WO2002103023A2 - Procede pour la production d'aldehydes c9 et d'alcools c9 par divinyl ether synthase - Google Patents

Procede pour la production d'aldehydes c9 et d'alcools c9 par divinyl ether synthase Download PDF

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WO2002103023A2
WO2002103023A2 PCT/EP2002/006723 EP0206723W WO02103023A2 WO 2002103023 A2 WO2002103023 A2 WO 2002103023A2 EP 0206723 W EP0206723 W EP 0206723W WO 02103023 A2 WO02103023 A2 WO 02103023A2
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alcohols
divinyl ether
cells
aldehydes
acid
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PCT/EP2002/006723
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German (de)
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WO2002103023A3 (fr
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Ivo Feussner
Michael Stumpe
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Ipk - Institut Für Pflanzengenetik Und Kulturpflanzen Forschung
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Publication of WO2002103023A2 publication Critical patent/WO2002103023A2/fr
Publication of WO2002103023A3 publication Critical patent/WO2002103023A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters

Definitions

  • the invention relates to a process for the preparation of C 9 aldehydes, C 9 alcohols and their esters, which is based on the reaction of polyunsaturated fatty acids with recombinant 9-lipoxygenase (9-LOX) and 9-divinyl ether synthase (9-DES).
  • 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.
  • Jasmonic acid is generated from hydroperoxides, such as 13-hydroperoxylinolenic acid, via an AU oxide synthase and an allen oxide cyclase dependent route. Jasmonic acid is involved in stress and disease resistance signal responses via the octadecanoid pathway.
  • 13-hydroperoxylinolenic acid can be cleaved by hydroperoxide lyase to form volatile aldehydes and traumatic acid.
  • Fatty acid hydroperoxide lyase catalyzes the cleavage of carbon-carbon bonds in polyunsaturated fatty acid hydroperoxides to produce short-chain C 6 or C 9 aldehydes and ⁇ -oxo acids of chain length C u or C 9 (Vick et al. ( 1976) Plant Physiol. 57: 780-788).
  • the short-chain volatile C 6 aldehydes contribute to the so-called
  • fragrance and aroma substances in a variety 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 cleaving (9S, 15Z, 12Z, 10E) -9-hydro ⁇ eroxy-15,12,10-octadecatrienoic acid by a 9-hydroperoxide -Lyase (9-HPL) is produced, 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.
  • the fatty acid hydroperoxides can be converted by divinyl ether synthases (DES) to conjugated ether fatty acids which contain an oxygen atom within the hydrocarbon chain.
  • DES divinyl ether synthases
  • the divinyl ethers of colenic acid and colenic acid are produced from linoleic acid or linolenic acid by sequential action of 9-lipoxygenase and a divinyl ether synthase specific for 9-hydroperoxides.
  • the molecular cloning of a tomato divinyl ether synthase is described by Itoh and Howe, J. Biol. Chem. (2001), 276: 3620-3627.
  • 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.
  • the elucidation of the physiological roles of enzymes in oxylipin biosynthesis and their products is also of interest for the further study of disease resistance.
  • the C 9 alcohols produced in this way can also 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 9 aldehydes (3Z) -nonenal or (3Z, 6Z) -nonadienal begins with the conversion of linoleic or ⁇ -linolenic acid with 9-lipoxygenase to (9S) -hydroperoxy derivatives. These can then be Hydroperoxide lyase (9-HPL) can be split into the C 9 - ⁇ -keto fatty acid and (3Z) -nonenal or (3Z, 6Z) -nonadienal.
  • the resulting aldehydes can then be converted enzymatically or chemically to the (2E) derivatives and / or reduced to the C 9 alcohols (3Z) -nonenol or (3Z, 6Z) -nonadienol.
  • the (3Z) -aldehydes generated from the reaction of (9S) -hydroperoxide derivatives of ⁇ -linolenic acid catalyzed by 9-hydroperoxide lyase have the disadvantage that the (3Z) double bond is acid-labile and therefore mixtures of (3Z) -, (2E) and (3E) isomers can arise. Such mixtures are undesirable for further processing into corresponding alcohols and esters and in particular for the use of the aldehydes, alcohols and esters as constituents of fragrances and flavorings.
  • the object of the present invention is 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.
  • a 9-divinyl ether synthase (9-DES) can be used in the second reaction step and the divinyl ethers thus produced can be used, for example, by mild acidification disintegrate into the corresponding (2E) -C 9 aldehydes.
  • a process for the preparation of C 9 aldehydes or C 9 alcohols or esters of C alcohols from polyunsaturated fatty acids of chain length C 18 is thus provided, the polyunsaturated fatty acids having double bonds at least at positions ⁇ 9 and ⁇ 12.
  • the method according to the invention comprises the following steps:
  • nucleic acid molecule which codes for a vegetable or microbial protein with the biological activity of 9-lipoxygenase
  • Divinyl ether synthase to divinyl ether g) the conversion of the divinyl ethers to (2E) -C 9 - aldehydes; h) optionally the reduction of the C 9 aldehydes to C 9 alcohols; i) optionally the reaction of the C 9 alcohols from step h) with an acid, in particular acetic acid, to give a corresponding ester.
  • the above-mentioned method according to the invention offers the advantage that the double bonds to the .beta.-hydroperoxides of fatty acids Have positions ⁇ 9 and ⁇ 12, with 9-divinyl ether synthase and the subsequent reaction of the divinyl ethers, only the stable (2E) isomer of the aldehyde formed is formed. In this way, disadvantageous product mixtures are avoided, and the aldehydes produced by the process according to the invention can be further processed with high purity to (2E) -C 9 alcohols or to corresponding esters and used as constituents of fragrances and flavorings.
  • the process according to the invention can now be used for the first time to prepare stable C 9 aldehydes or C 9 alcohols or esters of C 9 alcohols.
  • the disadvantages of the (3Z) -aldehydes produced by the reaction of certain fatty acids with 9-hydroperoxide lyase are overcome; instead, for the first time only a stable (2E) isomer can be obtained.
  • the transfer of the nucleic acid molecules from step a) or b) of the method according to the invention can optionally be dispensed with.
  • C 18 fatty acids are preferably used as polyunsaturated fatty acids in the present invention. All C 8 fatty acids can be implemented with a (9Z, 12Z) pentadiene system. Examples of C 18 fatty acids which can advantageously be used in the process according to the invention are ⁇ -linolenic acid and linoleic acid.
  • the method according to the invention additionally comprises the provision of additional ones Starting substrates in the plant cell, for example by exogenously adding the starting substrates.
  • the transformed cells are bacterial cells, yeast cells or algal cells.
  • the cells are cultivated and the C-aldehydes or C 9 alcohols or esters of the C 9 alcohols are prepared in one reactor.
  • a sufficient concentration of the substrates must also be ensured here, if necessary by providing additional starting substrates, for example by exogenous addition.
  • transgenic plant cells are cultivated (e.g. in the form of suspension or callus cultures) and as a production site for
  • Divinyl ethers are used, which are then reacted as described above to give the corresponding 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 9-DES from the potato or alternatively from tomato.
  • the conversion of the divinyl ethers to the corresponding C-aldehydes in step g) is preferably carried out by acidification, particularly preferably by mild acidification, for example with 20% HCl.
  • Mild acidification generally means that the acidification is gentle, e.g. with dilute acids, such as dilute hydrochloric acid or sulfuric acid, or with acetic acid.
  • acids in suitable dilutions are preferably used for the acidification, the person skilled in the art knowing or being able to determine in a simple manner which acid concentration or dilution is suitable for the acidification in step g) of the process according to the invention and which is not.
  • the aldehyde formed in step h) of the process according to the invention can be reduced enzymatically, preferably with alcohol dehydrogenase, or chemically, preferably with sodium borohydride, to a C 9 alcohol.
  • 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 is used as a component a polyunsaturated fatty acid with double bonds at least at the positions ⁇ 9 and ⁇ 12, in particular a C 18 fatty acid, particularly preferably ⁇ -linolenic acid or linoleic acid; 9-lipoxygenase and 9-divinyl ether synthase (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.
  • the enzyme components involved in the reaction can be present, for example, in purified form or in the form of a crude extract of
  • Another aspect of the present invention relates to methods for changing the content of divinyl ether which can be used as a precursor in the production of C 9 aldehydes or C 9 alcohols or esters of C 9 alcohols in a host cell.
  • the methods involve increasing the level of divinyl ether in a host cell.
  • the process involves the use of
  • Expression constructs for carrying out the expression of DNA sequences which encode a protein with the biological activity of a 9-divinyl ether synthase in a host cell It is particularly preferred to use expression constructs to change the divinyl ether content as a precursor for Cg aldehydes or C 9 alcohols or esters of C 9 alcohols in one
  • Plant cell or plant The method according to the invention is preferably used to change the content of divinyl ether in parts of plants, including leaves, roots, stems, flowers, fruits, seeds and seed oils, which are obtained from plant seeds.
  • the DNA sequences encoding 9-divinyl ether synthase are particularly preferably used to produce transgenic plants which have an increased production of divinyl ether in plant fruits and tissues. These divinyl ethers can in turn be converted into C 9 aldehydes or C 9 alcohols or esters of C 9 alcohols, for example, by mild acidification during workup. Such aldehydes are important components of characteristic aromas of fruits, vegetables and green leaves. So a 9- Divinyl ether synthase according to the invention can also be used for the production of products from transgenic plants with improved aroma characteristics.
  • the present invention also includes coexpressing a plant or other 9-divinyl ether synthase in plant tissue with a second gene that is involved in lipid peroxidation.
  • a 9-divinyl ether synthase 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 increase the content of divinyl ethers which are produced in the plant tissue.
  • Such an increase in the content of divinyl ether, which is subsequently converted into C 9 aldehydes can increase the "melon" or "cucumber" aroma in a vegetable product.
  • plant cells that contain a DNA sequence can be used for a
  • Coded divinyl ether synthase also used as a source of divinyl ether in reactions for the production of C 9 aldehydes or C 9 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 obtained by such methods.
  • 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 content of enzyme activity of lipoxygenase and divinyl ether synthase and an alcohol dehydrogenase.
  • the polyunsaturated fatty acid can vary and comprises 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, Cts fatty acids. Preferred examples include ⁇ -linolenic acid and linoleic acid.
  • Alcohol dehydrogenase 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.
  • NADH nicotin adenine dinucleotide
  • DNA sequence encoding a protein with the biological activity of a 9-LOX or a 9-DES can be isolated from natural sources or synthesized by conventional methods.
  • the present invention provides for the first time a DNA sequence which codes for a protein with the enzymatic activity of a 9-divinyl ether synthase from Solanum tuberosum.
  • the DNA sequence according to the invention is preferably selected from the group consisting of:
  • DNA sequences which comprise a nuclear acid sequence which encode the amino acid sequence given in SEQ ID NO: 2 or fragments thereof;
  • Another object of the present invention provides recombinant proteins with the enzymatic activity of a 9-divinyl ether synthase from Solanum tuberosum.
  • the proteins according to the invention preferably have the amino acid sequence given in SEQ ID NO: 2.
  • chimeric gene constructs be produced with the desired fusion of promoter and 9-LOX or 9-DES DNA sequence and possibly further regulatory and / or signal sequences, but, if desired, the person skilled in the art can additionally use routine techniques if desired introduce various mutations into the DNA sequence encoding the 9-LOX or 9-DES, which leads to the synthesis of proteins with possibly changed biological properties.
  • enzymes that are localized in certain compartments of the plant cell by adding corresponding signal sequences.
  • mutants can be produced which have an altered substrate or product specificity (Hornung et al. (1999), Proc. Natl. Acad. Sei. USA 96: 4192-4197). Furthermore, mutants can be produced which have a changed activity, temperature and or pH profile.
  • 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.
  • base exchanges can be carried out or natural or synthetic sequences can be added.
  • 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. For the expression of those contained in the recombinant nucleic acid molecules
  • Sequences for example, under the control of constitutive, but also inducible or tissue or development-specific regulatory elements, in particular
  • Promoters to be expressed in plant cells are expressed in plant cells. For example, while the cells are expressed in plant cells.
  • LOX or 9-DES DNA sequences in plant cells enables, for example, the use of tissue-specific, for example leaf or seed-specific, promoters to determine the content of divinyl ether in certain tissues, e.g. in leaf or seed tissue.
  • tissue-specific, for example leaf or seed-specific, promoters to determine the content of divinyl ether in certain tissues, e.g. in leaf or seed tissue.
  • Other suitable promoters e.g. Light-induced gene expression in transgenic
  • the promoter can be homologous or heterologous with respect to the plants to be transformed.
  • Suitable promoters are e.g. the 35S RNA promoter of the Cauliflower Mosaic
  • Constitutive, germination-specific, fruit-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 divinyl ether as a precursor for C 9 aldehydes or C alcohols or esters of C 9 alcohols in transgenic seeds are suitable, including in connection with the antisense or cosuppression technique.
  • the person skilled in the art can find suitable promoters in the literature or isolate them from any plants using routine methods. The same applies to the coding sequences.
  • transcription or termination sequence which serves to correctly end 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.
  • nucleic acid molecules contained in the vectors are linked to regulatory elements which ensure transcription and, if appropriate, translation in prokaryotic and eukaryotic cells.
  • nucleic acid sequences can be supplemented by enhancer sequences or other regulatory sequences.
  • regulatory sequences also contain, for example, signal sequences which ensure that the gene product is transported to a specific compartment.
  • Divinyl ether comes as a precursor of C 9 aldehydes or C 9 alcohols or esters of C 9 alcohols in the plant.
  • the invention relates to plants or their cells and parts in which the content of divinyl ether as a precursor for C 9 aldehydes or C 9 alcohols or esters of C 9 alcohols due to the presence and expression of the abovementioned Nucleic acid molecules are increased compared to wild type plants and thus have an increased resistance to bacteria or fungi.
  • the invention also relates to those plants in which the transfer of the abovementioned nucleic acid molecules leads to a reduction in the content
  • Divinylethem leads. Such a reduction can, for example, by the
  • Transfer of antisense constructs or by other suppression mechanisms, such as cosuppression, can be achieved.
  • the invention further relates to transgenic plant cells or such
  • Plants comprising plant cells and their parts and products in which the above-mentioned nucleic acid molecules coding for 9-DES are present integrated into the plant genome.
  • the invention also relates to plants in whose cells the aforementioned nucleic acid molecule is self-replicating
  • Form i.e. the plant cell contains the foreign DNA on an independent nucleic acid molecule (transient expression).
  • Alcohols or esters of C 9 alcohols are synthesized, it can in principle be any plant. It is preferably a monocot or dicot crop.
  • Examples of monocotyledonous plants are the plants belonging to the Avena genera
  • Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (corn) belong.
  • dicotyledonous crops are to name legumes, such as legumes and in particular alfalfa, soybean, tomato, sugar beet, potatoes, ornamental plants or trees and in particular oilseeds such as flax, sunflower and rapeseed.
  • 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 and medicinal plants and pasture grasses and fodder plants.
  • the cereals wheat, rye, oats, barley, rice, corn and millet, feed grain, sugar beet, soybean, sunflower, flax, tomato, potato, sweet grasses, feed grasses and clover are particularly preferred. 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 also 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.
  • the invention relates to host cells, in particular prokaryotic and eukaryotic cells which have been transformed or infected with a nucleic acid molecule or a vector described above, and cells which originate from such host cells and contain 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 host cells which, in addition to the nucleic acid molecules coding for LOX and DES, 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 an altered content of divinyl ether as a precursor of C 9 aldehydes or C 9 alcohols or esters of C 9 alcohols.
  • Nucleic acid molecules are integrated into the plant genome, ie stable transformants are generated.
  • a nucleic acid molecule according to the invention the presence and optionally expression of which in the plant cell causes an altered biosynthetic capacity, can be carried out in the plant cell or the plant be included as a self-replicating system.
  • the nucleic acid molecules according to the invention can be contained in a virus with which the plant or plant cell comes into contact.
  • Plant cells and plants with increased resistance to bacteria or fungi which, owing to the expression of a nuclear acid sequence according to the invention, have a modified, in particular increased content of divinyl ether as a precursor of C 9 aldehydes or C 9 alcohols or esters of C 9 alcohols , made by a process 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 nuclear acid sequence which codes for a protein with the biological activity of a 9-DES, and optionally operatively linked sequences that can serve as transcription, termination and / or polyadenylation signals in plant cells.
  • 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.
  • step a) of the above method can be modified such that the nuclear acid sequence which codes for a protein with the biological activity of a 9-DES is coupled to the 3 'end of the promoter in an antisense orientation.
  • 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).
  • cloning vectors 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. coli cells.
  • Transformed E. coli cells are grown in a suitable medium and then harvested and lysed, and the plasmid is recovered.
  • Analysis methods for characterizing the plasmid DNA obtained are generally used for restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods. 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.
  • transformation methods have been developed and established here over the past two decades. These techniques include transforming plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, diffusion of protoplasts, direct gene transfer of isolated DNA to protoplasts, injection and electroporation of DNA into plant cells Introduction of DNA using the biolistic methods as well as 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.
  • plasmids 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.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti or Ri plasmid, must be connected as a flank region to the genes to be introduced become.
  • 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 on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination.
  • 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
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants can then be regenerated from the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which may contain antibiotics or biocides for the selection of transformed cells.
  • Agrobacterium tumefaciens e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells
  • suitable medium which may contain antibiotics or biocides for the selection of transformed cells.
  • 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 transform the selection Allow cells versus 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 screening requirement. If the selection marker used after the
  • sequence specific recombinases are used, e.g. in the form of the retransformation of a recombinase-expressing starting line 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.
  • the plants thus obtained can then, if desired, by conventional methods, including molecular biological methods, such as PCR, blot analysis, or biochemical methods for the presence of the introduced DNA, which encodes a protein with the enzymatic activity of a 9-DES, or on Presence of 9-DES enzyme activity can be examined.
  • the person skilled in the art has a broad spectrum of molecular biological and / or biochemical methods for the analysis of the transformed plant cells, transgenic plants, parts of plants .
  • Food products and propagation material available eg PCR, Northern blot analysis for the detection of RNA specific for 9-LOX or 9-DES or for determining the level of accumulation of 9-LOX or 9-DES specific RNA, Southern blot analysis to identify 9-LOX or 9-DES coding DNA sequences or Western blot analysis to detect the 9-LOX or 9-DES encoded by the nucleic acid molecules.
  • the detection of the enzymatic activity of the 9-LOX or 9-DES can also be determined by a person skilled in the art using protocols available in the literature. Furthermore, one can, for example, lay out the seeds obtained by crossings on medium which contains the selection agent suitable for the selection marker transferred together with the 9-LOX or 9-DES DNA sequence, and on the basis of the germination capacity and the growth of the daughter generation (s ) and the segregation pattern allow conclusions to be drawn about the genotype of the respective plant.
  • Another object of the invention is to demonstrate uses of the 9-DES.
  • the present invention also encompasses every possible form of use of the nucleic acid molecule coding for 9-DES, its presence and, if appropriate, expression in plants, a change in the content of divinyl ether as a precursor of C 9 aldehydes or C 9 alcohols or esters of C 9 - alcohols.
  • the nucleic acid molecules coding for 9-DES can thus be used according to the invention to produce transgenic plants in which the content of 9-DES is higher or lower than natural, or in cell types or development stages in which the 9-DES is not normally found become. This causes a change in the content of divinyl ether in these cells, which in turn can be converted to C 9 aldehydes or C 9 alcohols or esters of C 9 alcohols.
  • the accumulation of divinyl ether can be an advantageous phenotype in plants used for food.
  • nucleic acid molecules coding for 9-DES 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.
  • nucleic acid molecules that encode 9-DES may also be desirable to reduce or eliminate the expression of nucleic acid molecules that encode 9-DES.
  • a nucleic acid molecule developed for the co-suppression of the 9-DES can be generated by linking a gene or gene fragment which encodes a 9-DES with plant promoter sequences.
  • a nucleic acid molecule developed for the expression of antisense RNA for all or part of the above-mentioned nucleic acid molecule can be generated 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 transformed into the plants by means of transformation are introduced, whereby the expression of the corresponding endogenous genes is reduced or eliminated.
  • Example 1 Expression of the recombinant proteins 9-lipoxygenase from Solanum tuber osoum
  • Amplified PCR fragments of cDNA sequences which code for a 9-lipoxygenase from Solanum tuberosoum were ligated into the expression vector QIAexpress pQE 30 (Qiagen, Hilden, Germany) and transformed with XL1-Blue cells using the pGEM R - T Easy Vector System II Kits (Promega, Madison, USA) pre-cloned. The cloning was then carried out into the expression strain E. coli SG13009 [ ⁇ REP4].
  • the E.co/ ⁇ 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
  • PCR conditions were as follows: 2 min at 94 ° C; 10 cycles 30 s at 94 ° C, 45 s at 51 ° C, 45 s at 72 ° C; 20 cycles 30 s at 94 ° C, 45 s at 51 ° C, 45 s at 72 ° C and an extension of the incubation time for 3 s / cycle; 10 min at 72 ° C.
  • the cDNA fragment obtained from the potato was 450 bp long and had a homology of over 95% to the sequence EST281141 from the tomato.
  • the following homologous primers for 3'RACE-PCR were derived from this sequence:
  • StAOS_3RACE (SEQ IDNO: 5):
  • a 5'- or 3'-RACE Ready cDNA library produced from RNA from elicited potato cell cultures using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, USA), was used as the template.
  • the PCR conditions for the 3'-RACE were as follows: 1 min at 94 ° C; 10 cycles 30 s at 94 ° C, 30 s at 58 ° C, 2 min at 72 ° C; 20 cycles 30 s at 94 ° C, 30 s at 55 ° C, 2 min at 72 ° C; 3 min at 72 ° C.
  • the fragment obtained was about 800 bp long and this size corresponded to the expected full 3 'end of the enzyme's cDNA.
  • the 5'-RACE was unsuccessful.
  • a primer for the cloning in pQE30 for the subsequent expression of the enzyme was derived from the sequence of the 3 'end.
  • This primer StDES3 'Hind3 contained a restriction site for HindIII:
  • StDES5 ⁇ BamHI (SEQ ID NO.8): 5 '- GGA TCC ATG TCT TCT TAT TCA GAG CTA TCA AAT C -3'
  • the PCR conditions for the complete cDNA fragment were as follows: 2 min at 94 ° C; 10 cycles 30 s at 94 ° C, 30 s at 61 ° C, 90 s at 72 ° C; 20 cycles 30 s at 94 ° C, 30 s at 64 ° C, 90 s at 72 ° C + an extension of the incubation time for 5 s / cycle; 2 min at 72 ° C.
  • the fragment obtained was intermediate cloned in pGEM-T easy and cut out of this vector with BamHI and HindIII.
  • the purified fragment was ligated into the pre-cut vector pQE30 and transformed into E. coli SG13009 [pRep4].
  • SEQ ID NO: 1 The complete cDNA sequence is shown in SEQ ID NO: 1, the 9-DES from the potato encoded by this DNA sequence is shown in SEQ ID NO: 2.
  • the culture was centrifuged for 15 min (4500 rpm, 4 ° C.) to harvest the cells.
  • the pellet was taken up in 25 ml of 50 mM sodium phosphate, pH 8.0 and completely dissolved.
  • the cells were disrupted using ultrasound (5 x 1 min, with a 30 s pause) on ice.
  • the cell lumps were centrifuged (4000 rpm, 15 min, 4 ° C) and the supernatant ultracentrifuged (37000 rpm, 1 h, 4 ° C).
  • the resulting pellet was dissolved in 10 ml of 50 mM sodium phosphate, pH 8.0, 1 M NaCl, 0.1% Triton and incubated on ice for 1 h.
  • the supernatant was mixed with 2 ml of Talon (Clontech), a co-affinity chromatography material (previously equilibrated with 50 mM sodium phosphate, pH 8.0, 1 M NaCl) and shaken overnight at 4 ° C.
  • this solution was placed on a Bakerbond filtration column (T.J. Baker, Philipsburg, USA) and gradually with 20 ml sodium phosphate (50 mM, pH 8), 1 M NaCl; 7 ml sodium phosphate (50 mM, pH 7), 1 M NaCl; 4 ml sodium phosphate (50 mM, pH 6), 1 M NaCl; 2 ml of sodium phosphate (50 mM, pH 5), 1 M NaCl washed.
  • the protein was eluted with 2 ml sodium phosphate (50 mM, pH 4), 1 M NaCl.
  • Dinitrophenylhydrazine (1 mg in 1 ml 1N HCl) can be converted to the corresponding dinitrophenyl hydrazones. After shaking for 1 h, these were extracted 3 times with 1 ml of hexane. The combined hexane phases were evaporated in a stream of nitrogen and taken up in 100 ⁇ l of solvent A.
  • Figure 1 Pathways of oxylipin biosynthesis. Linolenic acid and linoleic acid are converted into hydroperoxides by lipoxygenase (LOX).
  • LOX lipoxygenase
  • AOS AU oxide synthase
  • HPL hydroperoxide lyase
  • DES divinyl ether synthase
  • FIG. 2 HPLC analysis with Agilent 1000 with DAD detection at 380 nm for the conversion of colenic acid (see Example 7). The proof is here after reaction with DNPH to the corresponding hydrazone.

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Abstract

L'invention concerne un procédé pour la production d'aldéhydes C9, d'alcools C9, ainsi que de leurs esters. Le procédé selon l'invention est fondé sur la réaction d'acides gras polyinsaturés avec une 9-lipoxygénase (9-LOX) et une 9-divinyl éther synthase (9-DES) recombinées.
PCT/EP2002/006723 2001-06-19 2002-06-18 Procede pour la production d'aldehydes c9 et d'alcools c9 par divinyl ether synthase WO2002103023A2 (fr)

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DE10129338A DE10129338A1 (de) 2001-06-19 2001-06-19 Verfahren zur Herstellung von C9-Aldehyden, C9-Alkoholen durch Divinylethersynthase
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EP1621631A1 (fr) * 2004-07-28 2006-02-01 Georg-August-Universität Göttingen Procédé de production de C6 et/ou C9 aldéhydes et alcohols dans des plantes par divinyl ether synthase
WO2018219467A1 (fr) * 2017-06-01 2018-12-06 Symrise Ag Procédé pour la production d'un mélange aromatisant contenant des diénals insaturés

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1621631A1 (fr) * 2004-07-28 2006-02-01 Georg-August-Universität Göttingen Procédé de production de C6 et/ou C9 aldéhydes et alcohols dans des plantes par divinyl ether synthase
WO2018219467A1 (fr) * 2017-06-01 2018-12-06 Symrise Ag Procédé pour la production d'un mélange aromatisant contenant des diénals insaturés
US11485930B2 (en) 2017-06-01 2022-11-01 Symrise Ag Method for producing an aroma mixture containing unsaturated dienals

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