WO2001094606A2

WO2001094606A2 - Method for producing c9 aldehydes, c9 alcohols and esters thereof

Info

Publication number: WO2001094606A2
Application number: PCT/EP2001/006584
Authority: WO
Inventors: Ekkehart Berndt; Ivo Feussner
Original assignee: Ipk Institut Für Pflanzengenetik Und Kulturpflanzen Forschung
Priority date: 2000-06-09
Filing date: 2001-06-11
Publication date: 2001-12-13
Also published as: AU2001279652A1; WO2001094606A3; DE10028639C2; DE10028639A1

Abstract

The invention relates to a method for producing C9 aldehydes, C9 alcohols and esters thereof. Said method is based on the reaction of multiple unsaturated fatty acids with recombinant 9-lipoxygenase (9-LOX) and 13-hydroperoxide lyase (13-HPL).

Description

Process for the preparation of C - aldehydes, C - alcohols and their esters

The invention relates to a process for the preparation of C ₉ aldehydes, C ₉

Alcohols and their esters, which are based on the reaction of polyunsaturated fatty acids with recombinant 9-lipoxygenase (9-LOX) and 13-hydroperoxide lyase (13-HPL).

The breakdown of polyunsaturated fatty acids begins with the oxygenation of the (lZ, 4Z) pentadiene system of polyunsaturated fatty acids. This reaction is catalyzed by the enzyme lipoxygenase (EC 1.13.11.12), which is found in plants, animals and microorganisms. The oxygenated products, known as fatty acid hydroperoxides, are precursors for many important hormones, for example jasmonic acid, traumatic acid and taste and fragrance molecules in plants, for example hexenals, (3Z) -hexenol and nonenals.

Compounds such as jasmonic acid are produced from hydroperoxides, such as 13-hydroperoxylinolenic acid, via an allene oxide synthase and an alloxide cyclase-dependent route. Jasmonic acid is involved in stress and disease resistance signal responses via the octadecanoid pathway. Alternatively, 13-hydroperoxylinolenic acid can be cleaved by hydroperoxide lyase to form volatile aldehydes and traumatic acid.

Fatty acid hydroperoxide lyase (HPL) catalyzes the cleavage of carbon-carbon bonds in polyunsaturated fatty acid hydroperoxides to produce short-chain C ₆ or C ₉ aldehydes and ω-oxo acids of chain length C ₁₂ or C ₉ (Vick et al. ( 1976) Plant Physiol. 57: 780-788). The short-chain volatile C ₆ aldehydes contribute to the so-called "green fragrance notes" in a large number of plant leaves, vegetables and fruits. These fragrance notes are often referred to as "fresh grass". Other short-chain volatile aldehydes, such as (3Z, 6Z) nonadienal, which according to the prior art by cleavage of (9S, 15Z, 12Z, 10E) -9-hydroperoxy- 15,12,10-octadecatrienoic acid generated by a 9-hydroperoxide lyase (9-HPL) provide a melon or cucumber-like aroma and / or a corresponding taste contribution in fruits and vegetables. Such features are of great importance for fragrance and aroma substances, in particular for the food industry, but also for the cosmetics, pharmaceutical and chemical industries.

It is also believed that short chain aldehydes also play a role in pathogen resistance. For example, Croft et al. (1993) Plant Physiol. 101: 13-24, reports that the levels of (3Z) -hexenol and (2E) -hexenal increased during a hypersensitive response (HR) of the field bean. It has also been shown that (2E) -hexenal is an effective antibacterial.

The characterization of hydroperoxide lyases is of great importance for the further investigation of plant fatty acid metabolism and for the development of transgenic plants with increased sensory properties including aroma and taste. Plant mechanism studies may provide additional means to enhance, control, modify, or otherwise alter the sensory characteristics of plants that are important to the food industry. Furthermore, the elucidation of the physiological roles of HPL and its products is of interest for the further investigation of disease resistance.

The biosynthesis of C ₆ - and C ₉ - aldehydes and the associated C] ₂ - and C ₉ - ω-keto fatty acids from linoleic or linolenic acid is a widespread reaction in the plant kingdom (e.g. K. Matsui, Belgian J. Bot. 1998, 131, 50-62). Both substance groups can be subject to further reactions in the plant. The aldehydes are reduced by dehydrogenases to the corresponding alcohols and the fatty acids to ω-hydroxy fatty acids. The C ₉ alcohols thus produced can furthermore be reacted with acids, in particular acetic acid, to give the corresponding esters, in particular acetic acid esters, which are also of great industrial importance as fragrances and flavorings.

The synthesis of the C-aldehydes (3Z) -nonenal or (3Z, 6Z) -nonadienal begins with the conversion of linoleic or α-linolenic acid with 9-lipoxygenase to (9S) -hydroperoxy derivatives. These can then be cleaved by a 9-hydroperoxide lyase (9-HPL) into the C ₉ -ω-keto fatty acid and (3Z) -nonenal or (3Z, 6Z) -nonadienal. The resulting aldehydes can then be reduced enzymatically or chemically to the C ₉ alcohols (3Z) -nonenol or (3Z, 6Z) -nonadienol.

N.J. Bäte et al, Plant Physiology 117: 1393-1400 (1998) describe the molecular characterization of an Arabidopsis gene encoding a 13-hydroperoxide lyase. A clear HPL activity was observed when (13S, 9Z, 1 IE, 15Z) -13-hydroperoxy-9, l 1,15-octadecatrienoic acid was used as the substrate, while the activity with (13S, 9Z, 1 IE) -13-Hydroperoxy-9, l 1- octadecadienoic acid was about an order of magnitude lower. A. Geerts et al, Plant Physiology 105: 269-277 (1994) describes the expression of 9-lipoxygenase in wounded tubers of the potato.

International patent application WO 00/00627 discloses nucleic acid sequences. which code for 9-hydroperoxide lyases and 13-hydroperoxide lyases. According to this patent application, 13-hydroperoxide lyase catalyzes exclusively the formation of C ₆ - aldehydes and Cι ₂ -oxo acids from 13-hydroperoxide-Cι ₈ -

Fatty acid derivatives, while 9-hydroperoxide lyase catalyzes exclusively the formation of C ₉ aldehydes and C ₉ oxo acids from 9-hydroperoxide-C ₈ fatty acid derivatives. International patent application WO 00/22145 describes a hydroperoxide lyase gene from maize. Methods for improving disease resistance and for changing the content of aroma molecules in plants are also disclosed therein. These methods include expression of hydroperoxide lyase genes in plants, plant cells and plant tissues. A specificity of the hydroperoxide lyase from corn described there for certain hydroperoxide fatty acids is not described there.

The international patent application WO 99/58648 discloses the 13-hydroperoxide fatty acid lyase protein from guava and the gene which codes for this protein. Furthermore, this patent application describes the cleavage of a 13-hydroperoxide derivative of linolenic acid into n-hexanal and other C ₆ aldehydes and into a C ₂ oxocarboxylic acid catalyzed by the above-mentioned 13-HPL.

The processes described in the prior art thus describe the cleavage of 13 -hydroperoxide fatty acids by 13 -hydroperoxide lyase or 9-hydroperoxide fatty acids by 9-hydroperoxide lyase. Ie, the production of a C ₉ - aldehyde or alcohol from a C ₈ fatty acid via the corresponding hydroperoxide fatty acid derivative requires the cleavage of a 9-hydroperoxide fatty acid derivative with a 9-HPL according to the prior art.

It is an object of the present invention to provide a new, simple process for the preparation of C ₉ aldehydes, C ₉ alcohols and esters of C ₉ alcohols, in which the activity of a 9-hydroperoxide lyase is not required. Further objects of the invention will become apparent from the following description.

These tasks are solved by the subject matter of the independent claims.

Advantageous configurations are defined in the subclaims. Surprisingly, it has now been found that 13-hydroperoxide lyases (9S) convert hydroperoxide fatty acid derivatives which contain a further double bond in position Δ6. According to the invention, a method for producing Cp aldehydes or C alcohols or esters of C ₉ alcohols from polyunsaturated fatty acids is thus provided, the polyunsaturated fatty acids having double bonds at least at positions Δ6 and Δ9. The method according to the invention comprises the following steps:

a) the provision of a nucleic acid molecule which encodes a vegetable or microbial protein with the biological activity of 9-lipoxygenase; b) the provision of a nucleic acid molecule which encodes a vegetable or microbial protein with the biological activity of 13-hydroperoxide lyase; c) the transfer of the nucleic acid molecules from a) and b) to eukaryotic or prokaryotic cells, the coding sequence being under the control of suitable regulatory sequences, d) the expression of the recombinant 9-lipoxygenase and 13-hydroperoxide lyase in the cells; e) the conversion of the polyunsaturated fatty acids, which have at least double bonds at positions Δ6 and Δ9, with the recombinant 9-lipoxygenase to 9-hydroperoxides of the fatty acids; f) the conversion of the 9-hydroperoxides of the fatty acids with 13-hydroperoxide lyase to C ₉ -aldehydes; g) optionally reducing the C ₉ aldehydes produced to C ₉ alcohols; h) optionally the reaction of the C ₉ alcohols from step e) with an acid, in particular acetic acid, to give a corresponding ester; i) if appropriate, the recovery of the C ₉ aldehydes or C ₉ alcohols or esters of the C ₉ alcohols produced from the cells.

The polyunsaturated fatty acids, which have at least double bonds at positions Δ6 and Δ9, are reacted with recombinant 9-lipoxygenase to 9-hydroperoxides of the fatty acids in the process according to the invention. The latter are then converted to C ₉ aldehydes with 13-hydroperoxide lyase.

If a suitable level of 9-LOX or 13 -HPL activity is already present in the eukaryotic or prokaryotic cells, the transfer of the nucleic acid molecules from step a) or b) of the method according to the invention can optionally be dispensed with.

In the context of the present invention, such hydroperoxide lyases are referred to as 13-hydroperoxide lyases which have been described in the prior art as highly specific for 13-hydroperoxides of polyunsaturated fatty acids with at least double bonds at positions Δ6 and Δ9.

Hydroperoxidlyases, which according to the state of the art have no (pronounced)

Having specificity for 13 -hydroperoxide lyases or of which the implementation of 9-hydroperoxy fatty acids was known in the prior art does not fall under this definition.

Preferably used in the present invention as polyunsaturated

Fatty acids cis fatty acids used. Examples of C ₈ fatty acids which have a double bond in position Δ6 and can advantageously be used in the process according to the invention are γ-linolenic acid or stearidonic acid (6Z, 9Z, 12Z, 15Z-octadecatetraenoic acid). In the event that the above-mentioned polyunsaturated fatty acids, in particular γ-linolenic acid or stearidonic acid, are not present in sufficient concentrations in the plant cell, the process according to the invention and the process described below for the production of transgenic plants or plant cells with a modified, in particular increased, process Content of C ₉ aldehydes or C ₉ alcohols or their esters in a preferred embodiment additionally the provision of additional starting substrates in the plant cell. The provision of (additional) starting substrates can take place, for example, by exogenous addition or by co-expression of an acyllipid-Δ6-desaturase in the plant cell. Suitable DNA sequences which code for proteins with the enzymatic activity of an acyllipid-Δ6-desaturase are known to the person skilled in the art and can be found in the prior art. The DNA sequence which is transferred to and expressed in plant cells for the purpose of providing γ-linolenic acid in plants is preferably a DNA sequence from Boragio offlcinalis which codes for an acyllipid-Δ6 desaturase.

Following the transfer of the nucleic acid molecules to plant cells, complete plants are preferably regenerated from the transgenic cells, which serve as production sites for C-aldehydes or Cg-alcohols or esters of C ₉ -alcohols.

In an alternative embodiment of the present invention, the transformed cells are bacterial cells, yeast cells or algal cells. In this case, the cells are cultivated and the C ₉ aldehydes or C ₉ alcohols or esters of the C ₉ alcohols are prepared in one reactor. Of course, here too a sufficient concentration of the substrates may have to be used Provision of the starting substrates as described above can be ensured.

In a further embodiment, transgenic plant cells are cultivated (for example in the form of suspension or callus cultures) and used as a production site for C ₉ aldehydes or C alcohols or esters of C ₉ alcohols.

The nucleic acid molecule from step a) preferably encodes a protein with the biological activity of 9-lipoxygenase from the potato.

Furthermore, the nucleic acid molecule from step b) preferably encodes a 13-hydroperoxy lyase from Arabidopsis or alternatively from tomato, alfalfa or guava.

If desired, the aldehyde formed can be reduced in step e) of the process according to the invention enzymatically, preferably with alcohol dehydrogenase, or chemically, preferably with sodium borohydride, to a C ₉ alcohol.

As an alternative to the expression of the recombinant 9-lipoxygenase or 13 -hydroperoxide lyase in cells described above, the C ₉ -aldehydes or C ₉ -alcohols or esters of the C ₉ -alcohols can also be prepared in vitro in a reaction mixture which as components a polyunsaturated fatty acid with double bonds at least at the positions Δ6 and Δ9, in particular a C ₈ fatty acid, particularly preferably γ-linolenic acid or stearidonic acid; 9-lipoxygenase and 13 -hydroperoxide lyase (as crude extract or in pure form); and, if the production of an alcohol is desired, a reducing agent; and, if the production of an ester is desired, an acid, especially acetic acid; includes.

Another aspect of the present invention relates to methods for changing the content of C - aldehydes or C ₉ - alcohols or esters of the C - alcohols in a host cell. In general, the methods involve either increasing or decreasing the level of C ₉ aldehydes or C ₉ alcohols or esters of the C ₉ alcohols in a host cell. The method comprises the use of expression constructs for carrying out the expression in a host cell of DNA sequences which encode a protein with the biological activity of a 13 -hydroperoxide lyase. The use of expression constructs for changing the content of C-aldehydes or C ₉ alcohols or esters of the C ₉ alcohols in a plant cell or plant is particularly preferred. The process according to the invention is preferably used to change the content of C ₉ aldehydes, C ₉ alcohols or esters of C alcohols in plant parts including leaves, roots, stems, flowers, fruits, seeds and seed oils which are obtained from plant seeds ,

The 13-hydroperoxide lyase-encoding DNA sequences are particularly preferably used to produce transgenic plants which have an increased production of C ₉ aldehydes or C ₉ alcohols or esters of C alcohols in plant fruits and tissues. Such aldehydes are important components of characteristic aromas of fruits, vegetables and green leaves. Thus, a 13-hydroperoxide lyase can also be used according to the invention for the production of transgenic plants with improved aroma characteristics.

To increase lipid peroxidation in plant tissue, the present invention also includes coexpressing a plant or other 13-hydroperoxide lyase in plant tissue with a second gene that is involved in lipid peroxidation. For example, the coexpression of a 13 -hydroperoxide lyase in a plant tissue with a DNA sequence which codes for a lipoxygenase, in particular a 9-lipoxygenase, can increase the lipid peroxidation and thus the content of C - aldehydes which are produced in the plant tissue. increase. Such an increase in C -aldehydes can increase the "melon" or "cucumber" aroma in a plant. Furthermore, the plant cells containing a DNA sequence encoding a 13-hydroperoxide lyase can also be used as a source of C-aldehydes in reactions for the production of C-alcohols for use in flavorings. Such methods are known in the prior art and are described, for example, in US Pat. No. 5,695,973 and in international patent application WO 95/26413. A mixture of aldehydes and alcohols is generally obtained by such processes. The processes are usually carried out in a reaction mixture which contains at least one polyunsaturated fatty acid, a plant material with a relatively high level of enzyme activity of lipoxygenase and hydroperoxide lyase and an alcohol dehydrogenase.

The polyunsaturated fatty acid can vary and includes a single type of unsaturated fatty acid as well as mixtures of different unsaturated ones

Fatty acids. The fatty acids are generally, but not limited to, C ₁₈ fatty acids. Preferred examples include γ-linolenic acid and stearidonic acid. A prerequisite for the specificity of the 13 -hydroperoxide lyase for a 9-hydroperoxide fatty acid is a double bond at position Δ6.

Sources of an alcohol dehydrogenase suitable for the purposes of the present invention are preferably yeasts. Alcohol dehydrogenase catalyzes the conversion of an aldehyde into an alcohol. Yeast also provides a source of nicotin adenine dinucleotide (NADH) as a reducing agent.

The DNA sequence encoding a protein with the biological activity of a 9-LOX or a 13-HPL can be isolated from natural sources or synthesized by conventional methods. Using common molecular biological techniques (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) it is possible to prepare or produce desired constructs for the transformation of plant cells. The cloning, mutagenization, sequence analysis, restriction analysis and other biochemical-molecular biological methods commonly used for genetic engineering manipulation in prokaryotic cells are well known to those of ordinary skill in the art. Thus, not only can suitable chimeric gene constructs with the desired fusion of promoter and 9-LOX or 13-HPL-DNA sequence and possibly further regulatory and / or signal sequences be produced, but, if desired, the person skilled in the art can additionally use Routine techniques, various types of mutations into which the 9-LOX or 13-HPL coding DNA sequence are introduced, which leads to the synthesis of proteins with possibly changed biological properties. On the one hand, it is possible to generate deletion mutants in which the synthesis of correspondingly shortened proteins can be achieved by progressive deletion from the 5 'or from the 3' end of the coding DNA sequence. Furthermore, it is possible to specifically produce enzymes that are localized in certain compartments of the plant cell by adding corresponding signal sequences. Such sequences are described in the literature and are well known to those skilled in the art (see, for example, Braun et al. (1992) EMBO J. 11: 3219-3227; Wolter F. et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 846-850; Sonnewald U. et al. (1991) Plant J. 1: 95-106). Furthermore, the introduction of point mutations at positions at which a change in the amino acid sequence has an influence, for example on the enzyme activity or the regulation of the enzyme, is also conceivable. In this way, for example, mutants can be produced which are no longer subject to the regulatory mechanisms normally prevailing in the cell via allosteric regulation or covalent modification. Furthermore, mutants can be produced which have an altered substrate or product specificity (Hornung et al. (1999), Proc. Natl. Acad. Sci. USA 96: 4192-4197). Furthermore, mutants can be produced which have a changed activity, temperature and / or pH profile. For genetic engineering manipulation in prokaryotic cells, the recombinant nucleic acid molecules according to the invention or parts thereof can be introduced into plasmids which permit mutagenesis or a sequence change by recombining DNA sequences. With the help of standard methods (see, for example, Sambrook et al. (1989), vide supra), base exchanges can be carried out or natural or synthetic sequences can be added. To connect the DNA fragments to one another, adapters or linkers can be added to the fragments where necessary. Appropriate restriction sites can also be provided by means of enzymatic and other manipulations, or superfluous DNA or restriction sites can be removed. Where insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. Sequence analysis, restriction analysis and other biochemical-molecular biological methods are generally used as analysis methods.

In principle, any promoter active in plant cells can be used for the expression of the DNA sequences contained in the recombinant nucleic acid molecules in plant cells. For example, the 9-LOX or 13-HPL DNA sequences can be expressed in plant cells under the control of constitutive, but also inducible or tissue- or development-specific regulatory elements, in particular promoters. For example, while the use of an inducible promoter enables the specifically triggered expression of the 9-LOX or 13-HPL DNA sequences in plant cells, the use of tissue-specific, for example leaf or seed-specific, promoters offers the possibility of determining the content of C ₉ aldehydes or C ₉ alcohols or esters of C ₉ alcohols in certain tissues, for example in leaf or Seed tissue, changing. Other suitable promoters, for example, mediate light-induced gene expression in transgenic plants. The promoter can be homologous or heterologous with respect to the plants to be transformed. Suitable promoters are, for example, the 35S RNA promoter of the Cauliflower Mosaic Virus and the ubiquitin promoter from maize for constitutive expression. Suitable seed-specific promoters are, for example, the USP (Bäumlein et al. (1991), Mol. Gen. Genet. 225: 459-467) or the Hordein promoter (Brandt et al. (1985), Carlsberg Res. Commun. 50: 333-345).

Constitutive, germination-specific and seed-specific promoters are preferred in the context of this invention, since they are particularly suitable for the targeted increase in the content of C ₉ aldehydes or C ₉ alcohols or esters of C ₉ alcohols in transgenic seeds, including also in Connection with the antisense or cosuppression technique.

In any case, the person skilled in the art can find suitable promoters in the literature or isolate them from any plants using routine methods.

Furthermore, there is a transcription or termination sequence which serves to correctly terminate the transcription and can also be used to add a polyA tail to the transcript, which is assigned a function in stabilizing the transcripts. Such elements are described in the literature (e.g. Gielen (1989) EMBO J. 8: 23-29) and are interchangeable, e.g. the terminator of the octopine synthase gene from Agrobacterium tumefaciens.

In a preferred embodiment, the nucleic acid molecules contained in the vectors are linked to regulatory elements that the

Ensure transcription and, if necessary, translation in prokaryotic and eukaryotic cells.

If necessary, the nucleic acid sequences can be supplemented by enhancer sequences or other regulatory sequences. These regulatory sequences also include signal sequences, for example, which ensure that the gene product is transported to a specific compartment.

It is also an object of the invention to provide new transgenic plants, plant cells, plant parts, transgenic propagation material and transgenic harvested products which are characterized by a change in C ₉ aldehydes or C ₉ alcohols or esters of the wild type plants or cells Label C ₉ alcohols.

This object is achieved by the transfer of the nucleic acid molecules, which code for a plant or microbial protein with the biological activity of a 13-HPL, and their expression in plants. By transferring the above-mentioned nucleic acid molecules, it is now possible to use genetic engineering methods to modify plant cells to the extent that they have a new or modified 13 -HPL activity compared to wild-type cells and, as a result, there is a change in the C ₉ content - Aldehydes or C ₉ - alcohols or esters of C ₉ alcohols in the plant.

Thus, in one embodiment, the invention relates to plants or their cells and parts in which the content of C ₉ aldehydes or C ₉ alcohols or esters of C ₉ alcohols increases due to the presence and expression of the abovementioned nucleic acid molecules compared to wild type plants is.

However, the invention also relates to those plants in which the transfer of the abovementioned nucleic acid molecules leads to a reduction in the content of C ₉ aldehydes or C ₉ alcohols or esters of the C ₉ alcohols. Such a reduction can be achieved, for example, by the transfer of antisense constructs or by other suppression mechanisms, such as cosuppression. The invention further relates to transgenic plant cells or plants comprising such plant cells and their parts and products in which the above-mentioned nucleic acid molecules coding for 13-HPL are present integrated into the plant genome. The invention also relates to plants in the cells of which the abovementioned nucleic acid molecule is present in self-replicating form, ie the plant cell contains the foreign DNA on an independent nucleic acid molecule (transient expression).

In principle, the plants which have been transformed with the above-mentioned nucleic acid molecules and in which a modified amount of C ₉ aldehydes or C ₉ alcohols or esters of C alcohols are synthesized due to the introduction of such a molecule trade any plant. It is preferably a monocot or dicot crop.

Examples of monocotyledonous plants are the plants belonging to the genera Avena (oat), Triticum (wheat), Seeale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, sorghum (millet), Zea ( Corn). Among the dicotyledonous crops are to name legumes, such as legumes and in particular alfalfa, soybean, rapeseed, tomato, sugar beet, potatoes, ornamental plants or trees. Other useful plants can be, for example, fruit (in particular apples, pears, cherries, grapes, citrus, pineapple and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal, cotton, flax, sunflower and medicinal plants and pasture grasses and forage plants. The cereals wheat, rye, oats, barley, rice, corn and millet, feed grain, sugar beet, rapeseed, soybean, sunflower, flax, tomato, potato, sweet grasses are particularly preferred.

Forage grass and clover. It is self-evident that the invention relates in particular to conventional food or fodder plants. In addition to the plants already mentioned, peanut, lentil, broad bean, black beet, buckwheat, carrot, sunflower, Jerusalem artichoke, turnip, white mustard, turnip and stubble are worth mentioning. Oilseeds are particularly preferred.

The invention further relates to propagation material and harvest products of plants according to the invention, for example seeds, fruits, cuttings, tubers, rhizomes, etc., and parts of these plants, such as protoplasts, plant cells and calli.

In a further embodiment, the invention relates to host cells, in particular prokaryotic and eukaryotic cells, which have a cell as described above

Nucleic acid molecule or a vector have been transformed or infected, as well as cells derived from such host cells and containing the described nucleic acid molecules or vectors. The host cells can be bacteria, viruses, algae, yeast and fungal cells as well as plant or animal cells.

The invention also relates to those host cells which, in addition to the nucleic acid molecules according to the invention, contain one or more nucleic acid molecules which are transmitted by genetic engineering or natural means and which carry the genetic information for enzymes involved in the LOX-dependent catabolism of polyunsaturated fatty acids in plants.

The present invention is also based on the object of providing processes for the production of plant cells and plants which are distinguished by a changed content of C ₉ aldehydes or C alcohols or esters of the C ₉ alcohols.

This object is achieved by methods by means of which the production of new plant cells and plants which, owing to the transfer of the above-mentioned nucleic acid molecules coding for 13-HPL, result in a modified content of C ₉ aldehydes or C ₉ alcohols or esters of C ₉ - have alcohols, is possible. Various methods are available for producing such new plant cells and plants. On the one hand, plants or plant cells can be modified using conventional genetic engineering transformation methods in such a way that the new nucleic acid molecules are integrated into the plant genome, ie stable transformants are generated. On the other hand, a nucleic acid molecule according to the invention, the presence and optionally expression of which in the plant cell causes an altered biosynthesis, can be contained in the plant cell or the plant as a self-replicating system. For example, the nucleic acid molecules according to the invention can be contained in a virus with which the plant or plant cell comes into contact.

According to the invention, plant cells and plants which, owing to the expression of a nucleic acid sequence according to the invention, have a modified, in particular increased, content of Cg aldehydes or C ₉ alcohols or esters of C ₉ alcohols are produced by a process which comprises the following steps:

a) Production of a recombinant nucleic acid molecule, comprising the following components in a 5 '->3' orientation: regulatory sequences of a promoter active in plant cells, - operatively linked to a nucleic acid sequence which codes for a protein with the biological activity of a 13-HPL, and optionally operatively linked sequences that can serve as transcription, termination and / or polyadenylation signals in plant cells. b) transfer of the nucleic acid molecule from a) to plant cells. c) Provision of the starting substrates in the form of polyunsaturated fatty acids which have at least double bonds at the positions Δ6 and Δ9, in particular C ₈ fatty acids, particularly preferably γ-linolenic acid or stearidonic acid, in the plant cell. Step c) of the method according to the invention described above is of essential importance since the occurrence of the starting substrates such as, for example, γ-linolenic acid in the plant kingdom is restricted to a few plants, such as, for example, borage and the evening primrose.

Alternatively, one or more nucleic acid sequences according to the invention can be introduced into the plant cell or the plant as a self-replicating system.

As a further alternative, step a) of the above method can be modified such that the nucleic acid sequence which codes for a protein with the biological activity of a 13-HPL is coupled in antisense orientation to the 3 'end of the promoter.

In a further aspect of the present invention, one or more additional nucleic acid molecules can be introduced which code for proteins which catalyze the oxygenation of fatty acids to 9-hydroperoxide fatty acids (lipoxygenases).

To prepare the introduction of foreign genes into higher plants or their cells, a large number of cloning vectors are available, which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence can be introduced into the vector at a suitable restriction site. The plasmid obtained is then used for the transformation of E. co / z ^' cells. Transformed E. co / z ^' cells are grown in an appropriate medium and then harvested and lysed, and the plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical methods are generally used as the analysis method for characterizing the plasmid DNA obtained. molecular biological methods used. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences.

A prerequisite for the introduction of recombinant nucleic acid molecules and vectors in plant cells is the availability of suitable transformation systems. A wide range of transformation methods have been developed and established here over the past two decades. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, diffusion of protoplasts, the direct gene transfer of isolated DNA to protoplasts, the injection and electroporation of DNA into plant cells, the introduction of DNA by means of the biolistic Methods and other possibilities, whereby the person skilled in the art can easily determine the most suitable method. All transformation processes have been well established for many years and are undoubtedly part of the standard repertoire of the specialist in plant molecular biology, plant biotechnology and cell and tissue culture.

When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies to direct gene transfer. Simple plasmids, e.g. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is recommended. The usual selection markers are known to the person skilled in the art and it is not a problem for him to select a suitable marker.

Depending on the method of introducing desired genes into the plant cell, additional DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right one must be used Limitation, but often the right and left limitation of the T-DNA contained in the Ti or Ri plasmid as a flank region with the genes to be introduced. If Agrobacteria are used for the transformation, the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria by means of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. The intermediate vector can be found by means of a helper plasmid

Agrobacterium tumefaciens are transmitted (conjugation). Binary vectors can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria. The agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells. The use of T-DNA for the transformation of plant cells has been intensively investigated and has been sufficiently described in well-known overview articles and manuals for plant transformation.

For the transfer of the DNA into the plant cell, plant explants can inevitably be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants can then be regenerated from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells. Once the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker which gives the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin and others. The individually selected marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA. Alternative markers are also suitable for this, such as nutritive markers and screening markers (such as GFP, green fluorescent protein). Of course, selection markers can also be dispensed with entirely, but this is accompanied by a fairly high need for screening. If the selection marker used is to be removed again after the transformation and identification of successfully transformed cells or plants has been carried out, various strategies are available to the person skilled in the art. For example, sequence-specific recombinases can be used, for example in the form of the retransformation of a starting line expressing recombinase and outcrossing of the recombinase after removal of the selection marker (see, for example, Reiss et al. (1996) Proc. Natl. Acad. Sci. USA 93: 3094- 3098; Bayley et al. (1992) Plant Mol. Biol. 18: 353-361; Lloyd et al. (1994) Mol. Gen. Genet. 242: 653-657; Maser et al. (1991) Mol. Gen. Genet. 230: 170-176; Onouchi et al. (1991) Nucl. Acids Res. 19: 6373-6378). The selection marker can also be removed by cotransformation followed by outcrossing.

The regeneration of the transgenic plants from transgenic plant cells is carried out according to customary regeneration methods using conventional nutrient media and phytohormones. If desired, the plants obtained in this way can then be subjected to conventional methods, including molecular biological methods, such as PCR, blot analysis, or biochemical methods for the presence of the introduced DNA. which encode a protein with the enzymatic activity of a 13-HPL, or are examined for the presence of 13-HPL enzyme activity.

Regardless of the regulatory sequences used, which control the expression of the 9-LOX or 13-HPL sequences, the person skilled in the art has a wide range of molecular biological and / or biochemical methods for the analysis of the transformed plant cells, transgenic plants, parts of plants , Harvest products and propagation material available, e.g. PCR, Northern blot analysis for the detection of RNA specific for 9-LOX or 13-HPL or for determining the level of accumulation of 9-LOX or 13-HPL-specific RNA, Southern blot analysis for the identification of DNA sequences encoding 9-LOX or 13-HPL or Western blot analysis to detect the 9-LOX or 13-HPL encoded by the nucleic acid molecules. Of course, the detection of the enzymatic activity of the 9-LOX or 13-HPL can also be determined by a person skilled in the art using protocols available in the literature. Further you can e.g. lay out the seed obtained by crossings on medium containing the selection agent suitable for the selection marker transmitted together with the 9-LOX or 13-HPL DNA sequence, and on the basis of the germination capacity and the growth of the daughter generation (s) and the segregation pattern Draw conclusions about the genotype of the respective plant.

Another object of the invention is to demonstrate uses of 13-HPL.

This object is achieved by the uses according to the invention of 13-HPL for the production of plant cells and plants which are distinguished by a content of C ₉ aldehydes or C alcohols or esters of C ₉ alcohols which is different compared to wild type cells or wild type plants , solved. The present invention further encompasses every possible form of use of the nucleic acid molecule coding for 13-HPL, the presence of which and optionally expression in plants brings about a change in the content of C ₉ aldehydes or C alcohols or esters of C ₉ alcohols.

The nucleic acid molecules coding for 13-HPL can thus be used according to the invention to produce transgenic plants in which the 13-HPL content is higher or lower than natural or in cell types or development stages in which the 13-HPL is normally not being found. This causes a change in the content of C ₉ aldehydes or C ₉ alcohols or esters of the C ₉ alcohols in these cells.

The accumulation of C ₉ aldehydes or C ₉ alcohols or esters of C ₉ alcohols can be an advantageous phenotype in plants used for food.

For some applications it may be useful to introduce the 13-HPL into different cellular compartments or to facilitate their secretion from the cell. It is thus possible that the nucleic acid molecules coding for 13-HPL are modified in such a way that they are matched with suitable intracellular target sequences, such as transit sequences (K. Keegstra (1989) Cell 56: 247-253), signal sequences and the like be supplemented.

For some applications it may also be desirable to reduce or eliminate the expression of nucleic acid molecules encoding 13-HPL. To achieve this, a nucleic acid molecule developed for the cosupression of the 13-HPL can be generated by linking a gene or gene fragment which encodes a 13-HPL with plant promoter sequences. Alternatively, a nucleic acid molecule developed for the expression of antisense RNA for all or part of the above-mentioned nucleic acid molecule can be by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Both the genes for cosuppression and the antisense genes can be introduced into the plants by means of transformation, as a result of which the expression of the corresponding endogenous genes is reduced or eliminated.

The following examples serve to describe the present invention without restricting it in any way.

Examples

Example 1 :

Expression of the recombinant proteins 9-lipoxygenase from Solanum tuberosoum and 13-hydroperoxide lyase from Arabidopsis thaliana

Amplified PCR fragments of cDNA sequences which code for a 9-lipoxygenase from Solanum tuberosoum and a 13 -hydroperoxide lyase from Arabidopsis thaliana were ligated into the expression vector QIAexpress pQE 30 (Qiagen, Hilden, Germany) and transformed into XLl -Blue cells pre-cloned using the p GEM ^R -T Easy Vector System II kit (Promega, Madison, USA). The cloning was then carried out into the expression strain E. coli SG13009 [pREP4].

For the expression of the plant lipoxygenase or hydroperoxide lyase, the E. co / z strain was grown in LB medium at 37 ° C. up to an optical density at 600 nm of 0.6. The culture was then cooled on ice and expression induced by adding 1 mM IPTG. Expression took place at 10 ° C for 18 hours. Finally the bacteria were spun down at 4000 xg. Example 2:

Purification of Arabidopsis thaliana recombinant hydroperoxide lyase from E. coli

The Arabidopsis thaliana recombinant hydroperoxide lyase was purified according to the following protocol:

- Washing the bacterial pellet from Example 1 with 50 mM Na phosphate buffer at pH 8 - Resuspending the bacterial pellet in 5 volumes of 50 mM Na phosphate buffer

- Disrupt the bacteria in 50 mM Na-phosphate buffer by ultrasound treatment

- Centrifuge at 5,000 x g and discard the sediment

- Centrifuge the supernatant at 100,000 x g and discard the resulting supernatant

- Add 0.1% Triton X-100 to the sediment and incubate for 1 hour at 4 ° C

- Centrifugation at 100,000 x g and discarding the sediment

- Incubate the centrifugation supernatant with Talon Affϊnity Resin (Clontech, Palo Alto, USA) for 18 hours at 4 ° C

- washing the Talon-protein conjugate with:

20 column volumes of 50 mM Na phosphate pH 8 / 0.5 M NaCl 10 column volumes of 50 mM Na phosphate pH 7 / 0.5 M NaCl 10 column volumes of 50 mM Na phosphate pH 6 / 0.5 M NaCl - elution of the HPL with 3 column volumes of 50 mM Na phosphate pH 4.5 / 0.5 M NaCl Example 3:

Production of 9-hydroperoxides from γ-linolenic acid with recombinant 9-

Potato lipoxygenase

The production of 9-hydroperoxides from γ-linolenic acid was carried out according to the following protocol:

- Resuspend the bacterial pellet from 2 1 culture in 30 ml lysis buffer (50 mM Tris-HCl, pH 7.5, 10% (v / v) glycerol, 0.1% Tween 20, 0.5 M NaCl) - Disrupt the bacteria through ultrasound treatment

- Centrifugation at 14,000 x g and discarding the sediment

- Stir 10 ml of clarified bacterial homogenate with 20 mg fatty acid for 30 minutes at 4 ° C

- Extraction with diethyl ether - drying the extract under nitrogen and taking up in DiethyletheπHexan 93: 7

- Apply to a silica gel column and wash with diethyl ether: hexane 93: 7

- Elution of the LOX products with diethyl ether: hexane 80:20

- Calculation of the yield from the optical density at 234 nm (an extinction of 1 corresponds to 12.4 μg hydroperoxide with 1 ml measuring volume and one

Layer thickness of 10 mm)

Example 4: Reaction of the 9-hydroperoxide of γ-linolenic acid with recombinant

Hydroperoxide lyase from Arabidopsis thaliana and derivatization of the resulting aldehydes The reaction of the 9-hydroperoxide of γ-linolenic acid with recombinant HPL from Arabidopsis thaliana was carried out according to the following protocol:

- Final volume of the reaction mixture of 0.5 ml: 0.1 M Na phosphate

1 μg of purified HPL 5 μg of the hydroperoxide

- Reaction at room temperature for 30 minutes under argon

- Add 2 ml IM HC1 - Add 0.5 ml 2,4-dinitrophenylhydrazine (DNPH, 1 mg / ml in IM HC1)

- Shake for 1 hour at room temperature under argon

- extraction with hexane

Example 5: Detection of the derivatized aldehydes

The derivatized aldehydes from Example 4 were detected by separation by means of HPLC on a Phenomenex RP18 Jupiter 1 x 250 mm column (mobile phase acetonitrile: H ₂ O, gradient from 60:40 to 80:20, flow rate 0.05 ml / min) and Detection of the aldehyde derivatives at λ = 365 nm. The peaks were assigned to the individual products by means of pure substances carried along.

Claims

EXPECTATIONS

1. Process for the preparation of C ₉ aldehydes or C ₉ alcohols or esters of C ₉ alcohols from polyunsaturated fatty acids which have at least double bonds at positions Δ6 and Δ9, in particular C ₁₈ fatty acids, particularly preferably γ- Linolenic acid or stearidonic acid, comprising the following steps: a) the provision of a nucleic acid molecule which is suitable for a vegetable or microbial protein with the biological activity of

Encoded 9-lipoxygenase; b) the provision of a nucleic acid molecule which codes for a vegetable or microbial protein with the biological activity of 13-hydroperoxide lyase; c) the transfer of the nucleic acid molecules from a) and b) to eukaryotic or prokaryotic cells, the coding sequence being under the control of suitable regulatory sequences, d) the expression of the recombinant 9-lipoxygenase and 13 -hydroperoxide lyase in the cells; e) the implementation of polyunsaturated fatty acids, at least

Have double bonds at positions Δ6 and Δ9, with the recombinant 9-lipoxygenase to 9-hydroperoxides of the fatty acids; f) the conversion of the 9-hydroperoxides of the fatty acids with 13 -hydroperoxide lyase to C ₉ -aldehydes; g) optionally reducing the C ₉ aldehydes to Cg alcohols; h) optionally the reaction of the C ₉ alcohols from step e) with an acid, in particular acetic acid, to give a corresponding ester; i) if appropriate, the recovery of the C ₉ aldehydes or C ₉ alcohols or esters of the C ₉ alcohols produced from the cells.

2. The method according to claim 1, characterized in that following the transfer of the nucleic acid sequence to plant cells from the transgenic cells complete plants are regenerated, which as a production site for C ₉ aldehydes or C - alcohols or esters of C ₉ - Alcohols are used, and the starting substrates are provided in the form of polyunsaturated fatty acids which have at least double bonds at positions Δ6 and Δ9.

3. The method according to claim 1, characterized in that the transformed cells comprise bacterial cells, yeast cells and algae cells and the cultivation of the cells and the production of the C ₉ aldehydes or C ₉ all alcohols or esters of the C ₉ alcohols in a reactor takes place, and the starting substrates are provided in the form of polyunsaturated fatty acids which have at least double bonds at positions Δ6 and Δ9.

4. The method according to claim 1, characterized in that transgenic plant cells are cultivated and used as a production site for C ₉ aldehydes or C ₉ alcohols or esters of C ₉ alcohols, and the starting substrates in the form of polyunsaturated fatty acids have at least double bonds at positions Δ6 and Δ9.

5. The method according to any one of claims 2 to 4, wherein the

Starting substrates can be provided in the cell by expression of an acyllipid Δ6 desaturase.

6. The method according to any one of the preceding claims, characterized in that the nucleic acid sequence codes for a protein with the biological activity of 9-lipoxygenase from the potato.

7. The method according to any one of the preceding claims, characterized in that the nucleic acid sequence codes for a protein with the biological activity of 13 -hydroperoxide lyase from Arabidopsis.

8. The method according to any one of claims 1 to 6, characterized in that the nucleic acid sequence codes for a protein with the biological activity of a 13 -hydroperoxide lyase from tomato, alfalfa or guava.

9. The method according to any one of the preceding claims, characterized in that the reduction in step g) of claim 1 is carried out enzymatically, preferably with alcohol dehydrogenase.

10. The method according to any one of claims 1 to 8, characterized in that the reduction in step g) of claim 1 is carried out chemically, preferably with sodium borohydride.

11. A process for the preparation of C ₉ aldehydes or C ₉ alcohols or esters of C ₉ alcohols, comprising the reaction of the following components: a) at least one polyunsaturated fatty acid which has at least double bonds at positions Δ6 and Δ9 , in particular

C ι ₈ fatty acids, particularly preferably γ-linolenic acid or stearidonic acid; b) 9-lipoxygenase; c) 13-hydroperoxide lyase; d) optionally a reducing agent; and, e) optionally an acid, especially acetic acid.

12. The method according to claim 11, characterized in that a recombinant 9-lipoxygenase from the potato is used.

13. The method according to claim 11 or 12, characterized in that a recombinant 13 -hydroperoxide lyase from Arabidopsis is used.

14. The method according to claim 11 or 12, characterized in that a recombinant 13 -hydroperoxide lyase from tomato, alfalfa or guava is used.

15. The method according to any one of claims 11 to 14, characterized in that an enzymatic reducing agent, preferably an alcohol dehydrogenase is used.

16. The method according to any one of claims 11 to 14, characterized in that a chemical reducing agent, preferably sodium borohydride is used.

17. Process for the production of transgenic plants or plant cells with an increased content of C ₉ aldehydes or C ₉ alcohols or esters of the C ₉ alcohols, comprising the following steps: a) Production of a recombinant nucleic acid molecule comprising the following constituents in 5 '->3' orientation:

regulatory sequences of a promoter active in plant cells,

operatively linked to it a nucleic acid sequence which codes for a protein with the biological activity of a 13 -HPL, and optionally operatively linked sequences that can serve as transcription, termination and / or polyadenylation signals in plant cells. b) transfer of the nucleic acid molecule from a) to plant cells; c) Provision of the starting substrates in the form of polyunsaturated fatty acids which have at least double bonds at the positions Δ6 and Δ9, in particular C ₈ fatty acids, particularly preferably γ-linolenic acid or stearidonic acid, in the plant cell.

18. The method according to claim 17, wherein the starting substrates are provided by expression of an acyllipid-Δ6-desaturase in the plant cell.

19. The method of claim 18, wherein the acyllipid Δ6 desaturase is from Boragio offwinalis.

20. Use of 13 -hydroperoxide lyase for the production of plant cells or plants which have a content of C ₉ aldehydes or C alcohols or esters of C ₉ alcohols which is different compared to wild type cells or wild type plants.