DE102007053992A1 - Spectrally enhanced and nitrogen autotrophic photosynthesis - Google Patents

Abstract

The invention relates to transgenic photosynthetic organisms having an extended absorption spectrum and nitrogen autotrophy, which are optionally salt-resistant and / or capable of separating silicate from the medium and / or have an increased hydrocarbon content, and their use for the production of biofuels, and of nitrogen-containing biomass, in particular for Supply of animals and humans with essential amino acids, sequestration of CO2 from the atmosphere, amelioration of poor soils, treatment of effluents, expression of beneficial proteins, degradation of hydrophobic contaminants in natural habitats and mosquito control.

Description

The invention relates to transgenic photosynthetic organisms having an extended absorption spectrum and nitrogen autotrophy, which are optionally salt-resistant and / or capable of separating silicate from the medium and / or have an increased hydrocarbon content, and their use for the production of biofuels, and of nitrogen-containing biomass, in particular for Supply of animals and humans with essential amino acids, sequestration of CO ₂ from the atmosphere, amelioration of poor soils, treatment of effluents, expression of beneficial proteins, degradation of hydrophobic contaminants in natural habitats and mosquito control.

Of the five most important elements of life - carbon (C), hydrogen (H), oxygen (O), nitrogen (N) and phosphorus (P) - the first three are the primary photosynthetic producers capable of exploiting organic compounds To build up light energy from carbon dioxide and water, available in an amount that is limited only by the available light energy. Nitrogen, on the other hand, is a limiting factor in the vast majority of habitats, since molecular nitrogen (N ₂ ) is very inert and can only be converted into a form suitable for the synthesis of organic compounds at considerable expense, which i. A. by reduction to ammonia (NH ₃ ), which is then connected in the next step with a suitable carbon-containing skeleton, primarily by reaction with glutamate to glutamine.

The mentioned effort consists on the one hand in the high energy requirement of the reduction reaction as such, on the other hand in the chemical complexity and lability of the enzymes called "nitrogenases" which can catalyze such a reaction. Typically, nitrogenases contain iron-sulfur clusters, often involving unusual metallic cofactors such as molybdenum or vanadium, which are highly sensitive to oxidation by atmospheric oxygen. Only in anaerobic habitats, this problem does not arise, but anaerobes are severely limited by the low energy yield of anaerobic metabolic processes. On the other hand, energy-producing organisms are available in virtually any amount - limited solely by the light incidence and the efficiency of light absorption - but the highly productive photosynthesis of cyanobacteria (vulgo "blue-green algae"), eukaryotic algae and higher plants is inseparably more significant with the release Amounts of molecular oxygen (O ₂ ), which irreversibly damages the nitrogenases. Against this background, it can be seen that the ability to fix atmospheric nitrogen is a lesser evolutionary advantage due to the high cost than would be assumed by naive consideration, and is very disjoint, as the propensity to lose this ability is obviously large due to its limited value is.

In nature are basically two solutions of the Problems have been implemented.

Bacteria of the family Azotobacteraceae also fix nitrogen under aerobic conditions by activating the nitrogen fixation only at maximum growth, in which each available oxygen is consumed directly by the cells and thus creates a substantially oxygen-free environment in the cell interior. Since the nitrogen fixation is needed to overcome a limitation in anabolic processes, this makes economic sense. However, the efficiency is low due to the high energy consumption, to 1 g of converted carbohydrates are fixed at a maximum of 10 mg N ₂ .

photosynthetic By contrast, active organisms usually use a functional compartmentalization, wherein the nitrogen fixation takes place in an anaerobic compartment: nitrogen-fixing, Chain-forming cyanobacteria such as Nostoc or Anabaena form so-called Heterocysts, d. h., at a distance of about 12-15 cells The chain goes through one in each case of nitrogen deficiency Cell terminal differentiation with loss of photosynthetic pigments, Compaction of the walls, increase of cellular respiration to increase oxygen consumption and expression of nitrogenase. Under In the higher plants, there are some species with which prokaryotic nitrogen fixers live in symbiosis, with the the latter typically in anaerobic areas (Wurzelknöllchen the Fabaceae) and kept by the plant in return for the Supply of bound nitrogen supplied with photosynthetic products become. This compartmentalization is highly complex and is only at few species found. Typically, an alternative seems Method of nitrogen supply at nitrogen-poor sites, namely the carnivory, in a larger one Number of plant groups originated independently to be as the formation of stable symbioses. Leave it conclude that the complexity - expressed as the necessary amount of genetic information - from for symbiosis with fixers serving compartments with the known insect traps of Drosera, Nepenthes u. a. approximately is comparable.

The textbook example of a higher plant living in symbiosis with nitrogen fixers is the rootless water fern Azolla, which has its full complement nitrogen needs from the symbiosis with the cyanobacteria Anabaena azollae and Nostoc azollae, and thus is able to double its biomass in less than three days if the phosphate supply is sufficient. Azolla is thus capable of sequestrating nearly any amount of atmospheric carbon dioxide ( Brinkhuis & al., Nature 441: 7093, 2007 ). However, the utility of Azolla is zero and, moreover, Azolla is sensitive to salt (over 0.2% NaCl) and cold.

Especially Among the most important crops are only certain Fabaceae capable of forming symbioses with nitrogen fixers. Grains, however, are known to be the soil Deprive nitrogen compounds, and in fact is to Ensuring a high quality human diet a considerable amount of nitrogen is needed as essential amino acids only can be formed in plants. But also plants, primarily for the production of low-nitrogen or -free biomass be grown, z. B. for the production of biofuels need bound nitrogen to build up their tissues. At this point It should be noted that also chlorophyll itself a significant Contains nitrogen.

The nitrogen supply of the biosphere is thus, globally, a limiting factor for the photosynthetic formation of biomass, which can only be partially compensated by the use of artificial fertilizer. Moreover, the production of artificial fertilizer, the core of which remains the Haber-Bosch process (N ₂ + 3H ₂ on the platinum catalyst → 2NH ₃ ), requires a considerable amount of technically available energy, which must be provided otherwise. If this energy, as is customary today, is gained by burning fossil fuels on a large scale, the paradoxical situation arises that u. U. a larger amount of fossil carbon must be burned to carbon dioxide to provide energy for fixing a certain amount of carbon from atmospheric carbon dioxide, so that the total CO ₂ balance is positive, that is harmful to the environment.

by virtue of The complexity of the systems outlined above is current state of biotechnology in the foreseeable future not be possible, the compartmentalization of in symbiosis transfer live plants to crops. In appropriate Habitats it is possible to supply the nitrogen through To improve application of free nitrogen fixers; so was z. B. Anabaena in rice fields and led Nitrogen deficiency in the soil to improve growth. however nitrogen-fixing cyanobacteria require open water, This approach can therefore only in flood-resistant Plants are practiced. As an alternative approach was the direct Breeding of cyanobacteria proposed; so are cyanobacteria of the genus Spirulina for a long time in Mesoamerica and Central Africa used for human nutrition. However, need these cyanobacteria in turn specific environmental conditions, eg. B. a pH between 9 and 11, which is in view of the global Water acidification is becoming increasingly difficult, and excelling in the water With regard to their synthesis performances not: Yields are in the Range of 300 kg of biomass per hectare and year, compared with over 5 tons of biomass per hectare per year for many cereals. So remains as a large scale applicable alternative to Artificial fertilizer only the elaborate approach of crop rotation, in regular succession again and again in Symbiosis with natural nitrogen fixers living plants how legumes are interposed.

Under Conditions of nitrogen deficiency may be that of the plant Photosynthetic structures are not meaningfully recycled, and even with sufficient nitrogen supply is the rate limiting nitrogen uptake for the formation of new biomass. So Blackman & Matthaei observed that increase in light intensity over a given In addition to no further increase in photosynthesis leads or in extreme cases, the plant even by light stress can harm. Different groups of plants react in a different way to nitrogen deficiency conditions, cyanobacteria z. B. mainly by degradation of light collection pigments, eukaryotic plants favored by formation of carotenoids, on the one hand absorb light energy and thereby protect the cells on the other hand, since they are pure hydrocarbons, their synthesis even in severe nitrogen deficiency as an outlet for excess Energy can be used.

Without limitation to theory, it is believed that this chemical limitation is evolutionarily responsible for the fact that plants use only part of the solar spectrum, namely red and blue light, while green light in the range of λ = 500 to 600 nm remains virtually unused (cf. , 1 ) - this results in the characteristic vegetable green. Only cyanobacteria are able to use part of the blue-green light. The efficiency of the photosynthesis of higher plants is in the dimension of a maximum of about 4% of the total incident sunlight (record value measured in the tropical weed Eichhornia, the "water hyacinth"), the rest is reflected or converted into heat (ie dissipated).

A similar limitation applies to eu karyontic algae, which can often achieve impressive growth rates in nutrient-rich, ie polluted water. For the unicellular green alga Scenedesmus, a productivity of more than 50 tonnes per hectare per year was observed with a protein content of more than 50% of the dry matter ( Frohne & Jensen, "Systematics of the Plant Kingdom" 1985, Stuttgart: Fischer-Verlag, ISBN 3-437-30473-9, and references cited therein ).

It is assumed that the phenomenon of "global obscuration", d. H. the increasing reduction of the earth's surface reaching sunlight due to the increase fine-grained Particulates in the atmosphere, including the plants to Available light energy decreases. In this context It should be noted that the majority of the climate and Soil quality agriculturally usable areas of the mainland anyway in terms of light incidence rather unfavorable Regions lies.

The Breeding unicellular algae such as Chlorella as a source of food for the growing humanity has been much discussed in the past, However, there are no significant advantages: Chlorella also achieves one Use of incident sunlight of maximum about 3.8%; the Therefore, higher productivity is only observed on the better use of the - three-dimensional - habitat due.

Thus, if the highest possible production of general biomass, which is to be striven for sequestration of atmospheric carbon dioxide and providing today's common hydrocarbon-based fuels and fuels without positive total CO ₂ balance, and in particular the highest possible production of nitrogen-containing biomass Securing human nutrition is the dual problem that plants can only partially exploit solar performance and can not make better use of it due to the limitations of nitrogen supply. A biotechnical change aimed at increasing the efficiency of light absorption therefore did not lead to an increase in primary production, but only to the activation of protective mechanisms and possibly damage to the plant; By contrast, a biotechnological change aimed at fixing atmospheric nitrogen would have to unduly burden the energy balance of the plant and, in particular, lead to a depletion of reduction equivalents.

moreover are both of the possible changes already having unresolved technical problems: The synthesis of chlorophyll and the structure of photosynthetic Chlorophyll centers and suitable auxiliary structures are one complex process, where it is not clear what kind of intervention could be that increase the efficiency of light absorption would - there is no "gene for chlorophyll", rather, chlorophyll is synthesized by enzymatic reactions. The Nitrogen fixation is currently taking place with photosynthetic activity Plants by the oxygen sensitivity of the known nitrogenases and moreover the energy and reduction equivalent requirements prevents the procedure.

One general problem is the fact that freshwater is a scarce resource in many parts of the world today. The production of biomass is thereby hindered not insignificantly. The salt sensitivity of crops requires an energy-consuming Recovery of fresh water for irrigation.

One another significant environmental problem especially in the industrialized Countries today use low molecular weight Silicates as detergent additive. Many detergents form with divalent ones Cations, in particular calcium, sparingly soluble precipitates, which have no more washing activity and moreover tend to be cloudy, "dirty" rainfall to form on the laundry. To commercial detergent for Therefore, to make the use with "hard" water suitable, therefore added non-toxic chelators, traditionally phosphates. Because in nutrient-rich Water, however, the phosphate content (due to the sugar-phosphate backbone the nucleic acids) the limiting factor for the unfolding is photosynthetic life, the leads large-scale use of phosphate in detergents to ecological problems - over-fertilization and algae blooms. Low molecular weight silicates with cage form may be divalent cations without the use of phosphates tie; however, the silicate content of the water is itself a limiting factor for the development of diatoms, and use of low molecular weight silicates as detergent additive favors therefore diatom blooms, which likewise lead to ecological problems, even more so than Diatoms due to their silicified cell walls only for few planktivores are usable. By the polymerization of low molecular weight silicate to amorphous high molecular weight In-cell structures related to biological clarification effluents could avoid these effects become.

Parallel to this, many bad farmland, z. B. obtained by slash-clearing areas of the rainforest areas, by a lack of swellable high molecular weight, z. As linear and area, silicates, which causes organic and mineral fertilizers are washed out quickly and not the crop grew, but only contribute to the over-fertilization of the waters. The impoverishment of such a field soil is therefore not to be met by fertilization, after a few years it is unproductive and must be abandoned, whereby its condition makes a resettlement by the original plant community impossible.

• • unvollständige Lichtnutzung
• • ungenügende Stickstoffversorgung

auf biotechnischem Wege zu überwinden, bevorzugt dergestalt, dass der biotechnisch verwendete Organismus salzresistent ist und lösliches Silikat aus dem Wasser zu sequestrieren vermag.The object underlying the present invention was, in view of the problems presented, to find a way of limiting the production of biomass limiting factors, namely

• • incomplete use of light
• • insufficient nitrogen supply

biotechnically overcome, preferably such that the bioengineered organism is salt-resistant and able to sequester soluble silicate from the water.

• • ein fluoreszierendes Protein im Kontext des Photosyntheseapparates exprimiert wird, das imstande ist, Licht in einem normalerweise nicht oder nur geringfügig genutzten Wellenlängenbereich in Licht in einem von dem Photosyntheseapparat gut nutzbaren Wellenlängenbereich zu transformieren; und
• • Nitrogenase in einem subzellulären Kompartiment exprimiert wird, in dem ein anaerobes, mikroaerobes und/oder reduzierendes Millieu herrscht; sowie gewünschtenfalls
• • ein membranständiger Innentransporter, insbesondere ein Transporter für Natriumionen, und/oder
• • ein silikatpolymerisierendes Enzym
• • sowie optional weitere unterstütztende Produkte, z. B. oberflächenaktive Peptide zur Solubilisierung hydrophober Materialien,

in einer Zelle, typischerweise in einer photosynthetisch aktiven Zelle, exprimiert werden.According to the invention, this object is achieved in the present invention in that

• A fluorescent protein is expressed in the context of the photosynthetic apparatus capable of transforming light in a wavelength region which is normally unused or only slightly used into light in a wavelength range well usable by the photosynthetic apparatus; and
• • Nitrogenase is expressed in a subcellular compartment in which there is an anaerobic, microaerobic and / or reducing environment; as well as desired
• A membrane-bound internal transporter, in particular a transporter for sodium ions, and / or
• • a silicate-polymerizing enzyme
• • as well as optional other supported products, eg. B. Surface-active peptides for solubilizing hydrophobic materials,

in a cell, typically in a photosynthetically active cell.

One Aspect of the invention therefore relates to the use of fluorescent Proteins for expanding the usable spectrum of a photosynthetic active organism; another one, expression of nitrogenase in a reducing or anaerobic or microaerobic compartment a cell for the fixation of atmospheric nitrogen. It is obvious that each of these two aspects is also independent can be used by others, but without limitation assumed by theory on the basis of the discussion presented above, that they achieve optimal results in the combination. Both Aspects can be independent, but preferably in Combination with each other, with genetic determinants for Salt resistance and silicate polymerization are combined. Especially in the absence of nitrogenase expression, the combination with this but not exclusive, concerns another aspect The invention also extends the useful spectrum of a photosynthetic organism for the production of Carotenoids.

One Another aspect of the invention thus relates to a transgenic Eucyte, characterized in that they contain both a nitrogenase and a fluorescent protein, wherein at least one protein is selected under nitrogenase and a fluorescent protein in an organelle is contained with double membrane and preferably both a Nitrogenase and a fluorescent protein each in an organelle is contained with double membrane. It goes without saying that the organelle containing the nitrogenase with that containing the fluorescent protein may be identical, but does not have to be identical.

Of the The term "organelle with double membrane" is familiar to the expert and in the context of the present invention designates chloroplasts and Mitochondria and derived from these natural organelles including, without limitation, von Mitosomes, Hydrogenosomes, and Evolutionary Other Than these derived organelles as well as derivatives of chloroplasts, Mitochondria or derived from these natural organelles with or restricted to the wild type modified function. In particular, he refers to such subcellular organelles, their own genetic material have.

In a particular embodiment of the invention, the above-mentioned protein is encoded by a nucleic acid of the double-membrane organelle. Methods for genetically modifying double membrane organelles have been described in the prior art and are familiar to the person skilled in the art (see, for example, US Pat. Reputation S, Karcher D, Bock R: PNAS 104 (17): 6998-7002, 2007 ).

Of the The term "nucleic acid" is familiar to the person skilled in the art and in the context of the present invention denotes any type of single- or double-stranded DNA or RNA as well as hybrids, However, preferably those nucleic acids which are in the inventive Cell be kept stable or replicated and optionally be translated into amino acid sequences.

In another particular embodiment of the invention, the above-mentioned protein is encoded by a nucleic acid outside the double-membrane organelle. In this case, it is preferable that the protein comprises an amino acid sequence leading to the double mem ber organelle bran, in particular an amino-terminal signal sequence. Corresponding sequences that target any proteins to which they are linked to specific organelles, and their critical features have been extensively described in the art, including in the works of Nobel laureate Blobel. Basic publications are: Mold RM, Shackleton JB, Robinson C, "Transport of Proteins Into Chloroplasts, Requirements for the Efficient Import of Two Luminal Oxygen-evolving Complex Proteins Into Isolated Thylakoids", J Biol Chem. 266 (26): 17286-9, 1991 ; Schnell DJ, Blobel G, Pain D: "Signal peptide analogues derived from two chloroplast precursors interact with the signal recognition system of the chloroplast envelope", J Biol Chem. 266 (5): 3335-42, 1991 ; Pain D, Fast DJ, Murakami H, Blobel G: "Machinery for protein import into chloroplast and mitochondria", Genet Eng (NY). 1991; 13: 153-66, 1991 ; Pain D, Murakami H, Schnell DJ, Blobel G: "Identification and characterization of receptors for protein import into chloroplast and mitochondria", Indian J Biochem Biophys. 27 (6): 443-5, 1990 ,

It is readily possible for the skilled person, recombinant nucleic acids to produce the desired proteins in association with a corresponding directing sequence to produce these recombinant Transfer nucleic acids into the target cells and to express there. Furthermore, it is readily apparent to those skilled in the art possible to select successfully modified cells and in multicellular plants, whole plants thereof to regenerate. A selection can z. B. based on one of the same nucleic acid encoded resistance factor and / or by direct measurement of the fluorescence properties of the cell (Microscopy or flow cytometry).

A "Selection" of suitable cells or multicellular individuals takes place in the context of the present invention expediently basically by (1) setting a parameter of Phenotype selects, on the basis of which more suitable Cells or multicellular individuals of less suitable cells or multicellular individuals can be distinguished, (2) subsequently on a large number of cells or multicellular individuals, typically from previously one Genotype-affecting treatment-treated cells or multicellular Individuals, this parameter measures and (3) those cells or multicellular individuals, where the chosen parameter comes closest to this value, selects and further increases.

If desired, The selection can be at the same or a different parameter of the phenotype with the descendants of the selected Cells or multicellular individuals are carried out again. The measurement of the parameter of the phenotype can be either instrumentally done by taking a physical size or a chemical property of cells or multicellular individuals and those cells or multicellular individuals the selected parameter is closest to this value comes, selects and furthers, or evolutionary, by giving cells or multicellular individuals environmental conditions be exposed, among which those cells or multicellular Individuals where the chosen parameter is this value comes closest, better survive and / or yourself better multiply than the others. An instrumental selection can be done manually, semi-automatically or fully automatically; as complete automation as possible this is preferred for reasons of throughput. In each Case, it is preferable that the selected parameter with the expression of the same parameter compared to the wild type becomes.

Basically, the presence of transgenes by evolutionary selection on a located on the same nucleic acid construct resistance factor, eg. For example, a gene for a toxin detoxifying protein, and addition of the appropriate Noxe to the growth medium can be selected. The presence of fluorescent proteins can be instrumented by measuring the quantitative fluorescence properties, e.g. As the emission at a certain excitation wavelength and / or the temporal characteristics of the fluorescence, and the qualitative fluorescence properties, eg. , The intracellular distribution of fluorescence, with flow cytometry ("FACS") being a preferred method. The presence of any other molecules can similarly be selected using fluorescently labeled antibodies and determining the extent of their binding. The ability for nitrogen fixation can be selected evolutionarily, by growth under nitrogen deficiency conditions, or instrumentally by measuring the relative ¹⁵ N content for growth under a ¹⁵ N ₂ -containing atmosphere in the presence of organic ¹⁴ N ₂ compounds in the medium. Conveniently, such selection is made using high throughput pipetting equipment, e.g. In 96-well, 192-well or 384-well format. Salt resistance can be selected evolutionarily by growth under salt stress conditions or instrumentally by adding intracellular salt indicators. On silicate polymerization instrumentation, z. Example, by measuring the refraction of cells due to the formation of silicate bodies in the flow cytometry or by measuring the content of silicate, either by the skilled person known chemical detection method, by mass spectrometry and / or based on the inclusion of isotopically labeled silicate. Resistance to bioti or abiotic factors can be evolutionarily selected by growth in the presence of the respective noxa. Other parameters and selection options are possible and familiar to the person skilled in the art.

The Expression of the gene products of the invention is not particularly limited in principle; each appropriate combination of enhancers, promoters, Splice signals, polyadenylators, terminators, etc. can be used. Corresponding structures and their use are the Specialist familiar. To the desired expression can be selected as described above.

Become in the invention Eucyte several proteins expressed transgenically, so is any combination of localizations the coding nucleic acids inside and outside Organelles possible. To the desired expression each one of them can be selected as described above.

In a preferred embodiment, a nitrogenase is contained in an organelle having a reducing and / or microaerobic environment, in particular a mitochondrion-derived organelle such as a functional mitochondrion or a mitochondrion-derived organelle having reduced respiratory activity. It was shown ( Huang K, Manton KG: Frontiers in Bioscience 9: 1100-1117, 2004 ) that in mitochondria an overall reducing environment can be expected. The heterocysts of the blue-green algae Nostoc and Anabaena have also been shown to increase respiration, leading to a microaerobic environment inside the cell, in which oxygen-sensitive nitrogenase is also functional.

In a particular embodiment that contains the Nitrogenase-containing organelle an additional reducing Component, for example, one containing a hydroquinone.

In the embodiment in which the nitrogenase is contained in an organelle with a reducing and / or microaerobic environment, it is preferred that the nitrogenase is an oxygen-sensitive nitrogenase, in particular a nitrogenase containing an iron-sulfur cluster. Sequences of such nitrogenases are known in the art and e.g. Via the NIH database, online at http://www.ncbi.nih.gov to query. The isolation of genetic sequences from an organism, e.g. By screening or "screening" a cDNA library or genomic DNA library or by RT_PCR, will be apparent to those skilled in the art. In the context of the present invention, it may be preferable to synthesize such sequences. Corresponding processes are known from the prior art and are commercially available (for example from Sloning, Puchheim).

In another embodiment, the nitrogenase is an oxygen-insensitive nitrogenase, preferably the nitrogenase from Streptomyces thermoautotrophicus. Sequences of such nitrogenases are also known in the art and e.g. Via the NIH database, online at http://www.ncbi.nih.gov to query.

In In this embodiment, it is particularly preferred that the organelle containing the nitrogenase is photosynthetic is active, and particularly preferred that the organelle is a chloroplast is. Without limitation to theory, it is believed that the abundant availability of reduction equivalents in the active chloroplast for those catalyzed by the nitrogenase Reduction of nitrogen to ammonia is favorable.

The Nitrogenase catalyses a Haber-Bosch process Formation of ammonia, the i. A. reacted with other substances becomes. Typically, the primary ammonia formed in the next step with glutamate converted to glutamine. In a particular embodiment, the organelle therefore an additional transporter for products the nitrogenase and their immediate reaction products, preferably a glutamine transporter. In a further particular embodiment The Organell includes an additional transporter for Substances which are reacted in the organelle with ammonia, preferably one Glutamate or α-ketoglutarate transporters.

In a particular embodiment of the invention is the Fluorescent protein in a photosynthetic apparatus containing Organelle contained, preferably in a chloroplast. In this Embodiment, it is preferred that the fluorescent protein has an absorption maximum in a spectral range in which the light absorption of the photosynthetic apparatus of the organelle low is.

Of the Term "spectral region with low absorption" refers to this all wavelengths λ, where the molar extinction coefficient at most 20%, preferably at most 10% and more preferably at most 5%, e.g. At most 2% of the molar extinction coefficient measured at the absorption maximum is. Method of measuring the molar extinction coefficient at a certain wavelength, those skilled in the art.

The terms "molar extinction coefficient", "Absorption maximum" and "emission maximum" are also familiar to the person skilled in the art.

Especially in this case it is preferred that the fluorescent protein has an absorption maximum in the range of 500 to 600 nm and / or an absorption maximum in the Range of λ ≤ 420 nm, e.g. B. from λ = 350 to 420 nm, has.

Farther it is particularly preferred that the fluorescent protein has an emission maximum in a spectral region in which the light absorption of the organellium photosynthetic apparatus is high.

Of the The term "high absorption spectral range" refers to this all wavelengths λ, where the molar extinction coefficient more than 20%, preferably at least 30%, and more preferably at least 40% of the molar extinction coefficient measured at the absorption maximum is.

Especially in this case it is preferred that the fluorescent protein has an emission maximum in the range of λ = 420 to 500 nm or in the range of λ = 600 to 680 nm, and even more preferred that the fluorescent protein an emission maximum in the range of λ = 550 to 650 nm has. It is highly preferred that the fluorescent protein has an absorption maximum in the range of λ = 500 to 600 nm and an emission maximum in the range of 600 to 650 nm has.

at biological fluorophores is the wavelength distance Δλ between Absorbance and emission maxima usually low, typically less than 30 nm and often less than 20 nm. In addition the usable absorption range is often narrow. larger Wavelength distances, however, can be achieved Use of several by radiation-free resonance energy transfer (fluorescence resonance energy transfer, "FREE") coupled fluorophores bridge.

The Terms "radiation-free resonance energy transfer" and "FRET" the skilled worker and the art in detail explained. Radiation-free resonance energy transfer occurs by dipole-dipole interaction between fluorophores in spatial Neighborhood below a substance-specific minimum distance (so-called "Förster distance") in macromolecular dimensions, typically at a spatial proximity of the two fluorophores less than about 10 nm, when the emission spectrum of a Fluorophore overlaps with the absorption spectrum of another fluorophore. This energy transfer happens i. A. in the picosecond range and with very high efficiency. In this way, the light energy can therefore by using coupled fluorophores on larger ones Wavelengths are down-transformed. Conveniently, the fluorophores are covalent to a single molecule connected, ensuring a suitable structure of the molecule can be that their distance is significantly lower than the critical one Minimum distance for the energy transfer is.

In a particular embodiment has the fluorescent protein therefore at least two fluorophores with different absorption spectra, it being preferred that the fluorophores are within the fluorescent protein are capable of radiation-free resonance energy transfer. Likewise preferred is that the fluorescent protein to radiation-free resonance energy transfer to the photosynthetic apparatus of the organelle. exemplary For example, a fluorophore of the fluorescent protein can be selected be among the fluorophores of fluorescent proteins from Aequorea, the fluorophores of fluorescent proteins from Renilla, the fluorophores of fluorescent proteins from Discosoma and its derivatives. These Proteins and fluorophores as well as their structures and properties are familiar to the expert.

Numerous mutants and variations of these proteins are known in the art and are familiar to those skilled in the art, including BFP (blue fluorescent protein), CFP (blue-green fluorescent protein), GFP (green fluorescent protein), EGFP (green fluorescence enhanced protein), and YFP (yellow fluorescent protein). from Aequorea and the RFP family (red fluorescent proteins) from Discosoma and the GFP derivatives "Citrine", "dTomato", "FlAsH""mBanana","mCherry","mHoneydew","mOrange","mPlum"," mStrawberry "," mTangerine "," ReAsH "and" Sapphire "(developed in the laboratory of Roger Tsien, UCSD, and in the form of their coding DNA sequences available, for example, from the company Planet Gene in Menlo Park, California, USA) especially should be mentioned. These fluorescent proteins as well as their encoding nucleic acids are commercially available, the chemical structure and formation of the fluorophores is understood throughout (see, inter alia: Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D: Green fluorescent protein as a marker for gene expression, Science 263 (5148): 802-5, 1994 ; Chudakov D, Lukyanov S, Lukyanov K: "Fluorescent protein as a toolkit for in vivo imaging", Trends Biotechnol 23 (12): 605-13, 2005 ; Heim R, Cubitt A, Tsien R: "Improved green fluorescence", Nature 373 (6516): 663-4, 1995 ; Inouye S, Tsuji F, "Aequorea Green Fluorescent Protein: Expression of the Gene and Fluorescence Characteristics of the Recombinant Protein", FEBS Lett 341 (2-3): 277-80, 1994 ; Miesenböck G, De Angelis D, Rothman J: "Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins", Nature 394 (6689): 192-5, 1998 ; Morise H, Shimomura O, Johnson F, Winant J, "Intermolecular energy transfer in the bioluminescent system of Aequorea", Biochemistry 13 (12): 2656-62, 1974 ; Ormo M, Cubitt A, Kallio K, Gross L, Tsien R, Remington S: "Crystal structure of the Aequorea victoria green fluorescent protein", Science 273 (5280): 1392-5, 1996 ; Pédelacq J, Cabantous S, Tran T, Terwilliger T, Waldo G: "Engineering and characterization of a superfolder green fluorescent protein", Nat Biotechnol 24 (1): 79-88, 2006 ; Phillips G: "Green fluorescent protein - a bright idea for the study of bacterial protein localization", FEMS Microbiol Lett 204 (1): 9-18, 2001 ; Prasher D, Eckenrode V, Ward W, Prendergast F, Cormier M: "Primary structure of the Aequorea victoria green-fluorescent protein", Gene 111 (2): 229-33, 1992 ; Prendergast F, Mann K: "Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskålea", Biochemistry 17 (17): 3448-53, 1978 ; Shaner N, Steinbach P, Tsien R: "A guide to choosing fluorescent proteins", Nat Methods 2 (12): 905-9, 2005 ; Shelley R. McRae, Christopher L. Brown, Gillian R. Bushell: "Rapid purification of EGFP, EYFP, and ECFP with high yield and purity" , Protein Expression and Purification 41 (1): 121-127, 2005 ; Shimomura O, Johnson F, Saiga Y: "Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan Aequorea", J Cell Corp. Physiol 59: 223-39, 1962 ; Tsien R (1998): The Green Fluorescent Protein, Annu Rev Biochem 67: 509-44, 1998 ; Yang F, Moss L, Phillips G: "The molecular structure of green fluorescent protein", Nat Biotechnol 14 (10): 1246-51, 1996 ; Yuste R: "Fluorescence microscopy today", Nat. Methods 2 (12): 902-4; 2005 ; such as Pieribone V & Gruber D 2006: "Aglow in the Dark: The Revolutionary Science of Biofluorescence", Cambridge: Belknap Press, ISBN 0674019210 ; and Room M 2005: "Glowing Genes: A Revolution In Biotechnology", Buffalo, NY: Prometheus Books, ISBN 1591022533 ).

The Fluorescent proteins of the aequorea group have a tendency to non-covalent dimerization, which promotes intermolecular energy transfer. Without Restriction by theory is believed to be this Dimerization by hydrophobic interactions takes place, if desired to an integration of the fluorescent proteins into supermolecular Structures can be used. The formation of the fluorophores takes place autocatalytic without requirement for any external Factors and results chemically and physically extraordinarily robust fluorophores. Fusion with other proteins at the amino or Carboxyterminus impairs formation, activity and stability of the fluorophores not. It is therefore the expert readily possible, a large number of fluorophores and fluorophore combinations according to the invention use.

In an exemplary embodiment of the invention, the fluorescent protein comprises the fluorophores of the Tsien fluorescent proteins "mPlum" ( Shaner & al., Nat. Biotech. 2004 ) and "mTangerine" ( Wang & al., PNAS 2004 ). How out 2 and 3 As can be seen, it is thereby possible to obtain a full coverage in the range of about 550 to 600 nm and in the range of about 500 to 550 nm a broad coverage of the spectrum and to transform the absorbed energy in the usable for the chlorophylls a and b range.

In a particular embodiment of the invention is the transgenic Eucyte a cell of a cormophyte, preferably one free - swimming aquatic plant, preferably an aracee or lemnacee - in of the present invention synonymous - and especially prefers a species of one of the genera Spirodela, Lemna or Wolffia, or a fern, preferably of the genus Azolla; or a moss, preferably one of the genera Blasia, Clavicula or anthoceros; or a cell of a eukaryotic alga, preferably a green alga, especially a monadic or coccal Green alga, z. As a green alga of the genera Scenedesmus, Chlorella or Haematococcus.

Lemnaceae can be dried and used as animal feed; they are also suitable for human nutrition. The dry material typically contains 25-45% of proteins, 4-5% of fats and 8-10% of fiber. They grow in a temperature range of 6-33 ° C, but most preferably 20-28 ° C, at a pH of 5-9, preferably 6.5-7.5, in water of at least 30 cm depth. A phosphate content of 1 mg / l is ideal. Wild-type Lemnaceae require at least 60 mg / L of organic nitrogen compounds; for the plants according to the invention, this value can be greatly reduced, preferably completely dispensed with the addition of nitrogen compounds. Daily harvest is possible; typically, it is harvested so that less than 1 kg of plant material remains per m ² . In this way yields of 10-30 kg dry matter per hectare per year are possible.

method for the transfection of Lemnace cells and for the production of transgenic Lemnaceans are well known to those skilled in the art.

In a particular embodiment of the invention, the expression of the nitrogenase and a fluorescent protein is carried out by a physically contiguous nucleic acid. In the present invention, co-expression of several gene products from a contiguous nucleic acid is not particularly limited; it can Expression of multiple promoters, expression from a promoter using an "internal ribosome entry site", or any other method known in the art. Selection can be done as described above.

One Another aspect of the invention relates to a transgenic cyanobacterium, the at least one fluorescent protein as described in the previous aspect includes. In a preferred embodiment, the fluorescent protein is for radiation-free resonance energy transfer to the photosynthetic apparatus of the cyanobacterium. A selection on this can be as above described described, z. B. by measuring the fluorescence emission of chlorophyll on excitation with one not of the same, well but light wavelength absorbed by the fluorescent protein. It is particularly preferred that a fluorescent protein as a fusion protein with a component of the photosynthetic apparatus of the cyanobacterium is present. In a particular embodiment belongs the cyanobacterium of a heterocyst-forming cyanobacteria species, in particular one of the genus Nostoc or Anabaena belonging.

One Another aspect of the invention relates to a transgenic cell one of the preceding aspects, in which the content of hydrocarbons increased compared to the wild type, and in particular increased the content of carotenoids compared to the wild type is. It is preferred that the expression of nitrogenogenesis and / or a fluorescent protein from one physically with at least one genetic determinant related to the increased hydrocarbon content Nucleic acid takes place. The enzymes of carotenoid metabolism are characterized, and the skilled person is therefore able, if desired to increase their expression. Without restriction on the theory, however, it is assumed that anyway Formation of carotenoid-prone species such as Haematococcus inventive modification due to the increased Absorption of light energy in particular under light stress and therefore without further modifications increased amounts of carotenoids form when growth is blocked. Appropriately, is in such cells, the expression of the nitrogenase therefore among the Control of an externally regulated promoter, z. B. one by temperature or metal ion regulatable promoter. Such Control elements and their use are familiar to the skilled person. On the formation of carotenoids z. B. based on their fluorescence properties be selected.

In a particular embodiment of the invention the membrane of a transgenic cell according to one of the preceding Aspects an additional transporter for sodium ions, preferably a transporter for sodium ions, which is active Salt pumped out of the cell. It is preferred that the Transporter for sodium ions of a physically with the coding sequences for nitrogenase and / or fluorescent protein and / or a genetic determinant of increased hydrocarbon content contiguous nucleic acid is expressed. Appropriate molecules and their expression in heterologous Systems are described in the art and those skilled in the art familiar. On salt resistance can z. B. by growth under salt stress are selected as described above.

As described above, it is of dual advantage to polymerize silica and soluble low molecular weight silicates to substantially linear or planar structures. In a particular embodiment of the invention, the cell therefore additionally comprises a silicate-polymerizing enzyme, preferably a silicate-polymerizing enzyme from a radiolarian, a silicate-polymerizing enzyme from a diatom or a silicate-polymerizing enzyme from a connected amoeba. Here, it is preferred that the silicate-polymerizing enzyme is expressed by a nucleic acid physically related to the coding sequences for nitrogenase and / or fluorescent protein and / or a transporter for sodium ions and / or a genetic determinant for increased hydrocarbon content. An example of a silicate-polymerizing enzyme is in 5 shown; others are familiar to the person skilled in the art.

A Selection can z. B. by measuring the cellular refraction of light done as described above. The possibility of selection allows the skilled person in principle, suitable genes in foreign Identify genomes by their effects in the target cell, expediently by selection from a library. In a preferred embodiment, the identification takes place by the method often referred to as "shotgun cloning", by by known per se a cDNA library a competent organism isolated, transferred to an expression vector and thus expressed in the target cells, that each Target cell expressed an identifiable cDNA and by selection therefore suitable phenotypes can be identified. In the case of silicate polymerization, the competent organism is preferable an organism of a silicate polymerizing species, e.g. B. one Radiolaria, diatom or connected amoeba.

In a particular embodiment of the invention, the cell additionally comprises a resist tenzfaktor against heavy metals, in particular a reducing enzyme or a heavy metal binding biopolymer. Here, it is preferred that the resistance factor against heavy metals is expressed from a nucleic acid physically related to the coding sequences for nitrogenase and / or fluorescent protein and / or a silicate polymerizing enzyme and / or a transporter for sodium ions and / or a genetic determinant for increased hydrocarbon content.

For the expression of determinants and their selection is the same above been said; competent organisms are typically halophilic, preferably euryhaline organisms, especially halophilic, preferably euryhalins Plants.

Of the animal, z. B. human organism can not all amino acids synthesize itself, but is specific to certain amino acids, the so-called essential amino acids, to a supply of the same instructed with food. It is therefore of obvious benefit in the diet the essential amino acids to favor the nonessential quantitatively. The shares of the individual amino acids in the functional, "home-made" proteins of a cell are difficult to influence, but can be essential amino acids rich reserve proteins readily by expression of suitable nucleotide sequences in the cell. Such a nucleotide sequence is readily apparent to those skilled in the art isolate a natural source or fully synthetic produce.

In a particular embodiment of the invention carries the cell therefore additionally at least one further transgene, its product is rich in essential amino acids Polypeptide is. It is preferred that this is essential Amino acids rich polypeptide high molecular weight has, preferably a molecular weight of at least 50 kDa, preferred of at least 100 kDa and more preferably of at least 200 kDa, z. At least 500 kDa or at least 1 MDa.

Of the The term "essential amino acids" is familiar to the person skilled in the art and denotes in particular arginine, cysteine, glycine, glutamine, Isoleucine, leucines, lysines, methionine, phenylalanine, threonines, Tryptophan, tyrosine, histidine and valine, preferably histidine, Isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.

Of the Term "rich in essential amino acids" in the context of the present invention, a polypeptide under which Amino acids at least 40% (molar), preferably at least 50% (molar) and more preferably at least 60% (molar), e.g. B. at least 70% (molar) or at least 80% (molar), to the group of essential amino acids belong.

One Another significant environmental problem currently exists in the contamination of natural habitats hydrophobic substances, mainly medium and longer chain aliphatic and / or aromatic hydrocarbons. The one of them triggered damage to living organisms varied and ranging from physical damage the mechanical properties of large quantities of medium and longer-chain hydrocarbons (classic "oil spill") until the release of mutations and tumors due to Ability already small quantities in particular polycyclic Aromatics for intercalation between the nucleotides of the DNA (e.g. in case of contamination of the soil in the area poorly managed chemical, especially petrochemical operations). All these substances are poorly accessible to biodegradation, in particular partly due to their low solubility in the for living cells required aqueous environment. surfactants Substances (surfactants) are basically capable of being contiguous Layers of hydrophobic substances to distribute and degradation, in particular by biological and abiotic oxidation processes, accessible close. The production of sufficient quantities of such surface-active However, substances requires such an expenditure of energy and organic material that conventional microorganisms are overwhelmed with it. The invention Cells can be used to address this problem to solve.

In a particular embodiment of the invention expressed the cell therefore an additional surface active Peptide, it being preferred that it be the additional secreted surface-active peptide. In a preferred Embodiment comprises the surface active Peptide a head of at neutral pH charged amino acids, z. Arginine, lysine, glutamine, glutamate, asparagine or aspartate, and a tail of neutral pH uncharged, in particular hydrophobic amino acids, e.g. Leucine, isoleucine, valine, Alanine, phenylalanine and glycine.

It is preferred in this embodiment that the spatial form of the surface-active peptide favors the formation of micellar structures over the formation of membranous structures, and in particular, the spatial form of the surface-active peptide tapers from the beginning to the end of the hydrophobic tail. In a particularly preferred embodiment, the side chains of the amino acids terminad become smaller in the hydrophobic tail, ie the spatial size of the side chain of each amino acid in the tail is less than or equal to the spatial size of the side chain of the size of its head-side neighbor, and / or in the hydrophobic tail, the amino acids terminad higher repetitive, ie, the number of repeats of an amino acid is greater than or equal to the number of repetitions of their head-side neighbors. In a particular embodiment, at least one prolyl residue is located between the hydrophilic head region and the hydrophobic tail region. In an exemplary embodiment, the sequence of the peptide is as in FIG 6 shown. Other suitable surfactant peptides are described in the prior art and methods for their preparation are known to those skilled in the art.

One skilled in the art will recognize that for the secretion of a peptide from the cell, the sequence of the mature surface-active peptide with the sequence of suitable export sequences, e.g. B. amino-terminal amino acid sequences must be connected. Suitable sequences and methods for their incorporation into the peptides to be secreted, e.g. Example, by producing corresponding recombinant amino acids which encode such fusion peptides from an export sequence and a sequence to be exported, are described in the prior art and familiar to the expert. A selection for their expression is the expert z. B. instrumental by measuring the surface activity in the medium possible. Likewise, a selection of the inclusion of hydrophobic substances from the medium to the expert z. B. instrumental by measuring the incorporation of isotope-labeled hydrophobic substances, eg. B. ¹⁴ C or ³ H-labeled hydrocarbons in the material of the cell possible.

It is further preferred in this embodiment the cell secretes another polypeptide that interacts with the surfactant Peptide-formed micelles, and in particular, that the cell is capable of the surface-active peptide contain containing micelles, z. B. that she is capable of the mentioned further micelle-associated polypeptide-containing To record micelles. Preferably, it expresses an oxidase transgene, z. B. a mixed-functional oxidase, preferably an oxidase, the for the oxidation of aliphatic hydrocarbon chains and / or aromatic Hydrocarbon groups and / or polycyclic aromatic hydrocarbon systems is able. Corresponding oxidases are known in the art. A selection for their expression is the expert z. B. instrumental possible by tracking the substrate turnover.

In a particularly preferred embodiment is the surface-active peptide producing cell a potential heterotrophic algae, preferably an Euglenophyceae, a dinoflagellate or an amoeboid alga, z. B. an amoeboid alga one of the genera Chrysamoeba or Rhizochrys.

It It goes without saying that the corresponding modifications for the degradation of hydrophobic substances according to the conditions described with others throughout this application Modifications can be combined.

In The complex mechanism of a living cell can be individual Components or modifications of individual components previously not readily predictable effect. In a particular embodiment of the invention carries the cell therefore additionally has at least one additional genetic Change from the wild type.

In a preferred embodiment of the invention is a such genetic change is a chance mutation. Such Random mutations can, for. B. by short-wave irradiation, chemical mutagenesis, transposon mutagenesis or any other the known from the prior art and familiar to those skilled Procedures are produced. Selection can be as described above respectively.

In a preferred embodiment of the invention is a such genetic change the expression at least a gene from a foreign species, preferably a gene that Based on the effects that it unfolds in the cell, from a gene bank the foreign species was selected, for. By expression screening, z. By introducing a cDNA library of the foreign species. in this connection it is preferred that the foreign species be a different plant species is, in particular a species that is at least one biological Property that is to be transferred to the cell. These biological properties include without limitation on resistance to chemical and physical Environmental factors such. As pollutants, high or low temperatures and against diseases and pests.

One Another aspect of the invention relates to a plant which is a transgenic Eucyte embraces one of the previous aspects. The term "plant" denotes in this case all vegetative and generative stages of a photosynthetic Organism. Furthermore, this term is used here to also not photosynthetic organisms, but photosynthetic ones Endosymbionts such. B. Zoochlorellen or Zooxanthellen included, include. Such organisms are also in the field the invention, as long as they at least one inventive Contain cell as Endosymbionten.

One Another aspect of the invention relates to a process for the preparation a transgenic nonhuman organism, as in one of the previous aspects presented.

One Another aspect of the invention relates to a method for obtaining nitrogen-containing biomass, which is characterized in that you have an organism containing a cell after one of the previous ones Aspects under conditions where there is a lack of nitrogen the limiting factor is.

In In a typical application, the method of obtaining qualitative high quality animal feed and / or human food. It should be noted in particular that the inventive Providing protein-rich animal feed the need for hygienic reasons, eg. B. due to the possibility of transmission of prion-associated diseases ("mad cow disease" and the like) questionable recycling of slaughterhouse waste reduce in the form of animal meal or completely unnecessary could do.

In a typical embodiment, the inventive Organisms after an optional enrichment step by itself gained known method of filtration from their habitat, if desired, a cleaning, z. B. by washing, and then processed into a finished product, the Processing any combination of the prior art known steps such as drying, cutting, grinding, pressing and Briquetting may include. These extraction methods and their products are a part of the present invention.

A further aspect of the invention relates to a process for obtaining biomass from atmospheric CO ₂ , which comprises keeping an organism containing a cell according to one of the preceding aspects under growth-inhibiting conditions.

A further aspect of the invention relates to a process for the production of hydrocarbons from atmospheric CO ₂ , which comprises increasing an organism comprising a cell according to one of the preceding aspects, in particular a cell in which the content of hydrocarbons is increased compared to the wild-type and in which, in particular, the carotenoid content is increased over the wild-type, under growth-inhibiting conditions, preferably under growth-inhibiting conditions comprising an oversupply of light energy.

One Another aspect of the invention relates to a method for amelioration inferior soils, which is characterized in that you have an organism containing a cell after one of the previous ones Aspects under growth-inhibiting conditions with the inferior ones Bringing soil into contact. In a preferred embodiment the soils are heavy metal contaminated, in which embodiment it is particularly preferred that the cell additionally has a Resistance factor against heavy metals; salty, in which Embodiment, it is particularly preferred that the membrane the cell has an additional transporter for sodium ions includes; and / or low in swellable silicates, in which embodiment it is particularly preferred that the cell additionally silicate polymerizing enzyme. Without restriction The theory assumes that the silicate polymerization is particularly advantageous because the presence of linear and in particular flat silicates (phyllosilicates, "clay minerals ") the soil's ability to retain nutrients and increases trace elements.

One Another aspect of the invention relates to a method for processing of wastewater characterized by having one An organism containing a cell according to one of the previous aspects under growth-permeable conditions with the waste water brings in contact. In a preferred embodiment the wastewater is biologically contaminated; heavy metal burden, in which embodiment it is particularly preferred that the cell additionally has a resistance factor against heavy metals includes; salty, in which embodiment it is especially it is preferred that the membrane of the cell has an additional Transporter for sodium ions includes; and / or contain them low molecular weight silicates, in which embodiment it it is particularly preferred that the cell additionally silicate polymerizing enzyme.

One Another aspect of the invention relates to a method for obtaining a useful protein, preferably a recombinant useful protein, wherein the useful protein in an organism containing a novel Cell expressed, preferably recombinantly expressed.

The term "useful protein" in the context of the present invention refers to a protein which is recovered for further utilization, ie isolated from the other cell constituents, preferably having a purity of at least 90% by weight, preferably at least 95% by weight and more preferably at least 99% by weight, e.g. B. at least 99.9 wt .-% is recovered. Examples of useful proteins are familiar to the person skilled in the art and comprise a large number of biotechnologically and / or medically usable proteins, eg. B. antibodies. Conveniently, the protein is expressed in such a way that it accumulates in the cell and after harvesting and digesting the plant material is isolated from the same; Suitable expression systems and vectors are known in the art and will be apparent to those skilled in the art. Preferably, the protein is hereby provided with a label which facilitates its separation from the rest of the plant material, e.g. A terminal hexahistidinyl residue which, by virtue of its affinity for complexed nickel or copper ions, allows for simple isolation of large quantities of protein by methods well known in the art.

Becomes the beneficial protein expresses recombinantly, so it is beneficial from a nucleic acid that physically interacts with a transgene according to the invention (fluorescent protein and / or nitrogenase) encoding nucleic acid is. Without limitation to theory, it is believed that this is the burden on the cell expression of the useful protein stabilized in the population.

From Insects, especially of mosquitoes, transmitted Infectious diseases play a major medical role; By way of example, due to its particularly high importance, malaria is an example called, are more mosquito-borne diseases familiar to the skilled person. It has already been shown that a strong Growth of stagnant or slow flowing waters with free-swimming plants such. B. Azolla the development of mosquito larvae with special needs. Without limitation by theory, it is believed that the covering of the water surface with plant material the Respiration of the oxygen-dependent mosquito larvae with special needs.

One Another aspect of the invention therefore relates to a method for controlling of mosquitoes, in particular mosquitoes Genera Anopheles and Culex, in which one contains an organism a cell according to the invention with the habitat the mosquito larvae, especially the habitat oxygen-needy Stages of development of the mosquito larvae, brings into contact. In a preferred embodiment, the cell is hereby as previously described a cell of a free-swimming aquatic plant, preferably a cell of an aracee or lemnacee is and in particular one of the genera Spirodela, Lemna or Wolffia.

In In a particular embodiment, the method comprises in addition to fighting mosquitoes the recombinant expression of an insecticidally active protein, z. B. of Bacillus thuringiensis toxin. Corresponding proteins and methods for their expression are known to those skilled in the art familiar with the technique; others can be left if desired based on their specific insecticidal activity using identify methods known in the art.

One Another aspect of the invention relates to a method for degrading hydrophobic Impurities in natural habitats, in which one organism of the invention brings into contact with the habitat, especially one for expression surface-active peptides capable of the invention Organism, and more preferably an organism, the additional Oxidases expressed.

One Another aspect of the invention relates to a food for Animals or humans, characterized in that the raw material obtained by a method according to the invention or a erfindungsgmäße cell or comprises an organism according to the invention.

One Another aspect of the invention relates to a method for irrigation of farmland, where you have a invention Keeping organism in an open irrigation system. Without limitation to the theory, it is believed that the organism according to the invention by covering the water surface undesirable evaporation counteracts the growth of harmful organisms by occupying the ecological Niche hinders and at the same time the water with nutrients accumulates.

The Features of the claims are part of the description. The invention will be described below with reference to the figures and examples illustrated in more detail. The examples merely represent this exemplary embodiments and are by no means as limiting the invention. The expert recognizes that the items disclosed in the present specification numerous variations and variations are accessible. All of these modifications and variations are considered to be within the invention to be considered proper, provided they fall under the field of Claims fall. In particular, it is understood that if more than one additional feature is described, these features are present in separate cellular individuals can, as far as this is intrinsically possible; without Restriction to this is exemplary in this context surface-active peptides expressing cells and additional Called oxidase expressing cells.

Brief description of the figures:

1 shows the absorption spectra of isolated chlorophyll a and b known from the prior art (abscissa: wavelength λ of the incident light, ordinate: relative molar extinction). In the natural biological context, the absorption spectra are essentially comparable.

2 shows the absorption and emission spectra of the GFP derivatives "mPlum" and "mTangerine" (abscissa: wavelength λ of the incident light, ordinate: relative molar extinction).

3 is a superposition of the two spectra from the 1 and 2 ,

4 shows the annotated Nostoc nitrogenase amino acid sequence according to GenBank, which can be made by adding a mitochondrial import sequence or expression in mitochondrion to the basis of a mitochondrial nitrogenase according to the invention.

5 shows the annotated amino acid sequence of the silaffin protein from the diatom Cylindrotheca according to GenBank.

6 shows in 6a the sequence of an exemplary surfactant peptide in the mature, secreted form, ie after splitting off all possible transport signals, and its properties, as determined by the freely available EMBOSS software package ( http://www.emboss.org ): 6b - Kyte-Doolittle Hydropathy Chart; 6c - supervision of the N-terminus; 6d - schematic representation of the helix. Without wishing to be bound by theory, it is believed that the hydrophobic tail forms an approximately axially symmetric helical structure tapering towards the tail end and in the head is also an approximately axially symmetric distribution of the positive and negative charges.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

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• - http://www.emboss.org [0103]

Claims (122)

A transgenic eucyte, characterized in that it comprises both a nitrogenase and a fluorescent protein, wherein at least one protein selected from a nitrogenase and a fluorescent protein is contained in a double membrane organelle. Transgenic Eucyte according to claim 1, characterized in that wherein both a nitrogenase and a fluorescent protein, respectively contained in an organelle with double membrane. Transgenic Eucyte according to claim 1 or 2, characterized that a protein is selected from nitrogenase and a Fluorescent protein from a nucleic acid of the organelle is encoded with double membrane. Transgenic Eucyte according to one of claims 1 to 3, characterized in that a protein selected from Nitrogenase and a fluorescent protein from a nucleic acid encoded outside the organelle with double membrane. Transgenic Eucyte according to claim 4, characterized in that the protein comprises an amino acid sequence that binds to it directed to the organelle with double membrane. Transgenic Eucyte according to claim 5, characterized in that the amino acid sequence is an amino-terminal signal sequence is. Transgenic Eucyte according to one of claims 1 to 6, characterized in that nitrogenase in an organelle with reducing and / or microaerobic environment. Transgenic Eucyte according to one of claims 1 to 7, characterized in that nitrogenase in one of a mitochondrion derived organelle is included. Transgenic Eucyte according to one of claims 1 to 8, characterized in that nitrogenase in a functional Mitochondrion is included. Transgenic Eucyte according to claim 8, characterized in that the mitochondrion-derived organelle decreased one Has breathability. Transgenic Eucyte according to one of claims 7 to 10, characterized in that the organism containing the nitrogenase an additional reducing component. Transgenic Eucyte according to claim 11, characterized in that the additional reducing component is a hydroquinone contains. Transgenic Eucyte according to one of claims 7 to 12, characterized in that the nitrogenase is an oxygen-sensitive Nitrogenase is. Transgenic Eucyte according to claim 13, characterized in that the nitrogenase contains an iron-sulfur cluster. Transgenic Eucyte according to one of claims 1 to 6, characterized in that the nitrogenase is an oxygen-insensitive Nitrogenase is. Transgenic Eucyte according to claim 15, characterized in that the nitrogenase is a nitrogenase from Streptomyces thermoautotrophicus. Transgenic Eucyte according to claim 15 or 16, characterized that the organism containing the nitrogenase is photosynthetically active is. A transgenic eucyte according to any one of claims 1 to 17, characterized in that the organelle has an additional Transporter for products of nitrogenase includes. Transgenic Eucyte according to claim 18, characterized in that the extra transporter is a transmembrane Glutamine transporter is. A transgenic eucyte according to any one of claims 1 to 19, characterized in that a fluorescent protein in a Photosynthetic apparatus-containing organelle with double membrane is included. Transgenic Eucyte according to claim 20, characterized in that the fluorescent protein has an absorption maximum in a range in which the light absorption of the photosynthetic apparatus of the organelle with double membrane is low. Transgenic Eucyte according to claim 21, characterized in that the fluorescent protein has an absorption maximum in the range of λ = 500 to 600 nm. Transgenic Eucyte according to claim 21 or 22, characterized the fluorescent protein has an absorption maximum in the range of λ <420 nm. Transgenic Eucyte according to any one of claims 20 to 23, characterized in that the Fluorescent protein has an emission maximum in a range in which the light absorption of the photosynthetic apparatus of the organelle is high. Transgenic Eucyte according to claim 24, characterized in that the fluorescent protein has an emission maximum in the range of λ = 420 to 500 nm or in the range of λ = 600 to 700 nm. Transgenic Eucyte according to claim 25, characterized in that the fluorescent protein has an emission maximum in the range of λ = 600 to 680 nm. A transgenic eucyte according to any one of claims 21 to 26, characterized in that the fluorescent protein has an absorption maximum in the range of λ = 500 to 600 nm and an emission maximum in the range of λ = 600 to 650 nm. Transgenic Eucyte according to one of claims 20 to 27, characterized in that the fluorescent protein at least two Has fluorophores with different absorption spectra. Transgenic Eucyte according to claim 28, characterized in that at least two fluorophores within the fluorescent protein radiation-free resonance energy transfer are capable. A transgenic eucyte according to any one of claims 20 to 29, characterized in that the fluorescent protein to radiation-free Resonance energy transfer to the photosynthetic apparatus of the organelle is able. A transgenic eucyte according to any one of claims 20 to 30, characterized in that a fluorophore of the fluorescent protein is selected from the fluorophores of fluorescent proteins from Aequorea, the fluorophores of fluorescent proteins from Renilla, the fluorophores of fluorescent proteins from Discosoma and their Derivatives. Transgenic Eucyte according to one of the preceding claims, characterized in that it is a cormophyte cell. Transgenic Eucyte according to claim 32, characterized in that it is a cell of a free-swimming aquatic plant. Transgenic Eucyte according to claim 33, characterized in that she is a cell of an Aracee or Lemnacee. Transgenic Eucyte according to claim 34, characterized in that the plant belongs to one of the genera Spirodela, Lemna or Wolffia. Transgenic Eucyte according to claim 32, characterized in that she is a cell of a moss of one of the genera Blasia, Clavicula or Anthoceros or a fern of the genus Azolla. Transgenic Eucyte according to one of claims 1 to 31, characterized in that it is a cell of a eukaryotic Alga is. Transgenic Eucyte according to claim 37, characterized in that she is a cell of a green alga. Transgenic Eucyte according to claim 38, characterized in that a cell of a monadic or coccal green alga is. Transgenic Eucyte according to claim 39, characterized in that the monadic or coccal green alga of one of the genera Scenedesmus, Chlorella or Haematococcus. Transgenic Eucyte according to one of the preceding claims, characterized in that the expression of the nitrogenase and a fluorescent protein from a physically contiguous Nucleic acid takes place. Transgenic cyanobacterium, characterized in that it is at least a fluorescent protein as described in any of the claims 21 to 31. Transgenic cyanobacterium according to claim 42, characterized that the fluorescent protein leads to radiation-free resonance energy transfer to the photosynthetic apparatus of the cyanobacterium. Transgenic cyanobacterium according to claim 42 or 43, characterized characterized in that it contains a fluorescent protein as a fusion protein with a component of the photosynthetic apparatus of cyanobacterium is present. Transgenic cyanobacterium according to any one of claims 42 to 44, characterized in that it is a heterocyst forming Cyanobakterienspezies belongs. Transgenic cyanobacterium according to claim 45, characterized that it belongs to one of the genera Nostoc or Anabaena. Transgenic cyanobacterium according to any one of claims 42 to 44, characterized in that it further comprises an oxygen insensitive A nitrogenase as defined in claim 15 or 16. Transgenic cell after one of the preceding gone claims, characterized in that the content of hydrocarbons compared to the wild type is increased. Transgenic cell according to claim 48, characterized in that increased the content of carotenoids compared to the wild type is. Transgenic cell according to claim 48 or 49, characterized that the expression of the nitrogenase and / or a fluorescent protein of a physically having at least one genetic determinant related to the increased hydrocarbon content Nucleic acid takes place. Transgenic cell according to one of the preceding claims, characterized in that the outer membrane the cell has an additional transporter for sodium ions includes. Transgenic cell according to claim 51, characterized in that the transporter for sodium ions actively salt out the cell addition pumps. Transgenic cell according to claim 51 or 52, characterized that the transporter for sodium ions of a physically with the coding sequences for nitrogenase and / or Fluorescent protein and / or a genetic determinant for increased hydrocarbon content related Nucleic acid is expressed. Transgenic cell according to one of the preceding claims, characterized in that the cell additionally a silicate polymerizing enzyme includes. Transgenic cell according to claim 54, characterized in that the silicate-polymerizing enzyme is derived from a radiolarian. Transgenic cell according to claim 54, characterized in that the silicate-polymerizing enzyme is derived from a diatom. Transgenic cell according to claim 54, characterized in that the silicate-polymerizing enzyme from a connected amoeba comes. Transgenic cell according to one of claims 54 to 57, characterized in that the silicate polymerizing enzyme of physically associated with the coding sequences for nitrogenogenesis and / or Fluorescent protein and / or a transporter for sodium ions and / or a genetic determinant for enhanced Hydrocarbon content related nucleic acid is expressed. Transgenic cell according to one of the preceding claims, characterized in that the cell additionally a Resistance factor against heavy metals. Transgenic cell according to claim 59, characterized in that the resistance factor is a reducing enzyme. Transgenic cell according to claim 59, characterized in that the resistance factor is a heavy metal binding biopolymer. Transgenic cell according to one of claims 59 to 61, characterized in that the resistance factor against heavy metals of a physically coding sequences for Nitrogenesis and / or fluorescent protein and / or a silicate polymerizing Enzyme and / or a transporter for sodium ions and / or a genetic determinant related to increased hydrocarbon content Nucleic acid is expressed. A transgenic cell according to any one of claims 1 to 62, characterized in that the cell additionally at least carries another transgene whose product is essential Is amino acids rich polypeptide. Transgenic cell according to claim 63, characterized in that the polypeptide rich in essential amino acids is high Has molecular weight. Transgenic cell according to claim 64, characterized in that the essential amino acid-rich polypeptide has a molecular weight of at least 100 kDa. Transgenic cell according to one of claims 1 to 65, characterized in that it is an additional surface active Expressed peptide. Transgenic cell according to claim 66, characterized in that it secretes the additional surfactant peptide. Transgenic cell according to claim 66 or 67, characterized that the surface-active peptide heads out at neutral pH charged and a tail uncharged at neutral pH Includes amino acids. A transgenic cell according to any one of claims 66 to 68, characterized in that the spatial form of the surface-active Peptides against the formation of micellar structures favors the formation of membranous structures. Transgenic cell according to claim 69, characterized characterized in that the spatial form of the surface-active peptide tapers from the beginning to the end of the hydrophobic tail. Transgenic cell according to claim 70, characterized in that in the hydrophobic tail the side chains of the amino acids Terminad become smaller. Transgenic cell according to claim 70 or 71, characterized that in the hydrophobic tail the side chains of the amino acids termarily become more repetitive. A transgenic cell according to any one of claims 70 to 72, characterized in that between the hydrophilic head region and the hydrophobic tail region is at least one prolyl residue. A transgenic cell according to any one of claims 66 to 73, characterized in that it secretes another polyeptide, that with the surface active peptide according to one of the Claims 66 to 73 formed micelles associated exists. A transgenic cell according to any one of claims 66 to 74, characterized in that it is capable of accepting micelles. Transgenic cell according to claim 75, characterized in that it is capable of sequestering the surfactant peptide of claims 66 to 73 containing micelles. Transgenic cell according to claim 76, characterized in that it is capable of containing the further polypeptide of claim 74 To record micelles. Transgenic cell according to one of claims 75 to 77, characterized in that it transgenically expresses an oxidase. Transgenic cell according to claim 78, characterized in that it transgenically expresses a mixed-function oxidase. Transgenic cell according to claim 78 or 79, characterized that the oxidase for the oxidation of aliphatic hydrocarbon chains is able. Transgenic cell according to any one of claims 78 to 80, characterized in that the oxidase for the oxidation of aromatic Hydrocarbon groups is capable of. Transgenic cell according to claim 81, characterized in that the oxidase is capable of oxidizing polycyclic aromatic hydrocarbon systems is. A transgenic cell according to any one of claims 66 to 82, characterized in that it is a potentially heterotrophic alga is. Transgenic cell according to claim 83, characterized in that she is an Euglenophycee. Transgenic cell according to claim 83, characterized in that she is a dinoflagellate. Transgenic cell according to claim 83, characterized in that she is an amoeboid alga. Transgenic cell according to claim 86, characterized in that She belongs to one of the genera Chrysamoeba or Rhizochrys. Transgenic cell according to one of claims 1 to 87, characterized in that the cell additionally at least another genetic change bears the wild type. Transgenic cell according to claim 88, characterized in that a genetic change is a random mutation. Transgenic cell according to claim 88 or 89, characterized that a genetic change the expression at least another gene from a foreign species. Transgenic cell according to claim 90, characterized in that the gene from the effects that it unfolds in the cell a gene bank of the foreign species was selected. Transgenic cell according to claim 91, characterized in that the foreign species is a different plant species. Transgenic cell according to claim 92, characterized in that the other species has at least one biological property, which is to be transferred to the cell. Plant, characterized in that it has a transgenic Eucyte according to one of claims 32 to 41. Process for producing a transgenic non-human Organism, characterized in that the organism as in a of claims 1 to 94 is defined. Process for the production of nitrogen-containing biomass, characterized in that an organism containing a cell according to one of the Claims 1 to 93 under conditions in which nitrogen deficiency is the limiting factor. Process for the production of nitrogen-containing biomass according to claim 96, characterized in that the cell defines a cell as defined in any one of claims 63 to 93. Process for the production of nitrogen-containing biomass according to claim 97, characterized in that the cells for an optional Enrichment step by per se known methods of filtration wins from their habitat, if desired, a cleaning, z. B. by washing, subjected and then processed into a finished product, the processing being any combinations of off the state Technically known steps such as drying, cutting, grinding, May include pressing and briquetting. Process for the production of biomass from atmospheric CO ₂ , characterized in that an organism containing a cell according to one of claims 1 to 93 is kept under growth-inhibiting conditions. Process for the production of hydrocarbons from atmospheric CO ₂ , characterized in that an organism containing a cell according to one of Claims 48 to 50 is kept under growth-inhibiting conditions. Process for the production of hydrocarbons from atmospheric CO ₂ according to claim 100, characterized in that the growth-inhibiting conditions are the conditions of an excess supply of light energy. Process for the amelioration of inferior soils, thereby characterized in that one comprises an organism containing a cell according to any one of claims 1 to 93 under growth-permissive Conditions with the inferior soils. Method for the amelioration of inferior soils Claim 102, characterized in that the floors heavy metal loaded are. Method for the amelioration of inferior soils Claim 102 or 103, characterized in that the floors are salty. Method for the amelioration of inferior soils one of claims 102 to 104, characterized the soils are low in swellable silicates. Method for the amelioration of inferior soils one of claims 102 to 105, characterized that the organism has a cell according to one of the claims 51 to 93 contains. Process for the treatment of waste water, characterized in that an organism containing a cell according to one of claims 1 to 93 under growth-permeable conditions with the effluents brings in contact. Process for the treatment of wastewater according to claim 107, characterized in that the wastewater biologically are charged. Process for the treatment of wastewater according to claim 107 or 108, characterized in that the waste water heavy metal are loaded. Process for the treatment of waste water after a of claims 107 to 109, characterized in that the wastewater is saline. Process for the treatment of waste water after a of claims 107 to 110, characterized in that the effluents contain low molecular weight silicates. Process for obtaining a useful protein, characterized that the beneficial protein in an organism containing a cell after one of claims 1 to 93 is expressed. A process for obtaining a useful protein according to claim 112, characterized in that the useful protein expresses recombinantly becomes. Process for obtaining a useful protein according to claim 113, characterized in that the recombinant Nutzprotein a his Separation carries facilitating label. A process for obtaining a useful protein according to claim 114, characterized in that the label is a terminal hexahistinyl. Process for the degradation of hydrophobic impurities in natural Habitats, characterized in that you have an organism according to one of claims 66 to 87 with the habitat in Contact brings. Method of combating mosquitoes, characterized in that one contains an organism A cell according to any one of claims 1 to 93 having the habitat the mosquito larvae brings into contact. Method for controlling mosquitoes according to claim 117, characterized in that the organism as in one of the claims 33 to 35 is defined. Method for controlling mosquitoes after Claim 118, characterized in that the organism additionally expresses an insecticidally acting protein. Method for controlling mosquitoes after Claim 119, characterized in that the insecticidally active Protein is the Bacillus thuringiensis toxin. Foodstuffs for animals or humans, characterized that the raw material according to any one of claims 96 to 98 has been obtained or a cell or an organism according to one of Claims 1 to 93 comprises. Method for irrigating farmland, characterized that is an organism according to one of claims 32 to 37 in an open irrigation system.