatty acid desaturase (s), fatty acid acetylenase (s), lipoxygenase (s), triacylglycerol lipase (s), allene oxide synthase (s), Hydroperoxide lyase (s), fatty acid elongase (s), Δ4-desaturase (s), Δ5-desaturase (s), Δ6-desaturase (s), Δδ-desaturase (s), Δ9-desaturase (s), Δ12 Desaturase (s), Δ5 elongase (s), Δθ elongase (s) and Δ9 elongase (s).
9. Verfahren zur Herstellung eines Polypeptids mit Desaturase oder Elongase Aktivität umfassend die Schritte:9. A method of producing a polypeptide having desaturase or elongase activity comprising the steps of:
(a) Exprimieren eines Polynucleotids nach Anspruch 1 oder 2 in einer Wirtszelle; und(a) expressing a polynucleotide according to claim 1 or 2 in a host cell; and
(b) Gewinnen des Polypeptids, das von dem Polynucleotid kodiert wird, aus der Wirtszelle.(b) recovering the polypeptide encoded by the polynucleotide from the host cell.
10. Polypeptid, das von dem Polynucleotid gemäß Anspruch 1 oder 2 kodiert wird oder das erhätlich durch das Verfahren nach Anspruch 9 ist.A polypeptide encoded by the polynucleotide of claim 1 or 2 or obtainable by the method of claim 9.
1 1. Antikörper, der das Polypeptid nach Anspruch 10 spezifisch erkennt.1 1. Antibody which specifically recognizes the polypeptide according to claim 10.
12. Transgener, nicht-humaner Organismus umfassend das Polynucleotid nach Anspruch 1 oder 2, den Vektor nach einem der Ansprüche 3 bis 5 oder die Wirtszelle nach Anspruch 8.12. Transgenic, non-human organism comprising the polynucleotide according to claim 1 or 2, the vector according to any one of claims 3 to 5 or the host cell according to claim 8.
13. Transgener, nicht-humaner Organismus nach Anspruch 12, wobei der Organismus ein Tier, eine Pflanze oder ein multizellulärere Mikroorganismus ist.The transgenic non-human organism of claim 12, wherein the organism is an animal, a plant or a multicellular microorganism.
14. Verfahren zur Herstellung eines Stoffes, der die Struktur aufweist, die in der folgenden allgemeinen Formel I gezeigt wird14. A process for producing a substance having the structure shown in the following general formula I.
wobei die Variablen und Substiuenten die folgenden sindwhere the variables and substituents are the following
R1 = Hydroxyl, Coenzym A (Thioester), Lysophosphatidylcholin, Lysophospha- tidylethanolamin, Lysophosphatidylglycerol, Lysodiphosphatidylglycerol, Lysophosphatidylserin, Lysophosphatidylinositol, Sphingo- Base oder ein Radikal der Formel IlR 1 = hydroxyl, coenzyme A (thioester), lysophosphatidylcholine, lysophosphate tidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol, sphingolase or a radical of formula II
R2 = Wasserstoff, Llysophosphatidylcholin, Lysophosphatidylethanolamin, Lysophosphatidylglycerol, Llysodiphosphatidylglycerol, Lysophosphatidylserin, Lysophosphatidylinositol oder gesättigtes oder ungesättigtes C2-C24- Alkylcarbonyl,R 2 = hydrogen, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysodiphosphatidylglycerol, lysophosphatidylserine, lysophosphatidylinositol or saturated or unsaturated C 2 -C 24 -alkylcarbonyl,
R3 = Wasserstoff, gesättigtes oder ungesättigtes C2-C24-Alkylcarbonyl, oder R2 und R3 sind unabhängig voneinander ein Radikal der Formel Ia:R 3 = hydrogen, saturated or unsaturated C 2 -C 24 -alkylcarbonyl, or R 2 and R 3 are independently of one another a radical of the formula Ia:
n = 2, 3, 4, 5, 6, 7 or 9, m = 2, 3, 4, 5 oder 6 und p = 0 oder 3;n = 2, 3, 4, 5, 6, 7 or 9, m = 2, 3, 4, 5 or 6 and p = 0 or 3;
undand
wobei das Verfahren das Kultivieren von (i) einer Wirtszelle nach einem der Ansprüche 3 bis 5 oder (ii) eines transgenen, nicht-humanen Organismus nach Anspruch 12 oder 13 unter Bedingungen umfasst, die die Biosynthese des Stoffes erlauben.the method comprising cultivating (i) a host cell according to any one of claims 3 to 5 or (ii) a transgenic non-human organism according to claim 12 or 13 under conditions which allow the biosynthesis of the substance.
15. Verfahren zur Herstellung einer Öl-, Lipid- oder Fettsäurezusammensetzung umfassend die Schritte des Verfahrens nach Anspruch 14 und den weiteren Schritt des Formulierens des Stoffes als Öl-, Lipid- oder Fettsäurezusammensetzung.A process for the preparation of an oil, lipid or fatty acid composition comprising the steps of the process of claim 14 and the further step of formulating the substance as an oil, lipid or fatty acid composition.
16. Verfahren nach Anspruch 15, wobei die Öl-, Lipid- oder Fettsäurezusammensetzung weiter formuliert wird zu einem Arzneimittel, zu Kosmetik, zu einem Nahrungsmittel, zu einem Futtermittel, vorzugsweise Fischfutter, oder zu einem Nah- rungsergänzungsmittel. 16. The method of claim 15, wherein the oil, lipid or fatty acid composition is further formulated into a pharmaceutical, cosmetic, food, feed, preferably fish food, or dietary supplement.
17. Verwendung des Polynucleotids nach Anspruch 1 oder 2, des Vektors nach einem der Ansprüche 3 bis 5, der Wirtszelle nach Anspruch 6 oder 7, des Vektors oder der Wirtszelle nach Anspruch 8, des Polypeptids nach Anspruch 10 o- der des transgenen, nicht-humanen Organismus nach Anspruch 12 oder 13 für die Herstellung einer Öl-, Lipid- oder Fettsäurezusammensetzung.17. Use of the polynucleotide according to claim 1 or 2, the vector according to any one of claims 3 to 5, the host cell according to claim 6 or 7, the vector or the host cell according to claim 8, the polypeptide according to claim 10 or the transgenic, not Human organism according to claim 12 or 13 for the preparation of an oil, lipid or fatty acid composition.
18. Verwendung nach Anspruch 17, wobei die Öl-, Lipid- oder Fettsäurezusammensetzung einzusetzen ist als Arzneimittel, Kosmetik, Nahrungsmittel, Futtermittel, vorzugsweise Fischfutter, oder Nahrungsergänzungsmittel. 18. Use according to claim 17, wherein the oil, lipid or fatty acid composition is to be used as a medicament, cosmetics, food, feed, preferably fish feed, or dietary supplements.
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EP15201721.6A EP3066934A1 (en) | 2006-10-06 | 2007-10-04 | Method for producing polyunsatured fatty acids in transgenic organisms |
EP09175508.2A EP2177605B1 (en) | 2006-10-06 | 2007-10-04 | Delta-5 Desaturases and method for producing polyunsaturated fatty acids in transgenic non-human organisms |
EP09175509.0A EP2182056B1 (en) | 2006-10-06 | 2007-10-04 | Method for producing polyunsaturated fatty acids in transgenic non-human organisms |
PL09175508T PL2177605T3 (en) | 2006-10-06 | 2007-10-04 | Delta-5 Desaturases and method for producing polyunsaturated fatty acids in transgenic non-human organisms |
EP07820931A EP2054509A2 (en) | 2006-10-06 | 2007-10-04 | Processes for producing polyunsaturated fatty acids in transgenic organisms |
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PCT/EP2007/060554 WO2008040787A2 (en) | 2006-10-06 | 2007-10-04 | Processes for producing polyunsaturated fatty acids in transgenic organisms |
EP07820931A EP2054509A2 (en) | 2006-10-06 | 2007-10-04 | Processes for producing polyunsaturated fatty acids in transgenic organisms |
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EP09175508.2A Division EP2177605B1 (en) | 2006-10-06 | 2007-10-04 | Delta-5 Desaturases and method for producing polyunsaturated fatty acids in transgenic non-human organisms |
EP15201721.6A Division EP3066934A1 (en) | 2006-10-06 | 2007-10-04 | Method for producing polyunsatured fatty acids in transgenic organisms |
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EP09175509.0A Not-in-force EP2182056B1 (en) | 2006-10-06 | 2007-10-04 | Method for producing polyunsaturated fatty acids in transgenic non-human organisms |
EP07820931A Withdrawn EP2054509A2 (en) | 2006-10-06 | 2007-10-04 | Processes for producing polyunsaturated fatty acids in transgenic organisms |
EP09175508.2A Active EP2177605B1 (en) | 2006-10-06 | 2007-10-04 | Delta-5 Desaturases and method for producing polyunsaturated fatty acids in transgenic non-human organisms |
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EP (4) | EP3066934A1 (en) |
AU (1) | AU2007304229B2 (en) |
CA (2) | CA2665336C (en) |
ES (1) | ES2531391T3 (en) |
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- 2007-10-04 US US12/444,193 patent/US8710299B2/en active Active
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- 2007-10-04 ES ES09175508T patent/ES2531391T3/en active Active
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- 2007-10-04 WO PCT/EP2007/060554 patent/WO2008040787A2/en active Application Filing
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2013
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2016
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Also Published As
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US20190225948A1 (en) | 2019-07-25 |
CA2847007A1 (en) | 2008-04-10 |
EP3066934A1 (en) | 2016-09-14 |
US20160304841A1 (en) | 2016-10-20 |
US20220056423A1 (en) | 2022-02-24 |
ES2531391T3 (en) | 2015-03-13 |
CA2665336C (en) | 2016-01-05 |
WO2008040787A2 (en) | 2008-04-10 |
EP2182056B1 (en) | 2015-12-23 |
CA2847007C (en) | 2016-11-08 |
EP2177605B1 (en) | 2014-12-10 |
US20130340103A1 (en) | 2013-12-19 |
US20100088776A1 (en) | 2010-04-08 |
AU2007304229B2 (en) | 2013-09-19 |
CA2665336A1 (en) | 2008-04-10 |
US9382529B2 (en) | 2016-07-05 |
EP2182056A1 (en) | 2010-05-05 |
EP2177605A1 (en) | 2010-04-21 |
US11168308B2 (en) | 2021-11-09 |
US8710299B2 (en) | 2014-04-29 |
WO2008040787A3 (en) | 2008-06-12 |
US10308914B2 (en) | 2019-06-04 |
PL2177605T3 (en) | 2015-05-29 |
AU2007304229A1 (en) | 2008-04-10 |
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