DE10005973A1 - New plant nucleic acid encoding polyunsaturated fatty acid elongase, useful for producing transgenic organisms with increased content of long-chain unsaturated fatty acids - Google Patents

Abstract

Isolated nucleic acid (I), of plant origin, encoding a polypeptide (II) that extends C18 fatty acids, containing at least to double bonds, by at least two carbon atoms. Independent claims are also included for the following: (a) (II) encoded by (I); (b) gene construct containing a 1192 bp sequence (1; reproduced), functionally linked to regulatory signals; (c) vector containing (I) or the construct of (b); (d) organisms containing (I), the construct of (b) or the vector of (c); (e) method for producing polyunsaturated fatty acids (PUFAs) by growin organisms of (d); (f) oils, lipids, fatty acids or their fractions produced by method (e); and (g) oils, lipids and fatty acid compositions containing PUFAs and derived from transgenic plants. ACTIVITY : Hypocholesterolemic; cardiovascular. MECHANISM OF ACTION : None given.

Description

Field of the Invention

This invention relates to a new elongase gene with the sequence SEQ ID NO: 1 or its homologs, derivatives or analogs Gene construct that this gene or its homologues, derivatives or Analogs includes, as well as its use. The invention relates also vectors or organisms that have an elongase gene with the Sequence SEQ ID NO: 1 or its homologues, derivatives or analogs include.

The invention further relates to a method of manufacturing more polyunsaturated fatty acids and a method of incorporation of DNA in organisms that contain large amounts of oils and in particular Produce oils with a high content of unsaturated fatty acids. The invention also relates to an oil and / or a fatty acid composition with a higher content of polyunsaturated Fatty acids with at least two double bonds and / or one Triacylglycerol composition with a higher content of multiple unsaturated fatty acids with at least two double bonds.

Background of the Invention

Certain products and by-products of naturally occurring metabolic processes in cells are useful for a wide range of industries, including the feed, food, cosmetic, and pharmaceutical industries. These molecules, collectively referred to as "fine chemicals", also include lipids and fatty acids, among which an exemplary class is the polyunsaturated fatty acids. Polyunsaturated fatty acids (p oly u nsaturated f atty a cids, PUFAs) are example dropwise to infant formulas, in order to generate a higher nutritional value of these formulations. For example, PUFAs have a positive influence on the cholesterol level in the blood of humans and are therefore suitable for protection against heart diseases. Fine chemicals and polyunsaturated fatty acids (p oly u nsaturated f atty a cids PUFAs) can be isolated from animal sources such as fish, isolate or with micro-organisms by culturing microorganisms which have been developed so as large amounts of a plurality of one or Produce and accumulate or secrete desired molecules, produce on a large scale.

Particularly suitable microorganisms for the production of PUFAs are microorganisms like the algae Phaeodactylum tricornutum or Crypthecodinium species, ciliates, such as Stylonichia or Colpidium, mushrooms such as Mortierella, Entomophthora, Mucor. By Strain selection is a number of mutant strains of the ent speaking microorganisms that have been developed a number of desirable compounds, including PUFAs. The selection of strains with improved production of a However, certain molecule is a time consuming and difficult one Method.

Alternatively, the production of fine chemicals may suitably be carried out on a large scale through the production of plants designed to produce the above-mentioned PUFAs. Plants which are particularly suitable for this purpose are oil-fruit plants which contain large amounts of lipid compounds, such as rapeseed, canola, flax, soybeans, sunflowers, borage and evening primrose. However, other crop plants containing oils or lipids and fatty acids are also well suited, as mentioned in the detailed description of this invention. Using conventional breeding, a number of mutant plants have been developed that produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes. However, the selection of new plant varieties with improved production of a particular molecule is a time-consuming and difficult process or even impossible if the compound does not occur naturally in the corresponding plant, as in the case of polyunsaturated C ₂₀ fatty acids and those with longer carbon chains.

Summary of the invention

This invention provides a new nucleic acid molecule that for the modification of oils, fatty acids, lipids, of lipids originating compounds and most preferred to Her position of polyunsaturated fatty acids can be used.

Microorganisms such as Phaeodactylum, Colpidium, Mortierella, Ent omophthora, Mucor, Crypthecodinium and other algae and fungi and plants, especially oil crops, are commonly found in the industry to produce a variety of fine chemicals used on a large scale.

Assuming the availability of cloning vectors and techniques for genetic manipulation of the above Microorganisms and ciliates as disclosed in WO 98/01572, or Algae and related organisms such as Phaeodactylum tricornutum,  described in Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251, and Dunahay et al., 1995, Genetic transformation of diatoms, J. Phycol. 31: 10004-1012 and the quotes in it can the nucleic acid molecules according to the invention gen technological change these organisms are used so that they are better or more efficient producers of one or several fine chemicals. This improved production or efficiency of the production of a fine chemical can by a direct effect of the manipulation of an inventive Gene or by an indirect effect of this manipulation are caused.

Mosses and algae are the only known plant systems the significant amounts of polyunsaturated fatty acids, such as Arachidonic acid (ARA) and / or eicosapentaenoic acid (EPA) and / or Prepare docosahexaenoic acid (DHA). Therefore, nucleuses are suitable acid molecules from a moss, like Physcomitrella patens, come, especially for the modification of the lipid and PUFA Production system in a host, especially in micro organisms such as the microorganisms mentioned above, and Plants, such as oil crops, for example rape, canola, Flax, soy, sunflower, borage. Furthermore, Nuklein acids from the moss Physcomitrella patens for identification such DNA sequences and enzymes in other species, which are used for Modification of the biosynthesis of precursor molecules of PUFAs in appropriate organisms are used.

The moss Physcomitrella patens is a member of the moss. It is related to other mosses, such as Ceratodon purpureus, which in Absence of light can grow. Mosses such as Ceratodon and Physcomitrella, are very high at the DNA sequence and polypeptide levels homologous to each other, which is the use of heterologous screening from DNA molecules with from other mosses or organisms allows the probes to derive a consensus sequence that can be used for heterologous screening or for functio nelle commenting and prediction of gene functions in third Species. The ability to identify these functions e.g. B. can predict the substrate specificity of enzymes therefore be of significant importance. Furthermore, these Nucleic acid molecules as reference sequences for mapping others Mosses or to derive PCR primers.

These new nucleic acid molecules can encode proteins here as PUFA-specific elongases (PSEs or in the singular PSE). For example, these PSEs can be one Exercise a function that is related to metabolism (e.g. biosynthesis or degradation) of compounds that are used for lipid or fatty acid synthesis  necessary, such as PUFAs, are involved or involved in the trans membrane transport of one or more lipid / fatty acid compounds either participate in or out of the cell.

In this new application, the function becomes one of the sequences shown in more detail. We have a functional for the first time active plant gene isolated, which is used to produce long-chain polyunsaturated fatty acids, preferably with more than eighteen carbon atoms in the carbon skeleton of fat acid and / or at least two double bonds in the carbon fabric chain, is suitable. Therefore, here is from a PSE gene or -Protein speech. Other publications and patents open did not reveal or show a functionally active PSE gene, although there are various known patent applications which the Extension of saturated fatty acids with short or medium Chain (WO 98/46776 and US 5,475,099) or the extension or Production of long chain fatty acids, but then no more than have a double bond or too long chain fatty acid wax lead esters (see: WO 98/54954, WO 96/13582, WO 95/15387) demonstrate.

WO 99/64616, WO 98/46763, WO 98/46764, WO 98/46765 describe the production of PUFAs in transgenic plants and show the cloning and functional expression of corresponding desaturase activities, in particular from fungi, but do not show an inevitable PSE- coding gene and no functional PSE activity. The production of a trienoic acid with a C ₁₈ carbon chain has been shown and has been claimed on the basis of gamma-linolenic acid, but it has not hitherto been the production of very long-chain polyunsaturated fatty acids (with a C ₂₀ and longer carbon chain as well as trienoic acids and higher unsaturated types). taught.

To produce long-chain PUFAs, the polyunsaturated C ₁₈ fatty acids must be extended by at least two carbon atoms by the enzymatic activity of an elongase. The nucleic acid sequence according to the invention encodes the first plant elongase, which can extend C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms. After one round of elongation, this enzyme activity leads to C ₂₀ fatty acids, and after two, three and four rounds of elongation leads to C ₂₂ , C ₂₄ or C ₂₆ fatty acids. Longer PUFAs can also be synthesized with the elongase according to the invention. The activity of the elongase according to the invention preferably leads to C ₂₀ and / or C ₂₂ fatty acids with at least two double bonds in the fatty acid molecule, preferably with three or four double bonds, particularly preferably three double bonds in the fatty acid molecule. After the extension with the enzyme according to the invention has taken place, further desaturation steps can take place. Therefore, the products of the elongase lead activity and the possible further desaturation to preferred PUFAs with a higher degree of desaturation as docosadienoic, arachidonic acid, ω6-Eicosatriendihomo-γ-linolenic acid, Eicosapenten acid, ω3-eicosatrienoic, ω3-eicosatetraenoic, docosapentaen acid or docosahexaenoic acid. Substrates of the enzyme activity according to the invention are, for example, taxolic acid; 6,9-octadecadiene acid, linoleic acid, γ-linolenic acid, pinolenic acid, α-linolenic acid or stearidonic acid. Preferred substrates are linoleic acid, γ-linolenic acid and / or α-linolenic acid. The C ₁₈ fatty acids with 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.

Assuming cloning vectors for use in Plants and in plant transformation, such as those that are published in and cited there: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), Chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer to higher plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, ed .: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Trans fer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed .: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)), the nucleic acids according to the invention for the genetic engineering change of a broad spectrum Use plants to make them a better or more efficient one Producer of one or more products derived from lipids, like PUFAs. This improved production or efficiency of the Production of a product derived from lipids, such as PUFAs, can be by direct manipulation or an indirect effect Effect of this manipulation.

There are a number of mechanisms through which change takes place the yield, production of a PSE protein according to the invention and / or efficiency of producing a fine chemical from a Oil crop or a microorganism due to a ver modified protein can directly affect. The number or Activity of the PSE protein or gene can be increased so that larger quantities of these compounds are produced de novo,  because the organisms have this activity and ability to biosynthesize before the corresponding gene was introduced.

The introduction of a PSE gene into an organism or Cell can not only increase the biosynthesis flow to the final product, but also the corresponding triacylglycerol composition increase or create de novo. Likewise, the number or Activity of other genes involved in the import of nutrients required for Biosynthesis of one or more fine chemicals (e.g. fatty acids, polar and neutral lipids) are necessary, so that the concentration of these precursors, cofactors or intermediates connections within cells or within memory compartment is increased, increasing the ability of the cells to Production of PUFAs as described below is increased. Fatty acids and lipids are themselves fine chemicals desirable; by optimizing the activity or Increase the number of one or more PSEs involved in the bio Synthesis of these compounds are involved, or by destroying them the activity of one or more PSEs involved in the breakdown of these Compounds are involved, it may be possible to increase the yield, Production and / or efficiency of the production of fatty acids and To increase lipid molecules from plants or microorganisms.

The mutagenesis of the PSE gene according to the invention can also be increased a PSE protein with modified activities that cause the Production of one or more desired fine chemicals directly or indirectly affect. For example, the number or Activity of the PSE gene according to the invention can be increased, so that the normal metabolic waste or by-products of Cell (its amount may be due to overproduction the desired fine chemical is increased) efficiently exported before other molecules or processes within the Cell (which would lower the cell's viability) disrupt or disrupt the biosynthetic pathways of the fine chemical (which increases the yield, production or efficiency of production the desired fine chemical is reduced). Can also the relatively large intracellular amounts of the desired fines chemical itself may be toxic to the cell or enzyme return interfere with coupling mechanisms, such as allosteric regulation, for example, by increasing activity or Number of other downstream enzymes or detoxification enzymes of the PUFA pathway the allocation of the PUFA to the triacyl gylcerin fraction increase, one could the viability of Increase seed cells, which in turn leads to better development of Cells in culture or seeds that produce the desired fine produce chemical. The PSE gene according to the invention can also are manipulated so that the appropriate amounts of the different  Lipid and fatty acid molecules are produced. This can have a drastic effect on the lipid composition of the The cell membrane has and produces new oils in addition to opening newly synthesized PUFAs. Because every lipid type was different has physical properties, a change in the Lipid composition of a membrane significantly increases membrane fluidity change. Changes in membrane fluidity can affect the Transport of molecules across the membrane and on the Unver affect cell integrity, both of which are crucial Have an effect on the production of fine chemicals. In Plants can also change these changes paint like tolerance to ablotic and biotic stress situations, influence.

Biotic and ablotic stress tolerance is a common feature times that you can think of a wide range of plants, like corn, Wheat, rye, oats, triticale, rice, barley, soybean, earth nut, cotton, rapeseed and canola, cassava, pepper, sunflower and tagetes, solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bush plants (coffee, Cocoa, tea), salix species, trees (oil palm, coconut) and out permanent grasses and fodder crops, would like to inherit. This Field crops are another embodiment of the invention also preferred target plants for genetic engineering. Especially preferred plants according to the invention are oil fruit plants, such as Soybean, peanut, rapeseed, canola, sunflower, safflower, trees (Oil palm, coconut) or crops such as corn, wheat, rye, Oats, triticale, rice, barley, alfalfa, or bush plants (Coffee, cocoa, tea).

Thus, one aspect of the invention relates to isolated nucleotides acid molecules (e.g. cDNAs) comprising a nucleotide sequence, encoding a PSE or biologically active parts thereof, or Nucleic acid fragments that can be used as primers or hybridizations probes for detection or amplification of PSE-encoding Nucleic acids (e.g. DNA or mRNA) are suitable. Especially before Preferred embodiments, the nucleic acid molecule comprises one of the in sequence ID NO: 1 nucleotide sequences shown or the coding region or a complement of one of these nucleotides sequences. In other particularly preferred embodiments the isolated nucleic acid molecule according to the invention comprises a Nucleotide sequence attached to a nucleotide sequence as in the Sequence SEQ ID NO: 1 shown, or a part thereof hybridi or at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90% and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more is homologous to it. In other preferred embodiments  the isolated nucleic acid molecule encodes one the amino acid sequences shown in the sequence SEQ ID NO: 2. The preferred PSE gene according to the invention preferably also has at least one of the PSE activities described here.

In a further embodiment, the isolated code Nucleic acid molecule is a protein or a part thereof, wherein the protein or part thereof contains an amino acid sequence, which are sufficiently homologous to an amino acid sequence of the sequence SEQ ID NO: 2 is that the protein or part thereof is a PSE Maintains activity. Preferably the protein or the Part of it encoded by the nucleic acid molecule that Ability to build on cell membranes by metabolism Plants necessary connections or at the transport of molecules participate through these membranes. In one embodiment at least the protein encoded by the nucleic acid molecule about 50%, preferably at least about 60% and more before prefers at least about 70%, 80% or 90% and most strongly does at least about 95%, 96%, 97%, 98%, 99% or more homo log to an amino acid sequence of sequence SEQ ID NO: 2 In another preferred embodiment, the protein is a full length Physcomitrella patens protein, which is essentially homologous to an entire amino acid sequence of SEQ ID NO: 2 (that of the open reading frame shown in SEQ ID NO: 1).

In another preferred embodiment, the isolated one stirs Nucleic acid molecule from Physcomitrella patens and encodes Protein (e.g. a PSE fusion protein) that is a biologically active Domain containing at least about 50% or more homologous to an amino acid sequence of the sequence SEQ ID NO: 2 and the Ability to build on cell membranes by metabolism Plants necessary connections or at the transport of molecules participate through these membranes, maintains or at least one of the activities listed in Table 1, and includes also heterologous nucleic acid sequences that a heterologous poly encode peptide or regulatory proteins.  

Table 1

Fatty acid pattern of five transgenic yeast strains in Mol%. The proportions of the γ-linolen supplied and absorbed acid are highlighted in bold numbers that the ver longer products are underlined and those of the extended ones γ-linolenic acid by numbers in bold (last line).

In another embodiment, this is nucleic acid isolated molecule at least 15 nucleotides long and hybridizes under stringent conditions on a nucleic acid molecule that a Nucleotide sequence of SEQ ID NO: 1. Preferably corresponds the isolated nucleic acid molecule to a naturally occurring one Nucleic acid molecule. The isolated code is more preferred Nucleic acid molecule of naturally occurring Physcomitrella patens PSE or a biologically active part of it.

Another aspect of the invention relates to vectors, e.g. B. recombinant expression vectors containing at least one fiction contain appropriate nucleic acid molecule, and host cells into which these vectors have been introduced, in particular micro organisms, plant cells, plant tissues, organs or whole Plants. In one embodiment, such a host cell Store fine chemical compounds, especially PUFAs; to Isolation of the desired compound, the cells are harvested. The compound (oils, lipids, triacylglycerides, fatty acids) or the PSE can then be extracted from the medium or the host cell in plants there are cells that contain fine chemicals or store, most preferred cells from storage tissues, such as seed shells, tubers, epidermis and sperm cells become.

Yet another aspect of the invention relates to a genetic one altered plant, preferably an oil crop, as above mentions, particularly preferably a Physcomitrella patens plant, into which a PSE gene has been introduced or in which a PSE gene has been changed. In one embodiment, the genome is of Physcomitrella patens by introducing an invention  Nucleic acid molecule that is a wild-type or mutant PSE Sequence encoded, changed as a transgene. At a another embodiment is an endogenous PSE gene in the genome of Physcomitrella patens plant by homologous recombination with a changed PSE gene, d. H. functionally destroyed, been. In a preferred embodiment, the Plant organism to the genus Physcomitrella, Ceratodon or Funaria, with Physcomitrella being particularly preferred. At a preferred embodiment is the Physcomitrella plant too to produce a desired compound such as lipids and Fatty acids, with PUFAs being particularly preferred.

In yet another preferred embodiment, the moss Physcomitrella patens to demonstrate the function of a moss gens using homologous recombination based on the nucleic acids described in this invention can be used.

Yet another aspect of the invention relates to an isolated one PSE gene or part, e.g. B. a biologically active part, from that. In a preferred embodiment, the isolated one PSE or part of it on the metabolism of to build up Cell membranes in a microorganism or a plant cell necessary connections or on the transport of molecules via whose membranes participate. Another preferred Embodiment is the isolated PSE or part thereof sufficient homologous to an amino acid sequence of SEQ ID NO: 2, that this protein or part of it has the ability to work on the substance change from to building up cell membranes in microorganisms or Plant cells necessary connections or at the transport of Participating molecules across these membranes persists.

The invention also provides an isolated preparation of a PSE. In preferred embodiments, the PSE gene comprises an amino acid sequence of SEQ ID NO: 2. In a further preferred embodiment, the invention relates to an isolated full-length protein which is essentially homologous to a total amino acid sequence of SEQ ID NO: 2 (which is derived from that in SEQ ID NO: 1 shown open reading frame is encoded). In another embodiment, the protein is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80% or 90%, and most preferably at least about 95%, 96%, 97%, 98% , 99% or more homologous to an amino acid sequence of the sequence SEQ ID NO: 2. In other embodiments, the isolated PSE comprises an amino acid sequence which is at least about 50% homologous to one of the amino acid sequences of SEQ ID NO: 2 and on the metabolism of compounds necessary for the formation of fatty acids in a microorganism or a plant cell or in the transport of molecules across these membranes or has one or more of the PUFA-prolonging activities, which means the extension of desatured C ₁₈ carbon chains with double bonds at at least two sites is.

Alternatively, the isolated PSE can be an amino acid sequence which are encoded by a nucleotide sequence attached to hybridizes a nucleotide sequence of SEQ ID NO: 1, e.g. More colorful stringent conditions hybridized, or at least approximately 50%, preferably at least about 60%, more preferred at least about 70%, 80% or 90% and even more preferred at least about 95%, 96%, 97%, 98%, 99% or more homologous is to. It is also preferred that the preferred PSE Form one of the PSE activities described here have.

The PSE polypeptide or a biologically active part thereof can be operably linked to a non-PSE polypeptide so that a fusion protein is formed. With preferred off this fusion protein has an activity that differs from that of the PSE alone. With others preferred embodiment takes this fusion protein on the fabric alternation of compounds used to synthesize lipids and fat acids, cofactors and enzymes in microorganisms or plants are necessary, or for the transport of molecules via them Membranes part. In particularly preferred embodiments modulates the introduction of this fusion protein into a host the production of a desired connection by the cell Cell. In a preferred embodiment, these contain Fusion proteins also delta 4, delta 5 or delta 6 desaturase Activities alone or in combination.

Another aspect of the invention relates to a method for Manufacture of a fine chemical. This procedure includes either the cultivation of a suitable microorganism or the Cultivation of plant cells, tissues, organs or whole Plants comprising the nucleotide sequence of the invention SEQ ID NO: 1 or their homologues, derivatives or analogues or a Gene construct that is SEQ ID NO: 1 or its homologs, derivatives or analogs, or a vector comprising this sequence or the gene construct comprising the expression of a fiction brings about according PSE nucleic acid molecule, so that a fine chemical is produced. In a preferred embodiment the method further includes the step of recovering a cell that such an elongase nucleic acid sequence according to the invention ent holds, a cell with an elongase nucleic acid sequence,  a gene construct or a vector which expresses the expression of a bring about PSE nucleic acid according to the invention, transformed becomes. In a further preferred embodiment comprises this method further includes the step of recovering the fine chemical from culture. In a particularly preferred embodiment the cell belongs to the order of the ciliates, to microorganisms, like mushrooms, or to the plant kingdom, especially to oil fruits plants, particularly preferred are microorganisms or oil fruit plants.

Another aspect of the invention relates to methods for Modulation of the production of a molecule by a micro organism. These procedures involve bringing the Cell with a substance that has PSE activity or PSE nucleic acid expression modulated so that a cell-associated Activity relative to the same activity in the absence of Substance is changed. In a preferred embodiment is one or two pathways of the cell for lipids and modulates fatty acids, cofactors and enzymes or the trans port of compounds modulated over these membranes so that the Yield or the rate of production of a desired fine chemical is improved by this microorganism. The Substance that modulates PSE activity can be a substance be the PSE activity or PSE nucleic acid expression stimulated or as an intermediate in the fatty acid bio synthesis can be used. Examples of substances which stimulate PSE activity or PSE nucleic acid expression, are u. a. small molecules, active PSEs and PSEs encoding Nucleic acids that have been introduced into the cell. At play for substances that have PSE activity or expression inhibit, are u. a. small molecules and antisense PSE nucleic acid molecules.

Another aspect of the invention relates to methods for Modulation of the yields of a desired compound from a Cell comprising introducing a wild type or mutant PSE Gene that is either kept on a separate plasmid or in the genome of the host cell is integrated into a cell. At Integration into the genome can be random or by recombination such that the native gene is replaced by the incorporated copy, reducing production the desired compound is modulated by the cell, or by using a gene in trans so that the gene is associated with a functional expression unit, which at least one the Expression of a gene facilitating sequence and at least one  the polyadenylation of a functionally transcribed gene contains relieving sequence, is functionally linked.

In a preferred embodiment, the yields are modified. In another preferred embodiment the desired chemical multiplied, with unwanted disruptive Connections can be reduced. With one especially before preferred embodiment is the desired fine chemical Lipid or a fatty acid, a cofactor or an enzyme. At particularly preferred embodiment, this chemical is a polyunsaturated fatty acid. It is more preferred from selected from arachidonic acid (ARA), eicosapentaenoic acid (AEP) or Docosahexaenoic acid (DHA).

Detailed description of the invention

The present invention provides PSE nucleic acids and proteins molecules ready, involved in lipid and fatty acid metabolism, PUFA cofactors and enzymes in the moss Physcomitrella patens or on the transport of lipophilic compounds across membranes are divided. The compounds of the invention can be used for Modulation of the production of fine chemicals from organisms, for example microorganisms, such as ciliates, fungi, yeasts, Bacteria, algae, and / or plants, such as corn, wheat, rye, Oats, triticale, rice, barley, soybean, peanut, cotton, Brassica species, such as rape, canola and turnip, pepper, sun flower, borage, evening primrose and tagetes, solanaceae plants, such as potato, tobacco, eggplant and tomato, Vicia species, pea, Cassava, alfalfa, bush plants (coffee, cocoa, tea), salix species, Trees (oil palm, coconut) and perennial grasses and forage field crops, either directly (e.g. when overexpression or Optimization of a direct fatty acid biosynthesis protein Influence on the yield, production and / or efficiency of the Production of the fatty acid from modified organisms has) ver turn or can have an indirect impact, but still have an increase in yield, production and / or efficiency of Production of a desired connection or a decrease in desired connections (e.g. if the modulation of the substance alternating between lipids and fatty acids, cofactors and enzymes Changes in yield, production and / or efficiency of the Production or the composition of the desired compounds inside the cells, which in turn leads to the production of a or several fine chemicals). Aspects of Invention are further explained below.  

I. Fine chemicals and PUFAs

The term "fine chemical" is known in the art and includes Molecules that have been produced by an organism and Find applications in different industries like, but not limited to, the pharmaceutical, agricultural, food medium and cosmetic industry. These include connections Lipids, fatty acids, cofactors and enzymes etc. (such as described in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., Ed., VCH: Weinheim and literature references contained therein), Lipids, saturated and unsaturated fatty acids (e.g. arachidone acid), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, Vitamins, Pp. 443-613 (1996) VCH: Weinheim and the literature contained therein put; and Ong, A.S., Niki, E., & Packer, L. (1995) Nutrition, Lipids, Health and Disease Proceedings of the UNESCO / Confedera tion of Scientific and Technological Associations in Malaysia and the Society for Free Radical Research - Asia on the 1st-3rd Sept. 1994 in Penang, Malysia, AOCS Press (1995)), Enzymes and all others by Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086, and chemicals described therein. The Metabolism and uses of certain fine chemicals are explained further below.

The combination of different precursor molecules and biosynthesis enzyme leads to the production of various fatty acid molecules, what a decisive impact on the composition of the membrane Has. It can be assumed that PUFAs are not just simple in Triacylglycerol, but also be incorporated into membrane lipids.

The synthesis of membranes is a well characterized process on which a number of components, including lipids as Part of the bilayer membrane, are involved. The production of new ones Fatty acids, such as PUFAs, can therefore have new membrane properties create functions within a cell or organism.

Cell membranes serve a variety of functions in one Cell. First and foremost, a membrane limits the content a cell from the environment, thereby giving cell integrity gives. Membranes can also act as barriers to this Influx of dangerous or unwanted connections and also serve against the outflow of desired connections.  

More detailed descriptions and participations of membranes and the mechanisms involved see in: Bamberg, E., et al. (1993) Charge transport of ion pumps on lipid bilayer membranes, Q. Rev. Biophys. 26: 1-25; Gennis, R.B. (1989) Pores, Channels and Transporters, in: Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 270-322; and Nikaido, H., and Saier, H. (1992) Transport proteins in bacteria: common themes in their design, Science 258: 936-942, and that in everyone of these citations.

Lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding to sn-glycerin-3- Phosphate and the addition or modification of a polar Head group. Common lipids used in membranes include phospholipids, glycolipids, sphingolipids and phospho glycerides. Fatty acid synthesis begins with the conversion of Acetyl-CoA in either malonyl-CoA through the acetyl-CoA-carboxy lase or in acetyl-ACP by acetyl transacylase. To These two product molecules form a condensation reaction together acetoacetyl-ACP, which has a series of condensation, Reduction and dehydration reactions is converted, so that a saturated fatty acid molecule with the desired chains length is obtained. Production of unsaturated fatty acids these molecules are catalyzed by specific desaturases based, either aerobically using molecular oxygen or anaerobic (with regard to the synthesis of fatty acids in microorganisms see F.C. Neidhardt et al. (1996) E. coli and Salmonella. ASM Press: Washington, D.C., pp. 612-636 and contained therein References; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and the one included References, as well as Magnuson, K., et al. (1993) Micro biological reviews 57: 522-542 and the literature included put).

Precursors for PUFA biosynthesis are, for example, linoleic and linolenic acid. These C ₁₈ carbon fatty acids must be extended to C ₂₀ and C ₂₂ in order to obtain fatty acids of the Eicosa and Docosa chain type. With the help of various desaturases, such as enzymes, which have delta-6-desaturase, delta-5 and delta-4 desaturase activity, arachidonic acid, eicosa pentenoic acid and docosahexaenoic acid and various other long-chain PUFAs can be obtained, extracted and used for various purposes in food , Feed, cosmetic or pharmaceutical applications are used.

To produce long-chain PUFAs, as mentioned above, the polyunsaturated C ₁₈ fatty acids must be extended by at least two carbon atoms through the enzyme activity of an elongase. The nucleic acid sequence according to the invention encodes a first plant elongase which can extend C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms. After one round of elongation, this enzyme activity leads to C ₂₀ fatty acids, and after two, three and four rounds of elongation leads to C ₂₂ , C ₂₄ or C ₂₆ fatty acids. Longer PUFAs can also be synthesized with the elongase according to the invention. The activity of the elongase according to the invention preferably leads to C ₂₀ and / or C ₂₂ fatty acids with at least two double bonds in the fatty acid molecule, preferably with three or four double bonds, particularly preferably three double bonds in the fatty acid molecule. After elongation with the enzyme according to the invention, further desaturation steps can take place. Therefore, the products of the elongase activity and the possible further desaturation lead to preferred PUFAs with a higher degree of desaturation as docosadienoic, arachidonic acid, ω6-Eicosatriendihomo-γ-linolenic acid, eicosapentaenoic acid, ω3-eicosatrienoic, ω3-eicosatetraenoic, docosapentaenoic acid or docosahexaenoic acid. Substrates of this enzyme activity according to the invention are, for example, taxolic acid, 6,9-octadecadienoic acid, linoleic acid, γ-linolenic acid, pinolenic acid, α-linolenic acid or stearidonic acid. Preferred substrates are linoleic acid, γ-linolenic acid and / or α-linolenic acid. The C ₁₈ fatty acids with at least two double bonds in the fatty acid can be extended by the enzyme 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.

Furthermore, fatty acids have to be added to various Modifications transported and stored in the triacylglycerol storage can be incorporated lipid. Another important step in the Lipid synthesis is the transfer of fatty acids to the polar Head groups, for example by glycerol fatty acid acyltrans ferase (see Frentzen, 1998, Lipid, 100 (4-5): 161-166).

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 the references therein see in the following articles: Kinney, 1997, Genetic Engeneering, Ed .: 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: 611-641; Voelker, 1996, Genetic Engineering, Ed .: JK Setlow, 18: 111-13; 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, ed .: Murata and Somerville, Rockville, American Society of Plant Physiologists, 150-158, Murphy & Ross 1998, Plant Journal. 13 (1): 1-16.

Vitamins, cofactors and nutraceuticals such as PUFAs include one A group of molecules that higher animals no longer synthesize can and therefore have to take up or the higher animals not can produce more sufficiently themselves and thus additionally need to absorb, although easily from other organisms, such as Bacteria to be synthesized. The biosynthesis of these molecules in organisms that they can produce, such as bacteria, has been broadly characterized (Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, Pp. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley &Sons; Ong, A.S., Niki, E., & Packer, L. (1995) "Nutrition, Lipids, Health and Disease" Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia and the Society for Free Radical Research Asia, held on 1-3. Sept. 1994 in Penang, Malaysia, AOCS Press, Champaign, IL X, 374 S).

The molecules mentioned above are either biological themselves active molecules or precursors of biologically active substances that either as an electron carrier or as an intermediate in one Serve a variety of metabolic pathways. Have these connections in addition to their nutritional value, also a significant industrial value as dyes, antioxidants and catalysts or other ver work aids. (An overview of structure, activity and for industrial applications of these compounds see e.g. B. in Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996). Polyunsaturated Fatty acids have various functions and are beneficial to health Effects, for example in coronary heart disease, ent ignition mechanisms, child nutrition, etc. publications and references, including literature cited therein see, in: Simopoulos, 1999, Am. J. Clin. Nutr. 70 (3rd Suppl.): 560-569, Takahata et al., Biosc. Biotechnol. Biochem. 1998, 62 (11): 2079-2085, Willich and Winther, 1995, German Medical weekly 120 (7): 229ff.  

II. Elements and methods of the invention

The present invention is based, at least in part, on the Ent coverage of a new molecule, here called PSE nucleic acid and -Protein molecules is called, which have an effect on the Production of cell membranes in Physcomitrella patens and Exercise Ceratodon purpureus and, for example, exercise influenced by molecules across these membranes. With an off In the leadership form, the PSE molecules take their metabolism from to Structure of cell membranes in organisms such as microorganisms and Plants, necessary connections partially or indirectly influence the transport of molecules across these membranes. With a be preferred embodiment has the activity of the invention PSE molecules for regulating the production of membrane components and membrane transport have an impact on the production of desired fine chemical by this organism. At a particularly preferred embodiment is the activity of PSE molecules according to the invention modulated so that the yield, Production and / or efficiency of the production of the metabolic pathways of microorganisms or plants which the invention Regulate PSEs, are modulated and the efficiency of transportation of connections through the membranes is what either directly or indirectly the yield, production and / or efficiency the production of a desired fine chemical by micro organisms and plants modulated.

The term PSE or PSE polypeptide includes proteins that are on Metabolism of to build cell membranes in organisms, such as microorganisms and plants, necessary compounds or participate in the transport of molecules across these membranes. Examples of PSEs are in SEQ ID NO: 1 or their homologs, Derivatives or analogs disclosed. The terms PSE or PSE Nucleic acid sequence (s) include nucleic acid sequences that are one Encode PSE and in which a part is a coding region and also corresponding 5'- and 3'-untranslated sequence areas is. Examples of PSE genes are those in SEQ ID NO: 1 shown. The terms production or productivity are known in the art and include the concentration of Fermentation product (for example the desired fine chemical), which in a certain period of time and be certain fermentation volume is formed (e.g. kg product per Hour per liter). The term efficiency of production encompasses the time it takes to achieve a certain amount of production is necessary (e.g. how long the cell takes to erect a certain throughput rate of a fine chemical is required). The The term yield or product / carbon yield is in the art known and includes the efficiency of the conversion of the  Carbon source in the product (i.e. the fine chemical). This is usually expressed, for example, as kg of product per kg Carbon source. By increasing yield or production the connection is the amount of molecules obtained or the suitable obtained molecules of this compound in a be certain amount of culture increased over a specified period. The Terms biosynthesis or biosynthetic pathway are in the field Knows and include the synthesis of a compound, preferably an organic compound, through a cell from intermediate connections, for example in a multi-step and strong regulated process. The terms degradation or degradation route are in Known in the art and include splitting a connection, preferably an organic compound, through a cell in breakdown products (more generally, smaller or fewer complex molecules) for example in a multi-step and strong regulated process. The term metabolism is in the specialty known and encompasses all biochemical reactions, that take place in an organism. The metabolism of one certain compound (e.g. the metabolism of a fatty acid) then comprises the entirety of the biosynthesis, modification and Degradation pathways of this connection in the cell that this connection affect.

In another embodiment, the inventive PSE molecules like the production of a desired molecule a fine chemical, in a microorganism or in plants modulate. There are a number of mechanisms through which the Changing a PSR according to the invention, the yield, production and / or efficiency of production of a fine chemical a microorganism or plant strain that changed it Contain protein, can directly influence. The number or Activity of PSEs involved in the transport of fine chemical molecules involved inside or out of the cell can be increased so that larger amounts of these compounds across membranes trans be ported from which they are more easily extracted and into each other being transformed. Also fatty acids are triacylglycerols and / or lipids themselves desirable fine chemicals; by Optimizing activity or increasing the number of one or of several PSEs according to the invention which are involved in the biosynthesis of these Connections are involved, or by disrupting the activity one or more PSEs involved in breaking down these connections involved, it may be possible the yield, production and / or efficiency of production of fatty acid and lipid molecules from organisms, such as microorganisms or plants, to increase.  

The mutagenesis of the PSE gene according to the invention can also be done with PSEs bring about changed activities that affect production one or more desired micro fine chemicals indirectly affect organisms or plants. For example can PSEs according to the invention, the export of waste products are involved in a greater number or higher activity point so that the normal metabolic waste of the cell (whose Quantity may be due to overproduction of the desired one Fine chemical is increased) can be exported efficiently before which can damage molecules in the cell (which is viable cell would decrease) or the fine chemicals bio synthesis pathways (what the yield, production or Reduce the efficiency of the production of a desired fine chemical would). The relatively large intracellular amounts of the desired Fine chemicals themselves can also be toxic to the cell so that by increasing the activity or number of trans porters who can export these connections from the cell what can increase the viability of the cell in culture again leads to a larger number of cells in the culture, which produce the desired fine chemical. The fiction according to PSEs can also be manipulated so that the ent speaking amounts of different lipid and fatty acid molecules to be produced. This can have a significant impact on the Have lipid composition of the cell membrane. Because every lipid type has different physical properties, a Change in the lipid composition of a membrane the membrane change fluidity significantly. Changes in membrane fluidity can transport molecules across the membrane as well Integrity of the cell affect, each of which is significant Impact on the production of fine chemicals from micro organisms and plants in fermentation culture on a large scale stab. Plant membranes impart specific properties, such as tolerance to heat, cold, salt, dryness as well Tolerance to pathogens such as bacteria and fungi. Therefore, the Modulation of the membrane components has a fundamental effect the viability of the plants among the above Have stress parameters. This can result in changes in signal cascades or directly via the changed membrane composition (see for example: Chapman, 1998, Trends in Plant Science, 3 (11): 419-426) and signal cascades (see Wang 1999, Plant Physiology, 120: 645-651) or the cold tolerance, such as disclosed in WO 95/18222.

The isolated nucleic acid sequence according to the invention is in the genome of a Physcomitrella patens strain contained in the moss University of Hamburg collection is available. The nucleotide sequence of the isolated Physcomitrella patens cDNA and the derived ones  Amino acid sequences of the Physcomitrella patens PSEs are shown in SEQ ID NO: 1 and 2, respectively. There were computer analyzes performed that classify these nucleotide sequences as sequences fected and / or identified that are involved in the metabolism of cells proteins involved in membrane components or in the transport of ver encode proteins involved in cell membrane bonds. On EST with database entry no. 08_ck19_b07 of the inventor Part of the sequence shown in SEQ ID NO: 1. Meanwhile these ESTs renamed, resulting in the revised name: pp001019019f led. The partial poly peptide referred to as pp001019019f. The complete fragment Insert of the EST pp001019019f was sequenced to give the SEQ ID NO: 1, which shows the complete sequence of pp001019019f shows. It contains a complete, functionally active clone, which was found after specific expression in yeast, that he had the desired substrate specificity, as in the case game part is shown. Yeasts are also suitable according to the invention Organisms, for example as host cells for the invention appropriate genes, gene constructs or vectors.

The present invention also relates to proteins with an amino acid sequence that is essentially homologous to an amino acid sequence of SEQ ID NO: 2. As used here is a protein with an amino acid sequence that is essentially homologous to a amino acid sequence selected is at least about 50% homo log to the selected amino acid sequence, e.g. B. the entire selected amino acid sequence. A protein with an amino acid sequence that is essentially homologous to a selected amino acid sequence can also be at least about 50 to 60%, preferably at least about 60 to 70% and more preferred at least about 70 to 80%, 80 to 90% or 90 to 95% and most preferably at least about 96%, 97%, 98%, 99% or more homologous to a selected amino acid sequence.

The PSE according to the invention or the biologically active part or the fragment thereof can participate in the metabolism of compounds necessary for the construction of cell membranes in microorganisms or plants or in the transport of molecules across these membranes or one or more of the activities required for elongating C ₁₈ PUFAs have so that C ₂₂ or C ₂₄ PUFAs and related PUFAs are obtained.

Various aspects of the invention are further discussed in the following subsections.  

A. Isolated nucleic acid molecules

One embodiment of the invention is an isolated nucleic acid derived from a plant and encoding a polypeptide that extends C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms.

A further embodiment according to the invention is an isolated nucleic acid, comprising a nucleotide sequence which encodes a polypeptide which extends C ₁₈ fatty acids with at least two double bonds in the fatty acid and is selected from the group consisting of

• a) a nucleic acid sequence shown in SEQ ID NO: 1,
• b) a nucleic acid sequence which according to the degenerate genetic code from the sequence shown in SEQ ID NO: 1 comes from
• c) Derivatives of the sequence shown in SEQ ID NO: 1, which encodes polypeptides with at least 50% homology to the sequence which encodes the amino acid sequences shown in SEQ ID NO: 2, the sequences acting as C ₁₈ elongase.

The above-mentioned nucleic acid according to the invention comes from Organisms such as ciliates, fungi, algae or dinoflagellates, able to synthesize PUFAs, preferably from plants, particularly preferred from the genus Physcomitrella and am most preferred by Physcomitrella patens.

One aspect of the invention relates to isolated nucleic acid moles cool, the PSE polypeptides or biologically active parts thereof encode, as well as nucleic acid fragments for use as Hybridization probes or primers for identification or Amplification of a PSE-encoding nucleic acid (e.g. PSE-DNA) suffice. The term "nucleic acid molecule" as ver here DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules (e.g. mRNA) as well as DNA or RNA analogues, which by means of Nucleotide analogs are generated include. This term includes also those at the 3 'and 5' ends of the coding gene region located untranslated sequence: at least about 100 nucleotides the sequence upstream of the 5 'end of the coding region and at least about 20 nucleotides of the sequence downstream of the 3 'end of the coding gene region. The nucleic acid molecule can be single-stranded or double-stranded, but is preferred wise double-stranded DNA. An "isolated" nucleic acid molecule  is separated from other nucleic acid molecules that are in the natural source of the nucleic acid. An "isolated" Nucleic acid preferably has no sequences that the Nucleic acid in the genomic DNA of the organism from which the Nucleic acid originates, naturally flanking (e.g. sequences, which are located at the 5 'and 3' ends of the nucleic acid). At In various embodiments, the isolated PSE nucleus acid molecule for example less than about 5 kb, 4 kb, 3 kb, Contain 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences, which naturally contains the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates (e.g. a Flank Physcomitrella patens cell). An "isolated" Nucleic acid molecule, such as a cDNA molecule, can also be in the essentially free of other cellular material or culture be medium if it is made by recombinant techniques becomes, or free of chemical precursors or other chemicals be when it's chemically synthesized.

A nucleic acid molecule according to the invention, e.g. B. a nucleus acid molecule with a nucleotide sequence of SEQ ID NO: 1 or part of it can be used using molecular biology Standard techniques and the sequence provided here information to be isolated. For example, a P. patens cDNA can be isolated from a P. patens bank by using the full permanent SEQ ID NO: 1 or a part thereof as a hybridization probe as well as standard hybridization techniques (such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In addition, a nucleic acid molecule comprising one full can permanent sequence of SEQ ID NO: 1 or a part thereof Isolate polymerase chain reaction, wherein oligonucleotide primers, based on this sequence or parts thereof, ins special regions around His box motifs, see Shanklin et al. (1994) Biochemistry 33, 12787-12794 (e.g., a nucleic acid molecule comprising the full permanent sequence of SEQ ID NO: 1 or a part thereof Polymerase chain reaction using oligonucleotide primers are isolated that are based on these same Sequence of SEQ ID NO: 1 have been created). For example mRNA can be isolated from plant cells (e.g. by the Guanidinium thiocyanate extraction method by Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and reverse cDNA Transcriptase (e.g. Moloney MLV reverse transcriptase available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL) produce. Synthetic oligonucleotide primers for amplification  by means of a polymerase chain reaction on the basis of a create the nucleotide sequences shown in SEQ ID NO: 1. A The nucleic acid according to the invention can be used using cDNA or alternatively from genomic DNA as template and suitable oligo nucleotide primers according to standard PCR amplification techniques be amplified. The nucleic acid amplified in this way can be in a suitable vector can be cloned and by means of DNA sequence analysis can be characterized. Oligonucleotides that a PSE Nucleotide sequence can match by standard synthesis procedure, for example with an automatic DNA synthesis device, manufactured.

The cDNA shown in SEQ ID NO: 1 comprises sequences, the PSEs encode (i.e. the "coding region") and 5'-untrans latated sequences and 3'-untranslated sequences. Alternatively the nucleic acid molecule can only encode the coding region of the sequences in SEQ ID NO: 1 or can be whole genomic Contain fragments isolated from genomic DNA.

SEQ ID NO: 2 is identified by the same EST entry number Label identifies as SEQ ID NO: 1, making it easy can be correlated. For example, that is as PpPSE1 designated amino acid sequence in SEQ ID NO: 2 a translation the coding region of the nucleotide sequence of the nucleic acid molecule pp001019019f.

In a further preferred embodiment, a isolated nucleic acid molecule according to the invention a nucleotide acid molecule that is a complement of one of SEQ ID NO: 1 shown nucleotide sequences or a part thereof. On Nucleic acid molecule belonging to one of those shown in SEQ ID NO: 1 Nucleotide sequences is complementary, is one that is related to one of the nucleotide sequences shown in SEQ ID NO: 1 are sufficient is complementary that it is with one of those specified in SEQ ID NO: 1 Sequences can hybridize, creating a stable duplex ent stands.

Homologs of the new elongase nucleic acid sequence with the sequence SEQ ID NO: 1 means, for example, allelic variants at least about 50 to 60%, preferably at least about 60 to 70%, more preferably at least about 70 to 80%, 80 to 90% or 90 to 95% and more preferably at least about 95%, 96%, 97%, 98%, 99% or more homology to one in SEQ ID NO: 1 shown nucleotide sequences or their homologues, Derivatives or analogs or parts thereof. Another preferred embodiment comprises an isolated invention according nucleic acid molecule a nucleotide sequence attached to a  or part of the nucleotide sequences shown in SEQ ID NO: 1 hybridized thereof, e.g. B. hybridi under stringent conditions siert. Allelic variants include functional ones in particular Variants characterized by deletion, insertion or substitution of nucleotides from / in the sequence shown in SEQ ID NO: 1 can be obtained, but the intention is that the enzyme activity of the resulting synthesized proteins for the Advantageously maintain insertion of one or more genes becomes. Proteins that still have the enzymatic activity of elongase own, means proteins with at least 10%, preferably 20%, particularly preferably 30%, very particularly preferably 40% the original enzyme activity compared to that by SEQ ID NO: 2 encoded protein.

Homologs of SEQ ID NO: 2 also means, for example bacterial, fungal and plant homologs, shortened sequences, single-stranded DNA or RNA of the coding and non- coding DNA sequence.

Homologs of SEQ ID NO: 1 also means derivatives, such as wise promoter variants. The promoters upstream of the specified nucleotide sequences can be one or more Nucleotide exchanges, by insertion (s) and / or deletion (s) be modified without, however, the functionality or Activity of the promoters is disturbed. It is still possible that the activity of the promoters by modification of their sequence is increased or that it is completely replaced by more active promoters, even from heterologous organisms.

In addition, the nucleic acid molecule according to the invention can only have one Part of the coding region of one of the sequences in SEQ ID NO: 1 include, for example, a fragment that acts as a probe or Primer can be used, or a fragment containing a encoded biologically active section of a PSE. The one from the Cloning of the PSE gene from P, patens-determined nucleotide sequences enable the generation of probes and primers, those for the identification and / or cloning of PSE homologs in other cell types and organisms as well as PSE homologues from others Mosses or related species are designed. The probe / primer usually comprises essentially purified oligonucleotide. The Oligonucleotide usually includes a nucleotide sequence region the under stringent conditions at least about 12, preferably is about 16, more preferably about 25, 40, 50 or 75 consecutive nucleotides of a sense strand one of the in SEQ ID NO: 1 specified sequences, an antisense strand one the sequences specified in SEQ ID NO: 1 or its homologues, Derivatives or analogs or naturally occurring mutants thereof  hybridizes. Primer based on a nucleotide sequence of SEQ ID NO: 1 can be used in PCR reactions for cloning PSE homologs can be used. Probes based on the PSE Nucleotide sequences can be used for the detection of transcripts or genomic sequences that are the same or homologous proteins encode, are used. In preferred embodiments the probe also includes a label group attached to it, e.g. B. a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. These probes can be part of a Test kits for genomic markers to identify cells, that mis-express a PSE, for example by measuring one Amount of a PSE-encoding nucleic acid in a cell sample, e.g. B. Measure PSE mRNA levels, or to determine whether a genomic PSE gene is mutated or deleted can be used.

In one embodiment, the nucleotide according to the invention encodes acid molecule a protein or part thereof, the one Amino acid sequence that is sufficiently homologous to an amino acid sequence of SEQ ID NO: 2 is that the protein or part of which the ability to metabolize to build cells membranes in microorganisms or plants necessary ver bonds or the transport of molecules across these membranes to participate, maintains. As used here, the term refers to "sufficiently homologous" proteins or parts thereof, their amino acid sequences a minimal number of identical or equivalent Amino acid residues (e.g. an amino acid residue with a similar one Side chain, like an amino acid residue in one of the sequences of the SEQ ID NO: 2) to an amino acid sequence of SEQ ID NO: 2 point so that the protein or part of it on metabolism for building cell membranes in microorganisms or plants necessary connections or on the transport of molecules via these membranes can participate. Protein components of this Metabolic pathways for membrane components or membrane transport systems, as described here, can play a role in Production and secretion of one or more fine chemicals play. Examples of these activities are also here described. Thus, the "function of a PSE" either bears directly or indirectly to the yield, production and / or efficiency of the Production of one or more fine chemicals at. Examples for PSE substrate specificities of catalytic activity given in Table 1.

In a further embodiment, derivatives of the Nucleic acid molecule according to the invention proteins with at least about 50 to 60%, preferably at least about 60 to 70% and more preferably at least about 70 to 80%, 80 to 90%, 90 to 95% and most preferably at least about 96%,  97%, 98%, 99% or more homology to complete Amino acid sequence of SEQ ID NO: 2. The homology of the amino acid Sequence was made over the entire sequence area with the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5, 1989: 151-153).

Parts of proteins produced by the PSE nucleic acid according to the invention acid molecules are encoded, are preferably biological active parts of one of the PSEs. As used here, the term is meant to "biologically active part of a PSE", a section, e.g. Legs Domain / motive to include a PSE that is involved in the metabolism of the Structure of cell membranes in microorganisms or plants agile connections or the transport of molecules via them Membranes can participate or one given in Table 1 Has activity. To determine whether a PSE or a bio logically active part of it on the metabolism of to build up Ver. Necessary cell membranes in microorganisms or plants bonds or the transport of molecules across these membranes can take a test of enzymatic activity by be performed. These test procedures, as detailed in Example 8 described in the example part, are familiar to those skilled in the art.

Additional nucleic acid fragments that are biologically active Coding sections of a PSE can be done by isolation a part of one of the sequences in SEQ ID NO: 2 the encoded portion of the PSE or peptide (e.g., by recombinant expression in vitro) and determination of the activity of the encoded portion of the PSE or peptide.

The invention also encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in SEQ ID NO: 1 (and parts of this) due to the degenerate genetic code and thus encode the same PSE as that used by those in SEQ ID NO: 1 nucleotide sequences shown is encoded. At a another embodiment has an insulated according to the invention Nucleic acid molecule is a nucleotide sequence that contains a protein an amino acid sequence shown in SEQ ID NO: 2. At a further embodiment encodes the invention Nucleic acid molecule a full-length Physcomitrella patens protein, which results in an amino acid sequence of SEQ ID NO: 2 (which is derived from one in SEQ ID NO: 1 shown open reading frame is encoded) essentially is homologous.

In addition to the Physcomitrella patens- shown in SEQ ID NO: 1 Those skilled in the art will recognize PSE nucleotide sequences that DNA sequence poly morphisms that lead to changes in the amino acid sequences of the PSEs lead within a population (e.g. Physcomitrella  patens population) can exist. This genetic poly Morphisms in the PSE gene can occur between individuals within one Population due to natural variation exist. How As used herein, the terms "gene" and "recombinant" mean Gene "nucleic acid molecules with an open reading frame, the one PSE, preferably a Physcomitrella patens PSE, encoded. This natural variants usually result in a variance of 1 up to 5% in the nucleotide sequence of the PSE gene. All and all of these nucleotide variations and the resulting amino acid polymorphisms in PSE that are the result of natural variation and do not change the functional activity of PSEs, are intended to be included within the scope of the invention.

Nucleic acid molecules which correspond to the natural variants and non-Physcomitrella patens homologues, derivatives and analogs of the Physcomitrella patens PSE cDNA according to the invention can be used on the basis of their homology to the Physcomitrella patens PSE nucleic acid disclosed here using the Physcomitrella patens -cDNA or part thereof can be isolated as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. In another embodiment, an isolated nucleic acid molecule according to the invention is at least 15 nucleotides long and hybridizes under stringent conditions with the nucleic acid molecule which comprises a nucleotide sequence of SEQ ID NO: 1. In other embodiments, the nucleic acid is at least 25, 50, 100, 250 or more nucleotides in length. The term "hybridizes under stringent conditions", as used here, is intended to describe hybridization and washing conditions under which nucleotide sequences which are at least 60% homologous to one another usually remain hybridized to one another. The conditions are preferably such that sequences that are at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to one another usually remain hybridized to one another. These stringent conditions are known to the person skilled 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, non-limiting example of stringent hybridization conditions are hybridizations in 6 × sodium chloride / sodium citrate ( s odium chloride / s odium c itrate = SSC) at approximately 45 ° C., followed by one or more washing steps in 0.2 × SSC, 0 , 1% SDS at 50 to 65 ° C. The person skilled in the art is aware that these hybridization conditions differ depending on the type of nucleic acid and, if organic solvents are present, for example, with regard 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 an aqueous buffer with a concentration of 0.1 to 5 × 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. The hybridization conditions for DNA: DNA hybrids are preferably, for example, 0.1 × SSC and 20 ° C. to 45 ° C., preferably between 30 ° C. and 45 ° C. The hybridization conditions for DNA: RNA hybrids are preferably, for example, 0.1 × SSC and 30 ° C. to 55 ° C., preferably between 45 ° C. and 55 ° C. The above-mentioned hybridization temperatures are determined, for example, for a nucleic acid with a length of about 100 bp (= base pairs) and a G + C content of 50% in the absence of formamide. Those skilled in the art know how the required hybridization conditions are based on textbooks, such as that mentioned above or from the following textbooks Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames 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.

An isolated nucleotide according to the invention preferably corresponds acid molecule attached to a sequence under stringent conditions the SEQ ID NO: 1 hybridizes, a naturally occurring one Nucleic acid molecule. As used here, a "of course concerns occurring "nucleic acid molecule with an RNA or DNA molecule a nucleotide sequence that occurs naturally (e.g. a encodes natural protein). Encoded in one embodiment the nucleic acid is a naturally occurring Physcomitrella patens-PSE.

In addition to naturally occurring variants of the PSE sequence, those skilled in the art recognize those that may exist in the population further that changes by mutation into a nucleotide sequence of SEQ ID NO: 1 can be introduced, resulting in Changes in the amino acid sequence of the encoded PSE results without that the functionality of the PSE protein is impaired. For example, nucleotide substitutions that are "non-essential" amino acid residues for amino acid substitutions lead in a sequence of SEQ ID NO: 1. A "not- essential "amino acid residue is a residue that is in one Wildtypenzsequenz one of the PSEs (SEQ ID NO: 2) can be changed, without changing the activity of the PSE, whereas a "Essential" amino acid residue required for PSE activity is. Other amino acid residues (e.g. those in the Domain with PSE activity not conserved or only  are semi-preserved) but cannot be used for the activity essential and can probably be changed, without changing the PSE activity.

Accordingly, in another aspect of the invention relates to nucleic acid Molecules that encode PSEs ent the modified amino acid residues hold that are not essential for PSE activity. This PSEs differ in amino acid sequence from one Sequence in SEQ ID NO: 2 and still retain at least one of the PSE activities described here. The isolated nucleic acid In one embodiment, the molecule comprises a nucleotide sequence, which encodes a protein, the protein being an amino acid sequence with at least about 50% homology to an amino acid sequence of the SEQ ID NO: 2 includes and on the metabolism of to build cells membranes in P. patens necessary connections or on transport of molecules can participate through these membranes or one or has several of the activities listed in Table 1. The protein encoded by the nucleic acid molecule is preferred at least about 50 to 60% homologous to one of the sequences in SEQ ID NO: 2, more preferably at least about 60 to 70% homo log to one of the sequences in SEQ ID NO: 2, even more preferred at least about 70 to 80%, 80 to 90%, 90 to 95% homologous to one of the sequences in SEQ ID NO: 2 and most preferred at least about 96%, 97%, 98% or 99% homologous to one of the Sequences in SEQ ID NO: 2.

To determine the percentage homology of two amino acids sequences (e.g. one of the sequences of SEQ ID NO: 2 and one mutated form thereof) or of two nucleic acids Sequences for the purpose of optimal comparison with one another written (e.g., gaps in the sequence of a protein or a nucleic acid to be optimal Alignment with the other protein or nucleic acid to create). The amino acid residues or nucleotides on the ent speaking amino acid positions or nucleotide positions then compared. If a position in a sequence (e.g. one of the sequences of SEQ ID NO: 2) by the same amino acid residue or the same nucleotide as the corresponding site in the other sequence (e.g. a mutated form of the one from SEQ ID NO: 2 selected sequence), the molecules are on homologous to that position (i.e. amino acid or nucleic acid "Homology" as used here corresponds to amino acid or Nucleic acid "identity"). The percentage homology between the both sequences is a function of the number of identical ones Positions that are common to the sequences (i.e.% homology = Number of identical positions / total number of positions × 100).  

An isolated nucleic acid molecule that encodes a PSE a protein sequence of SEQ ID NO: 2 is homologous by Ein bring one or more nucleotide substitutions, additions or deletions generated in a nucleotide sequence of SEQ ID NO: 1 so that one or more amino acid substitutions, additions or deletions introduced into the encoded protein become. Mutations can occur in one of the sequences of SEQ ID NO: 1 using standard techniques such as site-specific mutagenesis and PCR-mediated mutagenesis. Preferably become conservative amino acid substitutions on one or several of the predicted non-essential amino acid residues manufactured. With a "conservative amino acid substitution" the amino acid residue is replaced by an amino acid residue with a similar side chain replaced. There are families in the field of amino acid residues with similar side chains. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. Aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, Cysteine), non-polar side chains, (e.g. alanine, valine, leucine, Isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, trypto phan, histidine). A predicted non-essential amino acid residue in a PSE is thus preferably by a other amino acid residue from the same side chain family exchanged. Alternatively, in another embodiment the mutations randomly over all or part the PSE coding sequence are introduced, e.g. B. by Saturation mutagenesis, and the resulting mutants can screened for the PSE activity described here, to identify mutants that maintain PSE activity. After mutagenesis of one of the sequences of SEQ ID NO: 1, this can encoded protein can be expressed recombinantly, and the activity the protein can e.g. B. using those described here Tests (see example part) can be determined.

In addition to the nucleic acid molecules that the above encoding PSEs described concerns another aspect of Invention isolated nucleic acid molecules, the "antisense" to it are. An "antisense" nucleic acid comprises a nucleotide sequence to a "sense" nucleic acid that encodes a protein, is complementary, e.g. B. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA Sequence. An antisense nucleic acid can therefore be above water Bind fabric bonds to a sense nucleic acid. The Antisense nucleic acid can encode an entire PSE  Strand or only complementary to part of it. At a Embodiment is an antisense nucleic acid molecule "antisense" to a "coding area" of the coding strand Nucleotide sequence encoding a PSE. The term "coding Area "refers to the area of the nucleotide sequence, the codons which are translated into amino acid residues (e.g. the entire coding area starting with the stop codon and ends, d. H. the last codon before the stop codon). At a Another embodiment is the antisense nucleic acid molecule "Antisense" to a "non-coding area" of the coding Strands of a nucleotide sequence that encodes PSE. The term "Non-coding region" refers to 5 'and 3' sequences that flank the coding area and not in amino acids be translated (i.e. which are also called 5'- and 3'-untrans latated areas).

Provided the PSE encoding disclosed here Sequences of the coding strand (e.g. those in SEQ ID NO: 1 sequences) can antisense nucleic acids according to the invention acids designed according to the rules of Watson-Crick base pairing become. The antisense nucleic acid molecule can be complementary to entire coding region of PSE mRNA, but is stronger preferably an oligonucleotide that only encode part of the the or non-coding region of PSE mRNA is "antisense". The antisense oligonucleotide can e.g. B. to the area that the Translation start site surrounded by PSE mRNA, be complementary. An antisense oligonucleotide can e.g. B. about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 and more nucleotides in length. A antisense nucleic acid of the invention can be used chemical synthesis and enzymatic ligation reactions using be constructed in the art of known methods. A Antisense nucleic acid (e.g. an antisense oligonucleotide) can e.g. B. chemically synthesized, naturally occurring Nucleotides or variously modified nucleotides ver are used, which are designed so that they the biological Molecular stability increase or physical stability that formed between the antisense and sense nucleic acids Increase duplexes, for example, phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples for modified nucleotides that are used to generate the antisense Nucleic acid can be used u. a. 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-ioduracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxy methylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, Dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,  N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxy carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentyl adenine, uracil-5-oxyacetic acid (v), wybutoxosin, pseudouracil, Queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, Uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3- N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. The anti Alternatively, sense nucleic acid can be used biologically an expression vector into which a nucleotide acid has been subcloned in the antisense direction (i.e. RNA, which is transcribed by the introduced nucleic acid to a target nucleic acid of interest in the antisense direction oriented what continues in the subsection below is described).

The antisense nucleic acid molecules according to the invention are usually administered to a cell or generated in situ, so that they are associated with the cellular mRNA and / or the genomic DNA encode, hybridize, or bind a PSE to thereby encode the Expression of the protein, e.g. B. by inhibiting transcription and / or translation. Hybridization can be done by conventional nucleotide complementarity to form a stable duplexes or z. B. in the case of an antisense nucleic acid molecule that binds DNA duplexes through specific changes effects occur in the large furrow of the double helix. The Antisense molecule can be modified to be specific a receptor or to one on a selected cell surface expressed antigen binds e.g. B. by binding the antisense Nucleic acid molecule to a peptide or an antibody, the binds to a cell surface receptor or an antigen. The Antisense nucleic acid molecule can also be used here described vectors are supplied to the cells. To achieve sufficient intracellular concentrations of antisense Molecules are vector constructs in which the antisense Nucleic acid molecule under the control of a strong pro caryotic, viral or eukaryotic, including vegetable, promoter is preferred.

In a further embodiment, the anti according to the invention is sense nucleic acid molecule is an α-anomeric nucleic acid molecule. On α-anomeric nucleic acid molecule forms specific double-stranded ones Hybrid with complementary RNA, the strands being unlike ordinary β units run parallel to each other. (Gaultier et al. (1987) Nucleic Acids Res. 15: 6625-6641). The antisense Nucleic acid molecule can also be a 2'-o-methyl ribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15: 6131-6148) or  a chimeric RNA-DNA analog (Inoue et al. (1987) FEBS Lett. 215: 327-330).

In a further embodiment, one according to the invention Antisense nucleic acid a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are single-stranded Can cleave nucleic acid, such as an mRNA, to which they join have complementary area. Thus ribozymes (e.g. Hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) for the catalytic cleavage of PSE mRNA Transcripts are used to thereby translate Inhibit PSE mRNA. A ribozyme with specificity for a PSE coding nucleic acid can be based on the nucleotide sequence a PSE cDNA disclosed here (i.e. 38_ck21_g07fwd in SEQ ID NO: 1) or on the basis of one in accordance with this Invention taught method to isolate heterologous sequence be designed. For example, a derivative of a tetra hymena-L-19-IVS-RNA can be constructed using the nucleotide sequence of the active site complementary to the nucleotide sequence which is to be cleaved in a PSE-encoding mRNA. See e.g. B. Cech et al., U.S. Patent No. 4,987,071 and Cech et al., U.S. Patent No. 5,116,742. Alternatively, PSE mRNA can be used for selection a catalytic RNA with a specific ribonuclease activity from a pool of RNA molecules can be used. See e.g. B. Bartel, D., and Szostak, J.W. (1993) Science 261: 1411-1418.

Alternatively, PSE gene expression can be inhibited by Nucleotide sequences complementary to regulatory Region of a PSE nucleotide sequence (e.g. a PSE promoter and / or enhancer) are so directed that triple helix structures are formed, which are the transcription of a Inhibit PSE genes in target cells. See generally Helene, C. (1991) Anticancer Drug Res. 6 (6) 569-84; Helene, C., et al. (1992) Ann. N. Y. Acad. Sci. 660: 27-36; and Maher. L. J. (1992) Bioassays 14 (12): 807-815.

B. gene construct

Another embodiment of the invention is a new gene constructed, which means an isolated nucleic acid derived from a plant and encoding a polypeptide that extends C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms, or the gene sequence of SEQ ID NO: 1, their homologs, derivatives or analogs that are operably linked to one or more regulatory signals, advantageously to increase gene expression. Examples of these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid. In addition to these new regulatory sequences, the natural regulation of these sequences can still be present before the actual structural genes and, if appropriate, have been genetically modified so that the natural regulation has been switched off and the expression of the genes has been increased. However, the gene construct can also have a simpler structure, ie that no additional regulatory signals have been inserted before the sequence SEQ ID NO: 1 or its homologs and the natural promoter with its regulation has not been deleted. Instead, the natural regulatory sequence has been mutated so that regulation no longer takes place and gene expression is increased. The gene construct can also advantageously comprise one or more so-called enhancer sequences which are operably linked to the promoter and which enable increased expression of the nucleic acid sequence. It is also possible to additionally insert advantageous sequences, for example further regulatory elements or terminators, at the 3 'end of the DNA sequences. The elongase genes can be present in the gene construct in one or more copies. It is advantageous for the insertion of further genes in organisms if further genes are present in the gene construct.

Advantageous regulatory sequences for the novel process are present for example in promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI ^q- T7, T5 , T3, gal, trc, ara, SP6, λ-P _R or λ-P _L 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, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV / 35S [Franck et al., 1980, Cell 21: 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter. In this context, inducible promoters are also advantageous, such as those in EP-A-0 388 186 (benzylsulfonamide-inducible bar), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracycline-inducible), EP-A -0 335 528 (abscisic acid inducible) or WO 93/21334 (ethanol or cyclohexenol inducible) described promoters. Other suitable plant promoters are the cytosolic FBPase promoter or the ST-LSI promoter of the potato (Stockhaus et al., EMBO J. 8, 1989, 2445), the phospho ribosylpyrophosphatamidotransferase promoter from Glycine max (Genbank accession number U87999) or the node-specific promoter described in EP-A-0 249 676. Particularly advantageous promoters are promoters which enable expression in tissues which are involved in fatty acid biosynthesis. Seed-specific promoters such as the usp, LEB4, phaseolin or napin promoter are particularly advantageous. Further particularly advantageous promoters are seed-specific promoters that can be used for monocotyledons or dicotyledons and in US 5,608,152 (napin promoter from rapeseed), WO 98/45461 (phaseolin promoter from Arobidopsis), US 5,504,200 (phaseolin promoter from phaseolus vulgaris), WO 91/13980 (Bce4 promoter from Brassica), by Baeumlein et al., Plant J., 2, 2, 1992: 233-239 99999 00070 552 001000280000000200012000285919988800040 0002010005973 00004 99880 (LEB4 promoter from legumes) , where these promoters are suitable for dicotyledons. The following promoters are suitable for example for monocotyledons lpt-2 or lpt-1 promoter from barley (WO 95/15389 and WO 95/23230), hordein promoter from barley and other suitable promoters described in WO 99/16890.

In principle it is possible to use all natural promoters with their Regulatory sequences, such as those mentioned above, for the new ver driving to use. It is also possible and advantageous additionally use synthetic promoters.

As described above, the gene construct can also contain other genes include that are to be introduced into the organisms. It is possible and beneficial, in the host organisms regulatory genes, such as genes for inducers, repressors or enzymes, which are caused by their enzyme activity in the regulation of one or more genes intervene in a biosynthetic pathway, introduce it and close it express. These genes can be heterologous or homologous Be of origin. The inserted genes can have their own Have promoter or under the control of the promoter the sequence SEQ ID NO: 1 or its homologues.

The gene construct advantageously comprises for the expression of the other present genes further 3'- and / or 5'-terminal Regulatory sequences to increase expression, which in Dependence on the selected host organism and the gene or the genes are selected for optimal expression.

As mentioned above, these regulatory sequences are intended to: specific expression of the genes and protein expression enable. This can, for example, depending on the host organism wise mean that the gene expresses only after induction or is overexpressed or immediately expressed and / or over is expressed.  

The regulatory sequences or factors may also be preferred have a beneficial effect on the expression of a have brought genes and thus increase them. In this way it is possible to use the regulatory elements strong transcription signals, such as promoters and / or enhancers, advantageously be amplified at the transcription level. It however, translation is still possible, for example by enhancing mRNA stability.

C. Recombinant Expression Vectors and Host Cells

Another aspect of the invention relates to vectors, preferably wise expression vectors containing a nucleic acid that encode a PSE (or part of it). As used here the term "vector" refers to a nucleic acid molecule that contains a can transport other nucleic acid to which it is bound is. A vector type is a "plasmid", what a circular double-stranded DNA loop is in the additional DNA Segments can be ligated. Another type of vector is a viral vector, with additional DNA segments in the viral genome can be ligated. Certain vectors can in a host cell into which they have been placed replicate autonomously (e.g. bacterial vectors with bacterial Origin of replication and episomal mammalian vectors). Other Vectors (e.g. non-episomal mammalian vectors) are used in the Introduction into the host cell in the genome of a host cell integrated and thereby replicated together with the host genome. In addition, certain vectors can express genes with to which they are functionally connected. These vectors are referred to here as "expression vectors". Usually have expression vectors that are used for recombinant DNA techniques are suitable, the form of plasmids. In the present Description "plasmid" and "vector" can be used interchangeably be used because the plasmid is the most commonly used Is vector shape. However, the invention is intended to address these others Expression vector forms, such as viral vectors (e.g. replication deficient retroviruses, adenoviruses and adeno-related viruses) that perform similar functions. Furthermore, the term Vector also other vectors known to those skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, Transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA.

The recombinant expression vectors according to the invention comprise a nucleic acid according to the invention or one according to the invention Gene construct in a form that is used to express the nuclein acid in a host cell, which means that the recombinant  Expression vectors of one or more regulations sequences selected on the basis of the expression to be ver turning host cells with the nucleotide to be expressed acid sequence is operatively linked. In one recombinant expression vector means "functional ver bound "that the nucleotide sequence of interest to the Regulatory sequence (s) is bound that the expression of the Nucleotide sequence is possible and they are bound to each other, so that both sequences are the predicted, attributed to the sequence Perform function (e.g. in an in vitro transcription / trans lation system or in a host cell if the vector is in the Host cell is introduced). The term "regulatory sequence" is said to be promoters, enhancers and other expression control elements (e.g., polyadenylation signals). These regulations sequences are e.g. B. described 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 Biotechnolgy, CRC Press, Boca Raton, Florida, ed .: Glick and Thompson, Chapter 7, 89-108 finally the references in it. Regulatory sequences include those that constitute the constitutive expression of a Control nucleotide sequence in many host cell types, and those which the direct expression of the nucleotide sequence only in control certain host cells under certain conditions. Those skilled in the art know that the design of the expression vector of Factors such as the selection of the host cell to be transformed, the extent of expression of the desired protein, etc. can. The expression vectors according to the invention can be found in hosts cells are introduced in order to thereby produce proteins or peptides, including producing fusion proteins or peptides, which are encoded by the nucleic acids as described here (e.g. PSEs, mutant forms of PSEs, fusion proteins, etc.).

The recombinant expression vectors according to the invention can for the expression of PSEs in prokaryotic or eukaryotic Cells. For example, PSE genes can be found in bacterial cells, such as C. glutamicum, insect cells (under Use of baculovirus expression vectors), yeast and other fungal cells (see Romanos, M.A., et al. (1992) "Foreign gene expression in yeast: a review ", Yeast 8: 423-488; by the Hondel, C.A.M.J., et al. (1991) "Heterologous gene expression in filamentous fungi ", in: More Gene Manipulations in Fungi, J.W. Bennet & L.L. Lasure, eds., Pp. 396-428: Academic Press: San Diego; and von den Hondel, C.A.M.J., & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F., 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, Pseudocohnilembus, Euplotes, Engelmaniella and Stylonychia, especially of the genus Stylonychia lemnae, with vectors after a transformation ver drive, as described in WO 98/01572, and cells 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, Florida, Chapter 6/7, pp. 71-119 (1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, ed .: Kung and R. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225 (and therein cited references)) or mammalian cells are expressed. Suitable host cells are also discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). The recombinant expression vector can alternatively, for example using T7 promoter Regulatory sequences and T7 polymerase, transcribed in vitro and be translated.

The expression of proteins in prokaryotes is usually done with Vectors containing constitutive or inducible promoters hold the expression of fusion or non-fusion control proteins. Fusion vectors add a series of amino acids to a protein encoded therein, usually the amino Terminus of the recombinant protein, but also at the C-terminus or fused within appropriate ranges in the proteins. These fusion vectors usually have three functions: 1) the Enhancing the expression of recombinant protein; 2) the Increasing the solubility of the recombinant protein and 3) the Support the purification of the recombinant protein by Effect as a ligand in affinity purification. In the case of fusion Expression vectors often become a proteolytic cleavage site the junction of the fusion unit and the recombinant Protein introduced so that the separation of the recombinant Protein from the fusion device after cleaning the fusion proteins is possible. These enzymes and their corresponding ones Recognition sequences include factor Xa, thrombin and entero kinase.

Typical fusion expression vectors include a. pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (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 to the recombinant target protein is fused. With an execution form is the PSE coding sequence in a pGEX expression vector cloned so that a vector is generated that merges encodes protein that transitions from the N-terminus to the C-terminus GST-thrombin Cleavage site X protein. The fusion protein can by Affinity Chromatography Using Glutathione Agarose resin can be cleaned. Recombinant PSE that does not participate GST is fused by cleavage of the fusion protein can be obtained with thrombin.

Examples of suitable inducible non-fusion E. coli Expression vectors are u. a. 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). The target gene expression from the pTrc vector relies on host RNA polymerase transcription from one Hybrid trp-lac fusion promoter. The target gene expression from the pET 11d vector is based on the transcription of a T7-gn10-lac Fusion promoter derived from a coexpressed viral RNA Polymerase (T7 gn1) is mediated. This viral polymerase is from the host strains BL21 (DE3) or HMS174 (DE3) from one resident λ prophage provided that a T7 gn1 gene under the transcription control of the lacUV 5 promoter.

Other vectors suitable in prokaryotic organisms are known to the person 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 M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ¹¹³ -B1,? gt11 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBDnebacter, in CBDn2146.

A strategy to maximize the expression of recombinant Protein is the expression of the protein in a host bacterium, its ability to proteolytically cleave the recombinant Protein is disturbed (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to change the Insert nucleic acid sequence into an expression vector the nucleic acid so that the individual codons for each amino Acid are those that are preferred for expression in one selected bacteria such as C. glutamicum can be used (Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). This The nucleic acid sequences according to the invention are changed through standard DNA synthesis techniques.  

In another embodiment, the PSE expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisiae include pYepSec1 (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 Process of constructing vectors that are ready for use in other fungi, such as filamentous fungi those that are described in detail in: from the Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et al., eds., pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi [J. W. Bennet & L.L. Lasure, eds., Pp. 396-428: Academic Press: San Diego]. Other suitable yeast vectors are examples example 2 µM, pAG-1, YEp6, YEp13 or pEMBLYe23.

Alternatively, the PSEs according to the invention can be used in insect cells expressed using baculovirus expression vectors become. Baculovirus vectors used to express proteins are available in cultured insect cells (e.g. Sf9 cells), include the pAc series (Smith et al. (1983) Mol. Cell Biol .. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

The above vectors only provide a small overview about possible suitable vectors. Other plasmids are that Known in the art and are described for example in: Cloning Vectors (ed. Pouwels, P.H., et al., Elsevier, Amsterdam- New York-Oxford, 1985, ISBN 0 444 904018).

In yet another embodiment, an invention appropriate nucleic acid in mammalian cells using a Mammalian expression vector expressed. Examples of mammalian Expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used in mammalian cells, the control functions of the expression vector often from viral regulatory elements provided. Commonly used promoters come from e.g. B. from Polyoma, Adenovirus2, Cytomegalievirus and Simian Virus 40. Other suitable expression systems for prokaryotic and eukaryotic cells see in chapters 16 and 17 of Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.  

In another embodiment, the recombinant mammalian Expression vector the expression of the nucleic acid preferably in control a specific cell type (e.g. tissue-specific Regulatory elements used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific Promoters include a. the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific Promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g. neurofilament Promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477), pancreas specific promoters (Edlund et al., (1985) Science 230: 912-916) and mammary-specific promoters (e.g. milk serum promoter; U.S. Patent No. 4,873,316 and European Patent registration publication No. 264,166). Also developmental regulated promoters are included, e.g. B. the hox promoters Mouse (Kessel and Gruss (1990) Science 249: 374-379) and the Fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546).

In a further embodiment, the inventive PSEs in single-cell plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3): 239-251 and therein cited references, and plant cells from higher Plants (e.g. spermatophytes, such as crops) are expressed become. Examples of plant expression vectors include those that are 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 ", Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721; Vectors for Gene Transfer in Higher plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, ed .: Kung and R. Wu, Academic Press, 1993, Pp. 15-38.

A plant expression cassette preferably contains Regulatory sequences that regulate gene expression in plants can control cells and are operatively connected so that each sequence its function, like termination of transcription, can fulfill, for example polyadenylation signals. Before pulled polyadenylation signals are those derived from Agro bacterium tumefaciens-t-DNA originate, like that as octopine synthase known gene 3 of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3  (1984) 83ff.) Or functional equivalents thereof, but also all other terminators are functionally active in plants suitable.

Because plant gene expression very often does not rely on transcription level restricted, contains a plant expression cassette preferably other operably linked sequences, such as Translation enhancers, for example the overdrive sequence, which is the 5'-untranslated tobacco mosaic leader sequence virus, which increases the protein / RNA ratio, contains (Gallie et al., 1987, Nucl. Acids Research 15: 8693-8711).

Plant gene expression must be functional with a suitable promoter linked to gene expression perform timely, cell or tissue specific ways. Preferred are promoters which have the constitutive expression induction (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those derived from plant viruses, such as 355 CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also US 5352605 and WO 84/02913) or plant promoters, such as that in US 4,962,028 described the small subunit of the Rubisco.

Other preferred sequences for use in function capable compound in plant gene expression cassettes Targeting sequences that are used to control the gene product appropriate cell compartments are necessary (see an over view in Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited therein), for example in the vacuole, the cell nucleus, all types of plastids, such as amyloplasts, Chloroplasts, chromoplasts, the extracellular space, the Mitochondria, the endoplasmic reticulum, oil body, Peroxisomes and other compartments of plant cells.

Plant gene expression can also be done chemically facilitate inducible promoter (see an overview 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 time-specific he follows. Examples of such promoters are salicylic acid inducible promoter (WO 95/19443), a tetracycline-induced bar promoter (Gatz et al. (1992) Plant J. 2, 397-404) and a Ethanol inducible promoter.

Also promoters working on biotic or ablotic stress React conditions are suitable promoters, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the heat-inducible hsp80 promoter  from tomato (US 5,187,267), the cold-inducible alpha amylase promoter from potato (WO 96/12814) or by Wound inducible pinII promoter (EP-A-0 375 091).

In particular, promoters are preferred which the Induce gene expression in tissues and organs in which lipid and oil biosynthesis takes place in sperm cells, such as the cells of the endosperm and the developing embryo. Suitable promoters are the Napingen promoter from rapeseed (US 5,608,152), the USP promoter from Vicia faba (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), the oleosin Arabidopsis promoter (WO 98/45461), the phaseolin promoter from Phaseolus vulgaris (US 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) and Promoters showing seed-specific expression in Mono cotyledonous plants such as corn, barley, wheat, rye, rice, etc. bring about. Suitable notable 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, wheat glutelin Gene, the corn zein gene, the oat glutelin gene, the sorghum Kasirin gene, the rye secalin gene).

Promoters which are also particularly suitable induce plastid-specific expression because plastids are the compartment in which the precursor as well as some end products of lipid biosynthesis are synthesized. Suitable Promoters, such as the viral RNA polymerase promoter described in WO 95/16783 and WO 97/06250, and the clpP- Arabidopsis promoter, described in WO 99/46394.

The invention also provides recombinant expression vector ready, comprising a DNA molecule according to the invention, that clones into the expression vector in the antisense direction is. d. H. the DNA molecule is so regulatory Sequence operably linked to expression (by Transcription of the DNA molecule) of an RNA molecule that mRNA "antisense" is made possible. There can be regulations sequences are selected that work with an in Antisense direction are linked to cloned nucleic acid and the continuous expression of the antisense RNA molecule in control a variety of cell types, for example viral ones Promoters and / or enhancers or regulatory sequences selected which are constitutive, tissue-specific or cell type Control specific expression of antisense RNA. The antisense  Expression vector can be in the form of a recombinant plasmid, Phagemids or attenuated virus are present in the anti sense nucleic acids under the control of a highly effective regulatory area are produced, its activity can be determined by the cell type in which the vector has been introduced. An explanation of the regulation of For gene expression using antisense genes, see Weintraub, H., et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1 (1) 1986.

Another aspect of the invention relates to host cells in which introduced a recombinant expression vector according to the invention has been. The terms "host cell" and "recombinant host cells "are used interchangeably here. Even understandably, these terms do not only apply to the specific one Target cell, but also the offspring or potential offspring come this cell. Because in successive generations certain modifications due to mutation or environmental influences these offspring are not necessarily with the Parental cell are identical, but are still of the size of the Term as used herein.

A host cell can be a prokaryotic or eukaryotic Be a cell. For example, a PSE can be found in bacterial cells such as C. glutamicum, insect cells, fungal cells or mammalian cells (such as Chinese hamster ovary (CHO) or COS cells), algae, Ciliates, plant cells, fungi or other microorganisms, such as C. glutamicum. Other suitable hosts cells are familiar to the person skilled in the art.

Vector DNA can be expressed in prokaryotic or eukaryotic cells using conventional transformation or transfection techniques bring in. The terms "transformation" and "transfection", Conjugation and transduction, as used here, are said to be one A variety of methods known in the art for one bring foreign nucleic acid (e.g. DNA) into a host cell finally calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, natural Competence, chemically mediated transfer, electroporation or Particle bombardment. Suitable procedures for transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (Molecular cloning: A Laboratory Manual., 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, eds .: Gartland and Davey, Humana Press, Totowa, New Jersey.  

It is known about the stable transfection of mammalian cells that depending on the expression vector used and Transfection technology only a small part of the cells the foreign DNA integrated into their genome. For identification and selection these integrants usually become a gene that selects one encoded marker (e.g. resistance to antibiotics) introduced into the host cells with the gene of interest. Before Selected selectable markers include those that have resistance against drugs such as G418, hygromycin and methotrexate, ver borrow, or in plants those that have resistance to a Apply herbicide such as glyphosphate or glufosinate. Further suitable markers are, for example, markers which genes encode the biosynthetic pathways of, for example, sugars or Amino acids are involved, such as β-galactodsidase, ura3 or ilv2. Markers containing genes such as luciferase, gfp or other fluorescence encoding genes are also suitable. These markers can be use in mutants in which these genes are not functional because, for example, using conventional methods have been deleted. Furthermore, markers, which are a nucleus encode acid that encodes a selectable marker into one Host cell can be introduced on the same vector as the those who encode a PSE, or can use a separate Vector can be introduced. Cells brought in with the For example, nucleic acids have been stably transfected identified by drug selection (e.g. survival Cells that have integrated the selectable marker, where die against the other cells).

To produce a homologously recombined microorganism made a vector that has at least a portion of a PSE Contains gene in which a deletion, addition or substitution has been introduced to thereby change the PSE gene, e.g. B. functionally disrupt. This PSE gene is preferred a Physcomitrella patens PSE gene, but it can be a homologue or analogue from a related plant or even from one Mammal, yeast or insect source can be used. At a preferred embodiment, the vector is designed such that the endogenous PSE gene is functional with homologous recombination is disrupted (i.e., no longer a functional protein encoded, also known as a knockout vector). Alternatively, you can the vector should be designed so that the endogenous PSE gene homologous recombination is mutated or otherwise changed, but still encodes a functional protein (e.g., the upstream regulatory area so be changed that this changes the expression of the endogenous PSE). To create a point mutation via homologous recombination can also use DNA-RNA hybrids known as chimeraplasty  from Cole-Strauss et al., 1999, Nucleic Acids Research 27 (5): 1323-1330 and Kmiec, Gene therapy, 19999, American Scientist, 87 (3): 240-247.

In the vector for homologous recombination is the changed one Section of the PSE gene at its 5 'and 3' end from additional Flanked nucleic acid of the PSE gene so that homologous recombi nation between the exogenous PSE gene that precedes the vector and an endogenous PSE gene in a microorganism or a plant is possible. The additional flanking PSE Nucleic acid is essential for successful homologous recombination the endogenous gene is long enough. Usually are in vector several hundred base pairs to DNA flanking kilobases (at both the 5 'and 3' ends) included (a description of vectors for homologous recombination see e.g. B. in Thomas, K.R., and Capecchi, M.R. (1987) Cell 51: 503 or the Rekombi nation in Physcomitrella patens based on cDNA in Strepp et al., 1998, Proc. Natl. Acad. Sci. USA 95 (8): 4368-4373). The vector is placed in a microorganism or a plant cell (e.g. by means of polyethylene glycol-mediated DNA), and Cells in which the introduced PSE gene with the endogenous PSE The gene is homologously recombined using in the art area of known techniques selected.

In another embodiment, recombinant organisms, how microorganisms are made, the selected systems contain, which a regulated expression of the introduced Enable gene. The inclusion of a PSE gene in a vector being brought under the control of the lac operon, enables z. B. expression of the PSE gene only in the presence from IPTG. These regulation systems are known in the art.

A host cell according to the invention, such as a prokaryotic or eukaryotic host cell, growing in culture or in a field, can be used to produce (i.e., express) a PSE. In plants, an alternative procedure can also be followed direct transfer of DNA into developing flowers Electroporation or gene transfer using Agrobacterium be applied. The invention therefore also provides methods for the production of PSEs using the inventive Host cells ready. In one embodiment, the ver drive the cultivation of the host cell according to the invention (in the a recombinant expression vector encoding a PSE has been brought in, or a gene has been introduced into their genome encoding a wild-type or modified PSE) in a suitable medium until the PSE has been produced. The  In another embodiment, the method comprises isolating the PSEs from the medium or the host cell.

Host cells, in principle for receiving the invention Nucleic acid, the new gene product according to the invention or of the vector according to the invention are suitable, are all pro caryotic or eukaryotic organisms. The more advantageous host organisms used wisely are organisms such as bacteria, Mushrooms, yeasts, animal or plant cells. More beneficial Organisms are animals or preferably plants or parts from that. Mushrooms, yeasts or plants are preferably used particularly preferred mushrooms or plants, very particularly preferred Plants, like oil crops, that have large amounts of lipid contain compounds such as rapeseed, evening primrose, canola, peanut, Flax, soy, safflower, sunflower, borage, or plants such as Corn, wheat, rye, oats, triticale, rice, barley, cotton, Cassava, pepper, tagetes, solanaceae plants, such as potato, Tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bush plants (coffee, cocoa, tea), salix species, trees (oil plant, Coconut) as well as perennial grasses and forage crops. Particularly preferred plants according to the invention are oil fruit plants, such as soybean, peanut, rapeseed, canola, sunflower, safflower, Trees (oil palm, coconut).

D. Isolated PSE

Another aspect of the invention relates to isolated PSEs and biologically active parts of it. An "isolated" or is "purified" protein or a biologically active part thereof essentially free of cellular material when DNA Recombination techniques is produced, or by chemical pre stages or other chemicals if it chemically synthesizes becomes. The term "essentially free of cellular material" includes PSE preparations in which the protein is cellular Components of the cells in which it is natural or recombinant is produced, is separated. In one embodiment the expression "essentially free of cellular material" PSE- Preparations with less than about 30% (based on the dry weight) non-PSE (here also as "contaminating protein" designated), more preferably less than about 20% non-PSE, even more preferably less than about 10% non-PSE and am most preferably less than about 5% non-PSE. If the PSE or a biologically active part thereof is produced recombinantly , he / she is also essentially free of culture medium, d. H. the culture medium makes up less than about 20%, more preferably less than about 10% and most preferred accounts for less than about 5% of the volume of the protein preparation  out. The term "essentially free of chemical precursors or other chemicals "includes PSE preparations in which the protein from chemical precursors or other chemicals is separated, which are involved in the synthesis of the protein. In one embodiment, the term "essentially includes free of chemical precursors or other chemicals "PSE- Preparations with less than about 30% (based on the dry weight) chemical precursors or non-PSE chemicals, stronger preferably less than about 20% chemical precursors or non- PSE chemicals, more preferably less than about 10% chemical precursors or non-PSE chemicals and most potent preferably less than about 5% chemical precursors or non- PSE chemicals. In preferred embodiments, iso gated proteins or biologically active parts thereof no ver impure proteins from the same organism where the PSE comes from. These proteins are usually made by recombinant expression for example of a Physcomitrella patens PSE in plants other than Physcomitrella patens or micro organisms, for example bacteria, such as C. glutamicum, fungi, such as Mortierella, yeast, such as Saccharomyces, or ciliates, algae or mushrooms.

An isolated PSE according to the invention or a part thereof can also be used in metabolism to build cell membranes in Physcomitrella patens necessary connections or on transport of molecules participating through these membranes or has one or several of the activities listed in Table 1. With preferred Embodiments include the protein or part thereof Amino acid sequence that is sufficiently homologous to an amino acid Sequence of SEQ ID NO: 2 is that the protein or part thereof the ability to metabolize to build cell membranes in Physcomitrella patens necessary connections or on Trans port of molecules to participate across these membranes. The part of the protein is preferably a biologically active one Part as described here. Another preferred Embodiment has a PSE according to the invention one of the in SEQ ID NO: 2 amino acid sequences shown. Another preferred embodiment, the PSE has an amino acid sequence, which is encoded by a nucleotide sequence which, for example under stringent conditions, to a nucleotide sequence of the SEQ ID NO: 1 hybridized. Yet another preferred one Embodiment, the PSE has an amino acid sequence that is one Is encoded nucleotide sequence that is at least about 50 to 60%, preferably at least about 60 to 70%, more preferably at least about 70 to 80%, 80 to 90%, 90 to 95% and more more preferably at least about 96%, 97%, 98%, 99% or even more homologous to one of the amino acid sequences of SEQ ID NO: 2  is. The preferred PSE according to the invention preferably has also at least one of the PSE activities described here. To the For example, a preferred PSE according to the invention comprises an amino acid sequence that is encoded by a nucleotide sequence that for example, under stringent conditions, to a nucleotide sequence of SEQ ID NO: 1 hybridizes and at the metabolism from to Structure of cell membranes in Physcomitrella patens necessary Connections or the transport of molecules across these membranes can participate or one or more of those listed in Table 1 activities.

In other embodiments, the PSE is essentially homologous to an amino acid sequence of SEQ ID NO: 2 and retains the functional activity of the protein of one of the sequences of the SEQ ID NO: 2, but their amino acid sequence differs due to natural variation or mutagenesis as detailed described in subsection I above. With another out The PSE is consequently a protein that is an amino acid sequence comprising at least about 50 to 60%, preferably at least about 60 to 70% and more preferably min at least about 70 to 80%, 80 to 90%, 90 to 95% and am most preferably at least about 96%, 97%, 98%, 99% or even more homologous to a complete amino acid sequence of the SEQ ID NO: 2 and is at least one of the PSE Activities. In another embodiment relates the invention a complete Physcomitrella patens protein, which is essentially homologous to a complete amino acid sequence of SEQ ID NO: 2.

Biologically active parts of a PSE include peptides comprising Amino acid sequences derived from the amino acid sequence of a PSE are derived, e.g. B. an amino acid shown in SEQ ID NO: 2 sequence or the amino acid sequence of a protein that results in a PSE is homologous, which is less amino acids than the full length PSE or have the full length protein homologous to a PSE and have at least one activity of a PSE. Usually comprise biologically active parts (peptides, e.g. peptides which are used for Example 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids long) with a domain or a motif at least one activity of a PSE. Moreover, others can biologically active parts in which other areas of the protein are deleted, produced by recombinant techniques and related to one or more of the activities described here to be examined. The biologically active parts of a PSE preferably comprise one / one or more selected domains / Motives or parts thereof with biological activity.  

PSEs are preferably made by recombinant DNA techniques manufactured. For example, one encoding the protein Nucleic acid molecule into an expression vector (as above described) cloned, the expression vector is in a host cell (as described above) and the PSE is expressed in the host cell. The PSE can then be replaced by a suitable cleaning scheme using standard protein cleaning techniques are isolated from the cells. As an alternative to recombinant expression can be a PSE, a polypeptide, or -Peptide chemically using standard peptide synthesis techniques be synthesized. In addition, native PSE can be derived from cells (e.g. endothelial cells) e.g. B. using an anti-PSE Antibody can be isolated by standard techniques can be produced, wherein a PSE or a fragment of it is used.

The invention also provides chimeric PSE proteins or PSE fusions proteins ready. As used herein, a "chimeric PSE- Protein "or" PSE fusion protein "a PSE polypeptide that funk is capable of being bound to a non-PSE polypeptide. A "PSE Polypeptide "refers to a polypeptide with an amino acid sequence, which corresponds to a PSE whereas a "non-PSE polypeptide" relates to a polypeptide with an amino acid sequence which corresponds to a Corresponds to protein, which is essentially not homologous to the PSE is, e.g. B. a protein that differs from PSE and comes from the same or a different organism. Within of the fusion protein the term "functionally linked" mean that the PSE polypeptide and the non-PSE polypeptide are so are fused together that both sequences the previous said to perform the function attributed to the sequence used. The non-PSE polypeptide can be attached to the N-terminus or the C-terminus of the PSE polypeptide must be fused. With an off the fusion protein is, for example, a GST-PSE Fusion protein in which the PSE sequences at the C-terminus of the GST sequences are fused. These fusion proteins can Facilitate cleaning of recombinant PSEs. Another Embodiment, the fusion protein is a PSE that is hetero has a logical signal sequence at its N-terminus. In particular Host cells (e.g. mammalian host cells) can express and / or secretion of a PSE using a heterologous Signal sequence can be increased.

A chimeric PSE protein or PSE fusion according to the invention protein is made by standard recombinant DNA techniques posed. For example, DNA fragments are different Encode polypeptide sequences according to conventional techniques in Reading frames ligated together, for example by smooth or  overhanging ends for ligation, restriction enzyme cleavage for Provide suitable ends, fill in cohesive ends, as required, treatment with alkaline phosphatase to avoid unwanted links, and enzymatic ligation be used. In a further embodiment, this can Fusion gene by conventional techniques, including DNA Automatic synthesizers to be synthesized. Alternatively, one PCR amplification of gene fragments using anchors primers are performed that have complementary overhangs between successive gene fragments that subsequently generate can be hybridized with one another and reamplified, so that a chimeric gene sequence is generated (see for example Current Protocols in Molecular Biology, ed. Ausubel et al., John Wiley & Sons: 1992). Furthermore, there are many expression vectors commercially available that already has a fusion unit (e.g. a GST polypeptide). A nucleic acid encoding PSE can be cloned into such an expression vector so that the Fusion unit is linked in frame with the PSE protein.

Homologs of PSE can by mutagenesis, e.g. B. by specific Point mutation or shortening of the PSE. The The term "homologue" as used here refers to a variant Form of PSE that acts as an agonist or antagonist of PSE activity works. An PSE agonist can be essentially the same Activity like that or part of biological activities maintain the PSE. An PSE antagonist can have one or several activities of the naturally occurring form of PSE for example competitive binding to a downstream or upstream element in the metabolic cascade for the cell membrane components that comprise the PSE, or by binding to a PSE, which is the transport of compounds across cell membranes mediates, inhibit, whereby the translocation is inhibited.

In an alternative embodiment, homologs of the PSE by screening combinatorial banks of mutants, e.g. B. Shortening mutants, the PSE in terms of PSE agonists or Antagonist activity can be identified. With an off a varied bank of PSE variants is implemented combinatorial mutagenesis at the nucleic acid level and encoded by a varied gene bank. A varied bank of PSE variants can e.g. B. by enzymatic ligation Mixtures of synthetic oligonucleotides in gene sequences be made so that a degenerate sentence of potential PSE sequences as individual polypeptides or alternatively as Set of larger fusion proteins (e.g. for the phage display) that contain this set of PSE sequences. It are a variety of processes used to manufacture banks  potential PSE homologs from a degenerate oligo nucleotide sequence can be used. The chemical Synthesis of a degenerate gene sequence can be done in a DNA Synthesized machines performed and then the synthetic gene be ligated into a suitable expression vector. The Ver using a degenerate set of genes enables the Provision of all sequences that match the desired sentence encode potential PSE sequences in a mixture. method for the synthesis of degenerate oligonucleotides are in the art known (see e.g. Narang, S.A. (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477).

In addition, banks of PSE fragments can be used to produce a varied population of PSE fragments for screening and used for the subsequent selection of homologs of a PSE become. In one embodiment, a bank of fragments the coding sequence by treating a double-stranded PCR fragment of a coding PSE sequence with a nuclease under conditions where double strand breaks only about done once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA, what sense / antisense pairs of different products with Double-strand breaks can include single-strand removal Sections from newly formed duplexes by treatment with S1 nuclease and ligating the resulting fragment library into one Expression vector can be generated. With this procedure a Expression bank can be derived, the N-terminal, C-terminal and encoded PSE internal fragments of various sizes.

There are several techniques for gene screening in the art products in combinatorial banks caused by point mutations or shortening have been made, and for screening from cDNA banks for gene products with a selected proprietary known. These techniques can be applied to the quick Customize screening of gene banks by combinatorial Mutagenesis of PSE homologs have been generated. The most common most used techniques for screening large gene banks, the can be subjected to high throughput analysis, usually involve cloning the library into replicable ones Expression vectors, transforming suitable cells with the resulting vector library and expressing the combinatorial Genes under conditions under which the detection of the desired Activity the isolation of the vector encoding the gene whose Product was proven relieved. Recursive ensemble muta genese (REM), a new technique that makes the frequency more functional Mutants increased in banks, in combination with the  Screening tests used to identify PSE homologs (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

In another embodiment, cell-based tests can be performed to analyze a varied PSE bank using methods known in the art can be used.

E. Uses and methods according to the invention

The nucleic acid molecules, proteins, protein described here homologa, fusion proteins, primers, vectors and host cells can be used in one or more of the following procedures used: identification of Physcomitrella patens and related organisms, mapping the genomes of organisms, related to Physcomitrella patens, identification and Localization of Physcomitrella patens sequences of interest, Evolutionary studies, determination of PSE protein areas that are required for the function is necessary, modulation of a PSE activity; Modulation of the metabolism of one or more cell membranes components; Modulation of the transmembrane transport of one or several compounds as well as modulation of cellular production a desired compound, such as a fine chemical. The PSE nucleic acid molecules according to the invention have a large number of uses. You can first use to identify a Organism as Physcomitrella patens or as a close relative of which are used. You can also use it to identify the Presence of Physcomitrella patens or a relative of which used in a mixed population of microorganisms become. The invention provides the nucleic acid sequences one Series of Physcomitrella patens genes ready; by probing the extracted genomic DNA of a culture of a unit or mixed population of microorganisms among stringent conditions with a probe covering an area a Physcomitrella patens gene or parts thereof that is unique to this organism, you can determine whether this organism is present. Physcomitrella patens itself does not become a commercial production multiple times unsaturated acids are used, but mosses are the only ones known plants that produce PUFAs. Therefore are suitable PSE-related DNA sequences especially for use in PUFA Production in other organisms.

Furthermore, the nucleic acid and protein according to the invention molecules serve as markers for specific areas of the genome. This is not only for mapping the genome, but also for  functional studies of Physcomitrella patens proteins net. To identify the genome area to which a specific DNA binding protein from Physcomitrella patens could bind the Physcomitrella patens genome, for example and the fragments are incubated with the DNA binding protein. Those who bind the protein can also use the Nucleic acid molecules according to the invention, preferably with light detectable markings to be probed; the binding of a such nucleic acid molecule to the genome fragment enables Localization of the fragment on the Physcomitrella genome map patens and relieved if this happens several times with different Enzymes is carried out, a rapid determination of the nucleus acid sequence to which the protein binds. The invention Nucleic acid molecules can also be sufficiently homologous to the Sequences of related types that these nucleic acid molecules as a marker for the construction of a genomic map related mosses can serve.

The PSE nucleic acid molecules according to the invention are suitable also for evolution and protein structure studies. The Metabolism and transport processes in which the fiction Appropriate molecules are involved by many pro used karyotic and eukaryotic cells; by ver equal to the sequences of the nucleic acid according to the invention molecules with those that have similar enzymes from other organisms encode the degree of evolutionary kinship of the organisms be determined. Such a comparison accordingly enables the determination of which sequence areas are conserved and which is not what when determining areas of the protein can be helpful, which are essential for the enzyme function. This type of determination is for protein engineering studies valuable and can give an indication of how much Mutagenesis the protein can tolerate without function to lose.

The manipulation of the PSE nucleic acid molecules according to the invention can be used to produce PSEs with functional differences lead the wild type PSEs. The efficiency or activity of this Proteins can be improved, they can be in larger numbers than usually present in the cell, or its efficiency or activity may be reduced. Improved efficiency or For example, activity means that the enzyme has a higher one Selectivity and / or activity, preferably at least one 10% higher, particularly preferably an at least 20% higher Activity, most preferably at least 30% higher Has activity than the original enzyme.  

There are a number of mechanisms through which change takes place the yield, production and / or a PSE according to the invention Efficiency of the production of a fine chemical, which a contains such modified protein, can directly influence. The extraction of fine chemical compounds from cultures of ciliates, algae or mushrooms on a large scale is signi savory improves when the cell makes the desired connections secreted because these compounds from the culture medium (in the opposite set for extraction from the mass of grown cells) easily can be cleaned. Otherwise the cleaning can be done improve when the cell connects in vivo in a specialty compartment with a kind of concentration mechanism saves. For plants that express PSEs, a increased transport for better distribution within the Plant tissue and organs. By enlarging the Number or activity of transporter molecules, which fine exporting chemicals from the cell, it may be possible Amount of fine chemical produced in the extracellular Medium is present to increase, making harvesting and cleaning or in plants a more efficient distribution can be facilitated. For efficient overproduction of one or more fine chemicals are increased amounts of cofactors, precursors and Interconnections required for the appropriate biosynthetic pathways Lich. By increasing the number and / or activity of Transporter proteins involved in the import of nutrients such as coal Substance sources (i.e. sugars), nitrogen sources (i.e. amino acids, Ammonium salts), phosphate and sulfur, can be involved the production of a fine chemical due to disposal all restrictions on the supply of nutrients in biosynthesis improve process. Fatty acids, such as PUFAs, and lipids, the PUFAs contain, are themselves desirable fine chemicals; by Optimize activity or increase the number of one or of several PSEs according to the invention which are involved in the biosynthesis of these Connections are involved, or by disrupting the activity one or more PSEs involved in breaking down these connections are involved, you can thus the yield, production and / or Efficiency of the production of fatty acid and lipid molecules in Ciliates, algae, plants, mushrooms, yeasts or other micro increase organisms.

The manipulation of one or more PSE genes according to the invention can also lead to PSEs with changed activities, which the production of one or more desired fine chemicals from algae, plants, ciliates or fungi. The normal biochemical metabolic processes lead e.g. B. for Production of a variety of waste products (e.g. hydrogen peroxide and other reactive oxygen species) that these metabolic processes  can actively interfere (e.g. nitrided peroxynitrite famously known tyrosine side chains, whereby some enzymes with Tyrosine can be inactivated in the active center (Groves, J. T. (1999) Curr. Opin. Chem. Biol. 3 (2); 226-235)). This waste products are usually eliminated, but those for cells used for large-scale fermentative production are used for the overproduction of one or more fine chemicals optimized and can therefore produce more waste products than common for a wild type cell. By optimizing the activity one or more PSEs according to the invention which are involved in the export of Waste molecules are involved, the viability of the Improve cell and have an efficient metabolic activity keep right. The presence of high intracellular amounts The desired fine chemical can actually be used for the cell be toxic so that by increasing the ability of the cell cell viability for the secretion of these compounds can improve.

The PSEs according to the invention can also be manipulated in such a way that the relative amounts of different lipid and fatty acid molecules to be changed. This can have a critical impact on the Have lipid composition of the cell membrane. Because every lipid type has different physical properties, one Change in the lipid composition of a membrane the membrane change fluidity significantly. Changes in membrane fluidity can affect the transport of molecules across the membrane, which, as explained above, is the export of waste products or the fine chemical produced or the import necessary Can modify nutrients. This changes the membrane Fluidity can also be critical to cell integrity influence; Cells with comparatively weaker membranes are more susceptible to ablotic and biotic stress conditions that can damage or kill the cell. By manipulating PSEs involved in the production of fatty acids and lipids for membrane construction are involved, so that the resulting membrane has a membrane composition that is suitable for in the cultures used to produce fine chemicals prevailing environmental conditions are more receptive, should a larger proportion of the cells survive and ver increase. Larger amounts of producing cells should appear in larger yields, higher production or efficiency of Manipulate the production of fine chemicals from culture.

The above mutagenesis strategies for PSEs leading to should lead to increased yields of a fine chemical not be restrictive; Variations on these strategies are that Easily seen by a specialist. Using these mechanisms  and with the help of the mechanisms disclosed here, the Nucleic acid and protein molecules according to the invention for generation of algae, ciliates, plants, animals, fungi or other micro organisms such as C. glutamicum can be used, the mutated Express PSE nucleic acid and protein molecules so that the Yield, production and / or efficiency of production of a ge desired connection is improved. This desired connection can be any natural product of algae, ciliates, Plants, animals, mushrooms or C. glutamicum, which the End products of biosynthetic pathways and intermediates, of course occurring metabolic pathways includes, as well as molecules that in Metabolism of these cells does not occur naturally, however are produced by the cells according to the invention.

A further embodiment according to the invention is a method for producing PUFAs, the method comprising growing an organism which comprises a nucleic acid according to the invention, a gene construct according to the invention or a vector according to the invention which encode a polypeptide which contains C ₁₈ fatty acids with at least two double bonds in the Fatty acid molecule extended by at least two carbon atoms under conditions under which PUFAs are produced in the organism. PUFAs produced by this method can be isolated by harvesting the organisms either from the culture in which they grow or from the field, breaking up and / or extracting the harvested material with an organic solvent. The oil containing lipids, phospholipids, sphingolipids, glycolipids, triacylglycerols and / or free fatty acids with a higher PUFA content can be isolated from this solvent. The free fatty acids with a higher PUFA content can be isolated by basic or acidic hydrolysis of the lipids, phospholipids, sphingolipids, glycolipids, triacylglycerols. A higher content of PUFAs means at least 5%, preferably 10%, particularly preferably 20%, very particularly preferably 40% more PUFAs than the original organism, which has no additional nucleic acid which encodes the elongase according to the invention.

The PUFAs produced by this process are preferably C ₂₀ or C ₂₂ fatty acid molecules with at least two double bonds in the fatty acid molecule, preferably three or four double bonds, particularly preferably three double bonds. These C ₂₀ or C ₂₂ fatty acid molecules 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.

An embodiment of the invention are oils, lipids or fat acids or fractions thereof by the above described Processes have been produced, particularly preferably oil, lipid or a fatty acid composition comprising PUFAs and from originate from transgenic plants.

Another embodiment of the invention is the use of the oil, lipids or fatty acid composition in feed agents, foodstuffs, cosmetics or pharmaceuticals.

This invention is further illustrated by the examples below illustrated, which are not to be taken as limiting should. The content of all in this patent application cited references, patent applications, patents and published patent applications are here by reference added.

Sample part example 1 General procedures a) General cloning procedures

Cloning methods, such as restriction cleavages, Agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids on nitrocellulose and nylon membranes, Ver binding of DNA fragments, transformation of Escherichia coli and yeast cells, bacterial growth and sequence analysis right combined DNA were performed as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994) "Methods in Yeast Genetics" (Cold Spring Harbor Laboratory Press: ISBN 0-87969-451-3). The transformation and breeding of algae, such as Chlorella or Phaeodactylum are carried out as be written by El-Sheekh (1999), Biologia Plantarum 42: 209-216; Apt et al. (1996) Molecular and General Genetics 252 (5): 872-9.

b) chemicals

The chemicals used were, unless otherwise stated in the text, in p. A. quality from Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Solutions were prepared from a Milli-Q water system water purification plant (Millipore, Eschborn) using pure pyrogen-free water, hereinafter referred to as H ₂ O. Restriction endonucleases, DNA-modifying enzymes and molecular biological kits were obtained from AGS (Heidelberg), Amersham (Braunschweig), Biometra (Göttingen), Boehringer (Mannheim), Genomed (Bad Oeynhausen), New England Biolabs (Schwalbach / Taunus), Novagen (Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden) and Stratagene (Amsterdam, Netherlands). Unless otherwise stated, they were used according to the manufacturer's instructions.

c) plant material

Plants of the Physcomitrella species were used for this study patens (Hedw.) B. S. G. from the collection of the Department for Genetic studies from the University of Hamburg used. They come from strain 16/14, which was developed by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire (England) collected and used by angels (1968, Am J Bot 55, 438-446) was subcultured from a spore. The plants were proliferated by means of spores and by means of regeneration of the gametophytes. The Protonema developed from the haploid spore to chloroplast-rich chloronema and low-chloroplast caulonema, which results after about 12 days Buds formed. These grew into gametophores with antheridia and archegonia. The diploid emerged after fertilization Sporophyte with a short Seta and spore capsule, in which the Meio spores mature.

d) plant breeding

The cultivation took place in an air-conditioned chamber at one Air temperature of 25 ° C and a light intensity of 55 micro mol-im-2 (white light; Phillips TL 65W / 25 fluorescent tube) and a light / dark change of 16/8 hours. The moss became either in liquid culture using Knop medium Reski and Abel (1985, Planta 165, 354-358) modified or on solid button medium using 1% oxoid agar (Unipath, Basingstoke, England).

The protonemata used for RNA and DNA isolation were described in aerated liquid cultures. The protonemata were all Crushed for 9 days and transferred to fresh culture medium.

Example 2 Isolation of total DNA from plants for Hybridization experiments

The details of the isolation of total DNA relate to the Auf working of plant material with a fresh weight of one Grams.  

CTAB buffer: 2% (w / v) N-acetyl-N, N, N-trimethylamionium bromide (CTAB); 100 mM Tris-HCl, pH 8.0; 1.4 M NaCl; 20mM EDTA.

N-lauryl sarcosine buffer: 10% (w / v) N-lauryl sarcosine; 100 mM Tris-HCl, pH 8.0; 20mM EDTA.

The plant material was triturated under liquid nitrogen in a mortar so that a fine powder was obtained and transferred to 2 ml Eppendorf tubes. The frozen plant material was then covered with a layer of 1 ml of decomposition buffer (1 ml of CTAB buffer, 100 ml of N-lauryl sarcosine buffer, 20 ml of β-mercapto ethanol and 10 ml of Proteinase K solution, 10 mg / ml) and for one hour incubated at 60 ° C with continuous shaking. The homogenate obtained was divided into two Eppendorf tubes (2 ml) and extracted twice by shaking with the same volume of chloroform / isoamyl alcohol (24: 1). For phase separation, centrifugation was carried out at 8000 × g and RT (= room temperature = ~ 23 ° C) for 15 min each. The DNA was then precipitated at -70 ° C for 30 minutes using ice cold isopropanol. The precipitated DNA was sedimented at 10,000 g for 30 min at 4 ° C. and resuspended in 180 ml TE buffer (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). For further purification, the DNA was treated with NaCl (1.2 M final concentration) and precipitated again at -70 ° C. for 30 min using twice the volume of absolute ethanol. After a washing step with 70% ethanol, the DNA was dried and then taken up in 50 ml H ₂ O + RNAse (50 mg / ml final concentration). The DNA was dissolved overnight at 4 ° C. and the RNAse cleavage was then carried out at 37 ° C. for 1 hour. The DNA was stored at 4 ° C.

Example 3 Isolation of total RNA and poly (A) ⁺ RNA from plants

Total RNA and poly (A) ⁺ RNA were isolated to examine transcripts. The total RNA was obtained from 9 T old wild-type protonemata by the GTC method (Reski et al., 1994, Mol. Gen. Genet., 244: 352-359).

Poly (A) ⁺ RNA was isolated using Dyna Beads® (Dynal, Oslo, Finland) according to the instructions in the manufacturer's protocol.

After determining the RNA or poly (A) ⁺ RNA concentration, the RNA was precipitated by adding 1/10 volumes of 3 M sodium acetate, pH 4.6, and 2 volumes of ethanol and stored at -70 ° C.

Example 4 Construction of the cDNA bank

The first strand synthesis was used to construct the cDNA library Use of mouse leukemia virus reverse transcriptase (Roche, Mannheim, Germany) and Oligo-d (T) primers, the second strand synthesis by incubation with DNA polymerase I, Klenow Enzyme and RNAse H cleavage at 12 ° C (2 hours), 16 ° C (1 hour) and 22 ° C (1 hour). The reaction was by incubation stopped at 65 ° C (10 min) and then transferred to ice. Double-stranded DNA molecules were created using T4 DNA polymerase (Roche, Mannheim) at 37 ° C (30 min) with smooth ends. The nucleotides were extracted by phenol / chloroform and Sephadex G50 centrifugation columns removed. EcoRI adapter (Pharmacia, Freiburg, Germany) were using T4 DNA ligase (Roche, 12 ° C, overnight) ligated to the cDNA ends and by Incubation with polynucleotide kinase (Roche, 37 ° C, 30 min) phosphorylated. This mixture was subjected to separation on a Subjected to low-melting agarose gel. DNA molecules over 300 bases pairs were eluted from the gel, phenol extracted, on Elutip- D-pillars (Schleicher and Schüll, dassel, Germany) concentrated and ligated to vector arms and in lambda-ZAPII phage or lambda ZAP Express phage using the Gigapack Gold kit (Stratagene, Amsterdam, The Netherlands) packed using material of the manufacturer and its instructions have been followed.

Example 5 DNA sequencing and computer analysis

cDNA banks, as described in Example 4, were used for DNA sequencing by standard methods, in particular by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany). The random sequencing was carried out after the preparative plasmid extraction from cDNA banks via in vivo mass section and retransformation of DH10B on agar plates (details on material and protocol from Stratagene, Amsterdam, Netherlands). Plasmid DNA was obtained from overnight grown E. coli cultures grown in Luria broth with ampicillin (see Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6)) , prepared on a Quiagen DNA preparation robot (Quiagen, Hilden) according to the manufacturer's protocols. Sequencing primers with the following nucleotide sequences were used:
5'-CAGGAAACAGCTATGACC-3 '
5'-CTAAAGGGAACAAAAGCTG-3 '
5'-TGTAAAACGACGGCCAGT-3 '

The sequences were made using standard software packages EST-MAX, commercially available from Bio-Max (Munich, German country) is delivered, processed and commented. A clone with weak homologies to known elongases became more detailed characterized.

Example 6 Identification of the PSE1 gene and analysis of the cDNA clones PpPSE1

The EST sequence: 08_ck19_b07, new name pp001019019f, was initially due to weak homology with known elongases considered as target gene among other candidate genes.

The BESTFIT program was used for the sequence comparison, d. H. the BLOSUM amino acid substitution matrices, with on Henikoff, S., and Henikoff. J.G. (1992) Amino acid substitution matrices from protein blocks, Proc. Natl. Acad. Sci. United States 89: 10915-10919.

The full length sequence of the clone with the database no. 08_ck19_b07 the inventor was used for comparison with the yeast elo1 peptide sequence. The following parameters were selected for comparison: cap weight: 8, average match: 2.912, length weight: 2, average mismatches: -2.003. The alignment shown in Fig. 1 resulted in 48.3% similarity and 38.5% identity.

The complete nucleotide sequence of the cDNA consisted of approximately 1200 bp. It contained an open reading frame of 873 bp, the 290 amino acids with a calculated molecular mass of 33.4 Da encoded. The protein sequence has only 38.5% identity and 48.3% similarity to the Saccharomyces PSE1 gene product cerevisiae, which is used to elongate fatty acids with medium Chain length in the yeast is required (Toke & Martin, 1996, Isolation and characterization of a gene affecting fatty acid elongation in Saccharomyces cerevisiae. Journal of Biological Chemistry 271, 18413-18422).

Example 7 Identification of genes using hybridization

Gene sequences can be used to identify homologous or Use heterologous genes from cDNA or genomic banks.

Homologous genes (ie full-length cDNA clones that are homologous or homologs) can be isolated via nucleic acid hybridization using, for example, cDNA libraries: Depending on the frequency of the gene of interest, 100,000 to 1,000,000 recombinant bacteriophages were plated and onto a nylon membrane transferred. After denaturing with alkali, the DNA was z. B. immobilized by UV crosslinking. The hybridization took place under highly stringent conditions. In aqueous solution, the hybridization and the washing steps were carried out at an ionic strength of 1 M NaCl and a temperature of 68 ° C. Hybridization probes were e.g. B. prepared by labeling using radioactive ( ³² P) nick transcription (High Prime, Roche, Mannheim, Germany). The signals were detected using autoradiography.

Partially homologous or heterologous genes that are related but not are identical, can be analogous to the Ver drive using low-stringent hybridization and Identify washing conditions. For aqueous hybridization the ionic strength was usually kept at 1 M NaCl, where the temperature was gradually reduced from 68 to 42 ° C.

Isolation of gene sequences that only lead to a single Domain of, for example, 10 to 20 amino acid homologies can be demonstrated using synthetic, radioactive perform labeled oligonucleotide probes. Radioactively marked Oligonucleotides were phosphorylated at the 5 'end two complementary oligonucleotides with T4 polynucleotide kinase manufactured. The complementary oligonucleotides were joined together hybridized and ligated so that concatems were formed. The double-stranded concatems was created, for example, by Nick radioactive transcription. The hybridization took place usually using under low-stringent conditions high oligonucleotide concentrations.

Oligonucleotide hybridization solution:
6 × SSC
0.01 M sodium phosphate
1 mM EDTA (pH 8)
0.5% SDS
100 µg / ml denatured salmon sperm DNA
0.1% low-fat dry milk

During the hybridization, the temperature became gradual to 5 to 10 ° C below the calculated oligonucleotide Tm or to to room temperature (means RT = ~ 23 ° C in all experiments, unless otherwise stated), followed by washing steps and autoradiography. The washing was extremely low Stringency performed, for example 3 washing steps below Using 4 × SSC. More details are as of  Sambrook, J., et al. (1989), "Molecular Cloning: A Laboratory Manual, "Cold Spring Harbor Laboratory Press, or Ausubel, F. M., et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons.

Example 8 Identification of genes of interest through Screening of expression banks with antibodies

There were cDNA sequences for the production of recombinant Protein used for example in E. coli (e.g. Qiagen QIAexpress pQE system). The recombinant proteins then became common via Ni-NTA affinity chromatography (Quiagen) affinity cleaned. The recombinant proteins were then used in production specific antibody using, for example Standard techniques are used to immunize rabbits. The antibodies were then analyzed using a Ni-NTA column, affinity that had been saturated with recombinant antigen purified as described by Gu et al., (1994) BioTechniques 17: 257-262 described. The antibody can then be used to screen for Expression cDNA libraries using immunological screening ver can be used (Sambrook, J., et al. (1989), "Molecular Cloning: A Laboratory Manual ", Cold Spring Harbor Laboratory Press, or Ausubel, F.M., et al. (1994) "Current Protocols in Molecular Biology ", John Wiley & Sons).

Example 9 Northern hybridization

For the RNA hybridization, 20 ug total RNA or 1 ug poly (A) ⁺ -RNA were gel electrophoresis in agarose gels with a strength of 1.25% using formaldehyde, as described in Amasino (1986, Anal. Biochem. 152 , 304) on separated, transferred by capillary attraction using 10 × SSC to positively charged nylon membranes (Hybond N + Amersham, Braunschweig), immobilized by means of UV light and 3 hours at 68 ° C. using hybridization buffer (10% dextran sulfate w / w Vol., 1 M NaCl, 1% SDS, 100 mg herring sperm DNA) prehybridized. The DNA probe was labeled with the Highprime DNA labeling kit (Roche, Mannheim, Germany) during the prehybridization using alpha- ³² P-dCTP (Amersham, Braunschweig, Germany). The hybridization was carried out after adding the labeled DNA probe in the same buffer at 68 ° C. overnight. The washing steps were carried out twice for 15 min using 2 × SSC and twice for 30 min using 1 × SSC, 1% SDS, at 68 ° C. The exposure of the closed filter was carried out at -70 ° C for a period of 1 to 14 T.

Example 10 Plasmids for plant transformation

Binary vectors such as pBinAR can be used for plant transformation can be used (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230). The construction of the binary vectors can be done by Ligation of the cDNA in sense or antisense orientation in T-DNA done. 5 'of the cDNA activates a plant promoter Transcription of the cDNA. There is a polyadenylation sequence 3 'away from the cDNA.

Tissue-specific expression can be determined using a achieve tissue-specific promoter. For example, the seed-specific expression can be achieved by the napin or the LeB4 or USP promoter 5 'of the cDNA becomes. Any other seed-specific promoter element can also be used. For constitutive expression in the whole The CaMV-355 promoter can be used in plants.

The expressed protein can be expressed using a signal peptides, for example for plastids, mitochondria or that Endoplasmic reticulum, in a cellular compartment to be conducted (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423). The signal peptide is 5 'in reading frame with the cDNA cloned to the subcellular localization of the fusion protein to reach.

Example 11 Agrobacterium transformation

The Agrobacterium-mediated plant transformation can Example using the GV3101- (pMP90-) (Koncz and Schell, Mol. Gen. Genet. 204 (1986) 383-396) or LBA4404- (Clontech) Agrobacterium tumefaciens strain. The Transformation can be done through standard transformation techniques (Deblaere et al., Nucl. Acids. Tes. 13 (1984), 4777-4788).

Example 12 Plant transformation

The Agrobacterium-mediated plant transformation can be found at Use of standard transformation and regeneration techniques are carried out (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc Central signature: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 pages, ISBN 0-8493-5164-2).  

For example, rapeseed can be made using cotyledons or hypocots transformation can be transformed (Moloney et al., Plant Cell Report 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 694-701). The use of antibiotics for that Agrobacterium and plant selection depends on that for that Transformation used binary vector and Agrobacterium Stem from. Rapeseed selection is commonly used of kanamycin as a selectable plant marker.

The Agrobacterium -mediated gene transfer in flax can be using, for example, one of Mlynarova et al. (1994) Plant Cell Report 13: 282-285 to lead.

The transformation of soy can be done using examples as one in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, US 5,376,543, US 5,169,770 (University Toledo) described technique can be performed.

Plant transformation using particles bombardment, polyethylene glycol-mediated DNA recording or is about silicon carbon fiber technology, for example described by Freeling and Walbot "The maize handbook" (1993) ISBN 3-540-97826-7, Springer Verlag New York).

Example 13 In vivo mutagenesis

The in vivo mutagenesis of microorganisms can be done by passage the plasmid (or other vector) DNA by E. coli or other microorganisms (e.g. Bacillus spp. or yeast, like Saccharomyces cerevisiae), where the skills that Maintain the integrity of their genetic information, is disturbed. Common mutator strains have mutations in the genes for the DNA repair system (e.g. mutHLS, mutD, mutT etc.; see Rupp, W. D. (1996) DNA repair for a reference mechanisms, in: Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington). These strains are known to the person skilled in the art. The Use of these strains is described, for example, in Greener, A., and Callahan, M. (1994) Strategies 7: 32-34. The transfer mutated DNA molecules in plants preferably follow Selection and test of the microorganisms. Become transgenic plants after various examples in the example part of this document generated.  

Example 14 Examination of the expression of a recombinant Gene product in a transformed organism

The activity of a recombinant gene product in the transformed Host organism was based on the transcription and / or the Translation plane measured.

A suitable method for determining the amount of Transcription of the gene (an indication of the amount of RNA, available for the translation of the gene product) is the performance of a Northern blot (as a reference point see Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York, or the sample section mentioned above), being a primer designed to match the gene of Interest binds with a detectable label (usually radioactive or chemiluminescent) is marked so that when the Total RNA of a culture of the organism extracted on a gel separated, transferred to a stable matrix and with it The probe is incubated, binding and the extent of binding Probe the presence and also the amount of mRNA for this gene displays. This information shows the degree of transcription of the transformed gene. Total cellular RNA can be derived from cells Tissues or organs with multiple procedures, all in the subject are known, such as that of Bormann, E.R., et al. (1992) Mol. Microbiol. 6: 317-326, prepared become.

To examine the presence or relative amount of this mRNA translated protein can use standard techniques such as a Western blot can be used (see e.g. Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In this process, the total cellular Proteins extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose and with a Probe, such as an antibody, specific to the desired one Protein binds, incubated. This probe is usually with one chemiluminescent or colorimetric marking, which is easy to prove. The presence and amount of observed mark shows the presence and amount of desired mutant protein present in the cell.  

Example 15 Analysis of the impact of the recombinant proteins on the production of the desired product

The impact of genetic modification in plants, fungi, Algae, ciliates or on the production of a desired ver binding (such as a fatty acid) can be determined by the modified microorganisms or the modified plant under suitable conditions (such as those described above) be grown and the medium and / or the cellular compo the increased production of the desired product (i.e. of lipids or a fatty acid) is examined. This analysis techniques are known to the person skilled in the art and include spectroscopy, Thin layer chromatography, staining methods of various types, enzymatic and microbiological processes as well as analytical Chromatography, such as high performance liquid chromatography (see for example Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery and purification", pp. 469-714, VCH: Weinheim; Belter, P.A., et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J.F., and Cabral, J.M.S. (1992) Recovery processes for biological Materials, John Wiley and Sons; Shaeiwitz, J.A., and Henry, J.D. (1988) Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to the methods mentioned above, plant lipids are made from Plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940, and Browse et al. (1986) Analytic Biochemistry 152: 141-145. The qualitative and quantitative lipid or fatty acid analysis is described with Christie, William W., Advances in Lipid Methodology, Ayr / Scotland: Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide - Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 S. (Oily 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.

In addition to measuring the end product of the fermentation it is also possible to add other components of the metabolic pathways analyze that to produce the desired connection used as intermediates and by-products to the  To determine overall production efficiency of the compound. The analysis methods include measurements of the amounts of nutrients in the medium (e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of the biomass composition and growth, analysis of production Common metabolites of biosynthetic pathways and measurements of Gases generated during fermentation. Standard Procedures for these measurements are in Applied Microbial Physiology; A Practical Approach, P.M. Rhodes and P.F. Stanbury, Ed., IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and the literature references specified therein.

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 film chromatography).

The unambiguous proof of the presence of fatty acid products can be analyzed using recombinant organisms Standard analysis methods are obtained: GC, GC-MS or TLC, as variously described by Christie and the literature (1997, in: Advances on Lipid Methodology, Fourth Ed .: Christie, Oily Press, Dundee, 119-169; 1998, gas chromatography mass spectrometry method, lipids 33: 343-353).

The material to be analyzed can be Grinding in a glass mill, liquid nitrogen and grinding or be broken up through other applicable procedures. The Material must be centrifuged after breaking open. The Sediment is in aqua dest. resuspended, 10 min at 100 ° C heated, cooled on ice and centrifuged again, followed by Extraction in 0.5 M sulfuric acid in methanol with 2% dimethoxy propane for 1 h at 90 ° C, resulting in hydrolyzed oil and lipid leads compounds that result in transmethylated lipids. This Fatty acid methyl esters are extracted into petroleum ether and finally a GC analysis using a capillary column (Chrome pack, WCOT fused silica, CP-Wax-52 CB, 25 m, 0.32 mm) a temperature gradient between 170 ° C and 240 ° C for 20 min and Subjected for 5 min at 240 ° C. The identity of the fat obtained Acid methyl ester must be made using standards that are out commercial sources are available (i.e. sigma) become.

For fatty acids for which no standards are available, the Identity via derivatization and subsequent GC-MS analysis to be shown. For example, the localization of fatty acids  with triple bond using GC-MS after derivatization with 4,4-dimethoxyoxazoline derivatives (Christie, 1998, see above) to be shown.

Example 16 Expression constructs in heterologous microbial Systems Strains, growth conditions and plasmids

The Escherichia coli strain XL1 Blue MRF 'kan (Stratagene) was used for subcloning the new elongase pPPSE1 from Physcomitrella patens. We used the Saccharomyces cerevisiae strain INVSc 1 (Invitrogen Co.) for the functional expression of this gene. E. coli was grown in Luria Bertini broth (LB, Duchefa, Haarlem, The Netherlands) at 37 ° C. If necessary, ampicillin (100 mg / liter) was added and 1.5% agar (w / v) was added for LB solid media. S. cerevisiae was at 30 ° C either in YPG medium or in complete minimal medium without uracil (CMdum; see: Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, Struhl, K., Albright, LB, Coen, DM, and Varki, A. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York) grown with either 2% (w / v) raffinose or glucose . For solid media, 2% (w / v) Bacto ^™ agar (Difco) was added. The plasmids used for cloning and expression were pUC18 (Pharmacia) and pYES2 (Invitrogen Co.).

Cloning and expression of a PUFA-specific elongase Physcomitrella patens

For expression in yeast, the P. patens cDNA clone ppPSE1 (former database sequence name: 08_ck19_b07, new name: pp001019019f), which encodes the PUFA-specific elongase (PSE1) gene, was first modified so that a BamHI restriction site and the yeast consensus sequence for highly efficient translation (Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-292) in addition to the start codon and a BamHI restriction site that flanked the stop codon. To amplify the open reading frame, a pair of primers complementary to its 5 'and 3' ends was synthesized.
ppex6: GGATCCACATAATGGAGGTCGTGGAGAGATTC
ppex6r: GGATCCTCACTCAGTTTTAGCTCCCTTTTGC

The PCR reaction was performed using plasmid DNA as a template in one Thermocycler (Biometra) with the Pfu-DNA (Stratagene) polymerase and the following temperature program: 3 min at 96 ° C, followed by 25 cycles of 30 s at 96 ° C, 30 s at 55 ° C and 3 min at 72 ° C, 1 cycle with 10 min at 72 ° C and stop at 4 ° C.

The correct size of the amplified DNA fragment of 890 bp was confirmed by agarose TBE gel electrophoresis. The amplified DNA was extracted from the gel with the QIAquick gel extraction kit (QIAGEN) and extracted into the SmaI restriction place of the dephosphorylated vector pUC18 using the Sure Clone Ligation Kit (Pharmacia) ligated using pUCPSE1 was obtained. After transforming E. coli XL1 Blue MRF ' a mini DNA preparation (Riggs, M.G., & McLachlan, A. (1986) A simplified screening procedure for large numbers of plasmid mini-preparation. BioTechniques 4, 310-313) to 24 ampicillin resistant transformants performed, and positive Clones were identified using BamHI restriction analysis. The sequence of the cloned PCR product was determined using Resequencing using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt) approved.

The plasmid DNA from pUC-PSE1 was also digested with BamHI and the ~ 900 bp fragment obtained into the BamHI restriction site of dephosphorylated yeast E. ligated coli shuttle vector pYES2, whereby pY2PSE1 was obtained. After the transformation of E. coli and DNA minipreparation from the transformates was the Orientation of the DNA fragment in the vector by HindIII cleavage checked. A clone was used for the DNA maxi preparation Nucleobond® AX 500 plasmid DNA extraction kit (Macherey-Nagel, Düringen) bred.

Saccharomyces INVSc1 was analyzed with pY2PSE1 and pYES2 using a modified PEG / lithium acetate protocol transformed (Ausubel et al., 1995). After selection on CMdum agar plates with 2% Four pY2PSE11 transformants (pY2PSE1a-d) and glucose were a pYES2 transformant for further breeding and functional Expression selected.  

Functional expression of elongase activity in yeast

Preculture

20 ml of CMdum liquid medium with 2% (w / v) raffinose were inoculated with the transgenic yeast clones (pY2PSEIa-d, pYES2) and grown for 3 T at 30 ° C, 200 rpm until an optical density at 600 nm (OD ₆₀₀ ) was reached by 1.5-2.

Main culture

For expression, 20 ml of CMdum liquid medium with 2% raffinose and 1% (v / v) Tergitol NP-40 with γ-linoleic acid (γ-18: 3) were mixed to a final concentration of 0.003% (w / v. ) enriched. The media were inoculated with the precultures to an OD ₆₀₀ of 0.05. Expression was induced at an OD ₆₀₀ of 0.2 with 2% (w / v) galactose for 16 h after which the cultures had reached an OD ₆₀₀ of 0.8-1.2.

Fatty acid analysis

The total fatty acids were extracted from yeast cultures and analyzed by gas chromatography. Of these, cells from 5 ml culture were harvested by centrifugation (1000 × g, 10 min. 4 ° C.) and washed once with 100 mM NaHCO ₃ , pH 8.0, in order to remove residual medium and fatty acids. To prepare the fatty acid methyl ester (FAMES), the cell sediments were treated with 1 M methanolic H ₂ SO ₄ and 2% (v / v) dimethoxypropane at 80 ° C. for 1 hour. The FAMES were extracted twice with 2 ml of petroleum ether, once with 100 mM NaHCO ₃ , pH 8.0 and once with distilled water and dried with Na ₂ SO ₄ . The organic solvent was evaporated under a stream of argon and the FAMES were dissolved in 50 ul petroleum ether. The samples were separated on a ZEBRON-ZB-Wax capillary column (30 m, 0.32 mm, 0.25 µm; Phenomenex) in a Hewlett Packard 6850 gas chromatograph with a flame ionization detector. The oven temperature was from 70 ° C (hold 1 min) to 200 ° C at a rate of 20 ° C / min. then programmed to 250 ° C (hold 5 min) at a rate of 5 ° C / min and finally to 260 ° C at a rate of 5 ° C / min. Nitrogen was used as the carrier gas (4.5 ml / min at 70 ° C). The fatty acids were identified by comparison with retention times of FAME standards (SIGMA).

Expression analysis

The fatty acid patterns of five transgenic yeast strains in mol% are shown in Table 1.

The ratios of the γ-linolen added and taken up acid are highlighted in bold numbers that the elongated products by red numbers and 06292 00070 552 001000280000000200012000285910618100040 0002010005973 00004 06173 γ-linolenic acid by bold numbers (last line).

The GC analysis of FAMES, which consists of total lipids of the yeasts transformed with pYES2 (i / control) and pY2PSE1 (ii-iv c + d / each transformed with pY2PSEIA, pY2PSEIB, pY2PSEIC, pY2PSEID) is shown in FIG. 2a-e shown. For the analysis, the transgenic yeasts were grown in the presence of γ-18: 3. Tab. 1 shows their fatty acid patterns in mol%. The uptake of γ-18: 3 is highlighted in bold numbers, the elongation product dihomo-γ-linolenic acid (20: 3 ^{? 8,11,14} ) is underlined and the ratio γ-18: 3 elongation product (also in in mol. -% by numbers printed in bold (last line) The structure and the mass spectra of the DMOX derivative of cis-Δ ^6,9,12 18: 3 can be seen from Fig. 3a + b The structure and the mass spectra of the DMOX derivative of Δ ^{8, 11, 14} 20: 3 can be seen from Fig. 4a + b.

The results show that γ-18: 3 has been incorporated in large amounts in all transgenic yeasts. All four transgenic yeast clones that were transformed with pY2PSE1 show an additional peak in the gas chromatogram, which was identified as 20: 3 ^{Δ 8, 11, 14} by comparison of the retention times. Gas chromatography / mass spectroscopy can provide additional support to confirm this identity. The proportion of elongated γ-18: 3, as shown in Table 1, was 23.7 to 40.5%. Furthermore, no significant elongation of palmitic acid (16: 0), palmitoleic acid (16: 1), stearic acid (18: 0) or oleic acid (18: 1 ^{Δ 9} ) was observed.

The identified products showed that the nucleotide sequence of PpPSE1 encodes a Δ ⁶ -selective fatty acid elongase from the moss Physcomitrella patens, which leads to the formation of new fatty acids in transgenic yeasts.

Further feeding experiments with various other fatty acids (e.g. linoleic acid (18: 2 ^{Δ 9.12?} ), Stearidonic acid (18: 4 ^{Δ 6,9,12,15} )) can be carried out to confirm the substrate ^{selectivity of} these elongase in more detail.

Example 17 Purification of the desired product from transformier organisms in general

Obtaining the desired product from plant material or Mushrooms, algae, ciliates, animal cells or from the supernatant of the cultures described above can be procedures known in the art. Will be the one you want Product not secreted from the cells, the cells can the culture is harvested by slow centrifugation, the Cells can be made using standard techniques such as mechanical force or Ultrasound treatment to be lysed. Organs of plants can mechanically separated from other tissues or other organs become. After homogenization, the cell debris are removed Centrifugation removed, and the supernatant fraction, which which contains soluble proteins is used for further purification of the desired connection. The product is made desired cells are secreted, the cells are slow Centrifugation removed from the culture, and the supernatant Fraction is kept for further cleaning.

The supernatant fraction from each cleaning procedure becomes one Chromatography with a suitable resin, which desired molecule either back on the chromatography resin is held, but not many impurities in the sample, or the impurities remain on the resin, the sample however not. These chromatography steps can be done if necessary can be repeated, using the same or different Chromato graphic resins are used. The specialist is in the selection suitable chromatography resins and their most effective application for a specific molecule to be purified. The ge purified product can be done by filtration or ultrafiltration concentrated and kept at a temperature at which is the maximum stability of the product.

There is a wide range of cleaning processes in the field known, and the above cleaning process should not be restrictive. These cleaning procedures are for example described in Bailey, J.E., & Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined by standard techniques of the field. To include high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin film chromatography, NIRS, enzyme test or microbiological. A For an overview of these analysis methods, see: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al.  (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, Pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A., et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

Equivalents

Those skilled in the art will recognize or can understand many equivalents of here described specific embodiments of the invention determine by using only routine experiments. These equivalents are intended to be encompassed by the claims.  

SEQUENCE LOG

Claims (21)

1. Isolated nucleic acid that comes from a plant and encodes a polypeptide that extends C ₁₈ fatty acids with at least two double bonds in the fatty acid by at least two carbon atoms. 2. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide that extends C ₁₈ fatty acids with at least two double bonds in the fatty acid molecule, selected from the group consisting of

• a) a nucleic acid sequence shown in SEQ ID NO: 1,
• b) a nucleic acid sequence which, according to the degenerate genetic code, comes from the sequence shown in SEQ ID NO: 1,
• c) Derivatives of the sequence shown in SEQ ID NO: 1, which encodes polypeptides with at least 50% homology to the sequence which encodes the amino acid sequences in SEQ ID NO: 2, the sequence acting as a C ₁₈ elongase.

3. The isolated nucleic acid sequence according to claim 2, wherein the Sequence comes from a plant. 4. Isolated nucleic acid sequence according to claim 2 or Claim 3, wherein the sequence is from Physcomitrella. 5. Amino acid sequence derived from an isolated nucleic acid sequence according to one of claims 2 to 4. 6. gene construct comprising an isolated nucleic acid with the Sequence SEQ ID NO: 1 according to any one of claims 2 to 4, wherein the nucleic acid is functional with one or more Regulation signals is connected. 7. gene construct according to claim 6, the gene expression by the Regulation signals is amplified. 8. A vector comprising a nucleic acid according to claim 2 or one Gene construct according to claim 6.   9. organism comprising at least one nucleic acid after Claim 2, a gene construct according to claim 6 or one The vector of claim 8. 10. The organism of claim 9, wherein the organism is a micro organism, an animal or a plant. 11. Organism according to claim 9 or 10, wherein the organism is a transgenic plant. 12. A method for producing PUFAs, the method comprising growing an organism comprising a nucleic acid according to claim 2, a gene construct according to claim 6 or a vector according to claim 8, encoding a polypeptide containing C ₁₈ fatty acids with at least two double bonds extended in the fatty acid molecule by at least two carbon atoms under conditions under which PUFAs are formed in the organism. 13. The method according to claim 12, wherein the PUFAs produced by the method are C ₂₀ or C ₂₂ fatty acid molecules with at least two double bonds in the fatty acid molecule. 14. The method according to claim 13, wherein the C ₂₀ or C ₂₂ fatty acid molecules are isolated from the organism in the form of an oil, lipid or a free fatty acid. 15. The method according to any one of claims 12 to 14, wherein the Organism a microorganism, an animal or a plant is. 16. The method according to any one of claims 12 to 15, wherein the Organism is a transgenic plant. 17. The method according to any one of claims 12 to 16, wherein the C ₁₈ fatty acid is a fatty acid with three double bonds in the molecule. 18. Oil, lipids or fatty acids or a fraction thereof provided by the method according to any one of claims 12 to 17th 19. Oil, lipid or fatty acid composition comprising PUFAs and comes from transgenic plants.   20. oil, lipid or fatty acid composition according to claim 19, the PUFAs originating from transgenic plants which have a The nucleotide sequence of claim 2. 21. Use of the oil, lipid or fatty acid composition in Feed, food, cosmetics or pharmaceuticals.