DE102007041862A1 - Microbiological production of isoprenoids - Google Patents

Abstract

The present invention relates to a cell which has been genetically engineered with respect to its wild type such that it is capable of forming isoprenoids included in defined compartments. The invention also relates to a method for producing a genetically modified cell, the genetically modified cell obtainable by this method, a method for producing isoprenoids, the isoprenoids obtainable by this method, isolated nucleic acids and isolated polypeptides.

Description

Field of the invention

The The present invention relates to a wild type genetically modified cell, a method of production a genetically modified cell, by this Method available, genetically modified Cell, a process for producing isoprenoids by this process obtains isoprenoids, isolated nucleic acids as well as isolated polypeptides.

State of the art

To The terpenoids include compounds that are based on isoprene build up. Thus, the terpenoids belong to the subgroup the terpenes, whose carbon atoms can always be divided by 5, but also derived from the terpenes compounds in which carbon atoms later be removed in the biosynthesis, and their Consequently, carbon atoms can not be divided by 5. terpenoids form important features for the identification of plants, as a particular ingredient pattern characteristic of a certain plant is. There are more than 40,000 terpenoids known around 8,000 of them belong to the subgroup of terpenes.

Terpenoids are formed from the two precursors isopentenyl diphosphate (IPP = isopentenyl pyrophosphate) and dimethylallyl diphosphate (DMAPP = dimethylallyl pyrophosphate), whereby the provision of these two precursors can take place via the mevalonate pathway or via the deoxyxylulose metabolic pathway. Since the terpenoids are based on C ₅ -carbon compounds as subunits, terpenoids usually have a number of carbon compounds that are divisible by five. Accordingly, terpenoids are divided into the following subclasses: hemiterpenes _(C5, such as isoprene), monoterpenes (C _10, such as farnesyl), sesquiterpenes (C _15), diterpenes (C ₂₀ such as Taxol), triterpenes _(C30, such as as squalene), tetraterpenes (C _40, such as lycopene) and polyterpenes with more than 40 carbon atoms, such as rubber.

Lots Terpenoids are of great economic interest as natural products, for example, as feed additives (lycopene), in skin or Hair care products (Q10) or as resistant polymers (natural rubber). For example, cis-polyisoprene comes in rubber, while trans-polyisoprene is a major constituent of gutta-percha is. Gutta-percha is formed when drying the Milky juice of tropical tree species Palaquium gutta. Chicle, won from the pear apple tree, provides a 1: 2 mixture of trans and cis polyisoprene represents.

The Provision of the above occurring as natural products isoprenoids as a component of feed, skin or hair care products or resistant polymers requires on the one hand the Formation of terpenoids by appropriate organisms and the subsequent isolation of the affected by the microorganism formed terpenoids. In general, however, both have availability of the terpenoids forming organism as well as the method of Isolation of terpenoids has a decisive influence on the Value of terpenoids. As many of the terpenoids described above also be provided by chemical or biotechnological means can therefore always be weighed from an economic point of view, whether a given terpenoid advantageously by a natural Organism or by chemical or biotechnological means should be produced.

numerous the aforementioned terpenoids, such as on cis-polyisoprene based rubber, are too complex, as they are based on chemical Way, or their production chemically is against cost reasons the production by natural organisms does not make sense. The production of such complex isoprenoids by natural Organisms, especially by plants, is however with the large one Disadvantage linked to the availability of these plants often depends on factors caused by the Manufacturers can not be affected, such as drought as well as parasitic infestation or infections of the plants.

Description of the invention

Of the Present invention was based on the object resulting from the State of the art resulting disadvantages in connection with the production of terpenoids, but especially in the context of complex ones Terpenoids, such as polyisoprenes to overcome.

Especially the present invention was based on the object, a method to provide terpenoids with which it is possible is to also make complex terpenoids such as polyisoprenes, the economy of the production of terpenoids possible not by factors such as climatic conditions, parasitic infestation or Infections depends.

One Another object of the present invention was to provide a method of providing terpenoids, with which it is possible to use terpenoids as possible consistent chemical quality, especially with as constant as possible, chemical composition manufacture.

a Contribute to the solution of the above-mentioned tasks a cell which is genetically engineered relative to its wild type modified to include isoprenoids included in defined compartments to be able to form.

Completely surprising, but not less advantageous, could be found be that cells, especially bacteria or yeast cells, can be modified by recombinant methods such that they include isoprenoids included in defined compartments make money. The recombinant modification of the cell Causes cells to undergo recombinant modification either no or barely detectable levels of isoprenoids although they were able to form or that prior to recombinant modification Isoprenoids were able to form but were unable to to include these isoprenoids in defined compartments in the cell after carrying out the recombinant modification now are able to form isoprenoids and these in defined To include compartments of the cell.

As used herein, the term "isoprenoid" is understood to mean a structurally heterogeneous group of natural products derived biosynthetically from the two C ₅ compounds IPP and DMAPP, such isoprenoids being formed by the multiplication of isoprene (= 2). The term "isoprenoid", however, also includes natural degradation and rearrangement products of the actual isoprenoids, in which it may no longer be necessary to recognize directly all the original isoprene units and their derivatives The number of carbon atoms can no longer be divisible by 5 ("irregular structure"). Particularly preferred isoprenoids according to the invention are the terpenoids, with the terpenoids again being the polyisoprene, in particular poly-cis-isoprene, poly-trans-isoprene or mixtures of poly-cis-isoprene and poly-trans-isoprene, the most preferred isoprenoids. In this connection, it is particularly preferred that the above-mentioned polyisoprenes have more than 55 carbon atoms, more preferably more than 100 carbon atoms, more preferably more than 1,000 carbon atoms, more preferably more than 10,000 carbon atoms, and most preferably more than 100,000 carbon atoms each Molecule, wherein preferably a number of carbon atoms of 1,000,000, more preferably of 500,000 carbon atoms per molecule is not exceeded.

Under A "wild-type" cell is preferably a cell whose genome is in a state as naturally originated through evolution. The term is used both for used the entire cell as well as for individual genes. The term "wild type" therefore does not apply in particular to such Cells or genes whose gene sequences are at least partially modified by humans using recombinant techniques have been.

The term "defined compartments of a cell", as used herein, preferably means certain organelles in the cytoplasm of a cell, which organelles may optionally be surrounded by a biomembrane According to a particularly preferred embodiment of the cell according to the invention defined compartments around so-called inclusion bodies, which usually have a size in the range of 1 × 10 ⁴ to 1 × 10 ¹² Å ³ (cubic angstroms), more preferably in a range of 1 × 10 ⁵ to 1 × 10 ¹⁰ Å ^3, and most preferably in a range of 1 × 10 ⁶ to 1 × 10 ⁸ Å ³ , and appear in electron micrographs as less electron-dense, milky-white, irregularly flattened, shaped organelles in the cytoplasm. Frequently, such inclusion bodies have a cylindrical structure.

The Cells according to the invention can be prokaryotes or eukaryotes. These may be mammalian cells (such as cells from humans) to plant cells or around Microorganisms such as yeasts, fungi or bacteria act as microorganisms particularly preferred and bacteria and yeasts most preferred are.

Particularly suitable bacteria, yeasts or fungi are those bacteria, yeasts or fungi which are deposited in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany, as bacterial, yeast or fungal strains. Bacteria suitable according to the invention belong to the genera under
http://www.dsmz.de/species/bacteria.htm
Yeasts which are suitable according to the invention belong to those genera which are listed under
http://www.dsmz.de/species/yeasts.htm
are listed and suitable fungi according to the invention are those under
http://www.dsmz.de/species/fungi.htm
are listed.

Particularly according to the invention preferred cells are those of the genera Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Lactobacillus, Lactococcus, Candida, Pichia, Kluveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Burkholderia and Clostridium, Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Saccharomyces cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Ralstonia eutropha, especially Ralstonia eutropha H16, Pseudomonas aeroginosa, Acinetobacter calcoaceticus and Pichia pastoris are particularly preferred.

According to one particularly preferred embodiment of the invention This cell is able to do more compared to its wild type to form isoprenoids included in the defined compartments. It is inventively preferred that the Genetically modified cell so genetically modified is that they are in a defined time interval, preferably within of 2 hours, more preferably within 8 hours and on most preferably within 24 hours, at least 2 times, especially preferably at least 10 times, moreover preferably at least 100 times, moreover, more preferably at least 1,000 times and most preferably at least 10,000 times more isoprenoids and in the defined compartments of the cell as the wild type of the cell. The increase of education and inclusion the isoprenoids can be determined, for example, by that the cell according to the invention and the wild-type cell each separately under the same conditions (same cell density, same Nutrient medium, same culture conditions) for a certain time interval in a suitable nutrient medium be cultivated and then the amount of in the Inclusion bodies contained isoprenoids is determined.

The Formulation "that the genetically modified Cell is genetically engineered to do more Isoprenoid forms and in the defined compartments of the cell includes as the wild type of the cell "also concerns the Case that the wild type of the genetically modified cell either no isoprenoids at all or no detectable Forms quantities of isoprenoids or forms isoprenoids, but not in defined compartments, especially in to include the inclusion bodies described above able, and detectable only after the genetic modification Quantities of these components formed and in the defined compartments the cell can be included.

According to one particularly preferred embodiment of the invention In this cell, expression is the expression, and thus also the activity, at least one enzyme involved in the production of isoprenoids, preferably in the preparation of polyisoprenes with the above mentioned number of carbon atoms, in comparison increased to the wild type, which is preferably in this enzyme an enzyme selected from the group consisting of a Rubber binding protein, such as the "small rubber binding protein" encoded by the srpp3-tk gene, a rubber elongation factor ("rubber elongation factor ") such as the" rubber "encoded by the ref2-hb gene elongation factor ", and a prenyltransferase, wherein the increase in the activity of a prenyltransferase is particularly preferred. Further preferred is the combined Increasing the activity of a prenyltransferase and a gum-binding protein, a prenyltransferase and a Gum elongation factor and a prenyltransferase, a Gum-binding protein and a rubber elongation factor.

A suitable gene for a gum-binding protein, whose expression according to the invention can preferably be increased in a microorganism is, for example, the srpp gene from Hevea brasiliensis, which inter alia by Soo Kyung Oh et al. in "Isolation, Characterization and Functional Analysis of a Novel cDNA Clone Encoding a Small Rubber Particle Protein from Hevea brasiliensis", The Journal of Biological Chemistry, Vol. 274 (24), pages 17, 132-17, 138 (1999) has been described. Another suitable gene for a gum-binding protein is the ghs gene, which is derived from In Jeong Kim et al. in "A novel cDNA from Par thenium argentatum Gray enhances the rubber biosynthetic activity in vitro ", Journal of Experimental Botany, Vol. 55 (396), pages 377-385 (2004) , has been described.

A suitable gene for a gum elongation factor whose expression according to the invention can preferably be increased in a microorganism is, for example, the ref gene from Hevea brasiliensis, which inter alia Elisabeth Goyvaerts et al. in "Cloning and Sequencing of the cDNA Encoding the Rubber Elongation Factor of Hevea brasiliensis", Plant Physiology, Vol. 97, pp. 317-321 (1991) has been described.

The present invention therefore relates in particular to recombinant microorganisms, in particular recombinant bacterial or yeast cells, in which the activity of a prenyltransferase is increased. In this connection, it is preferred that the cell according to the invention has an increased activity of an enzyme E ₁ in comparison with its wild-type, which catalyzes the transfer of an isopentenyl diphosphate unit to an allylic diphosphate initiator.

The term "increased activity of an enzyme" as used above in connection with the enzyme E ₁ and in the following statements in connection with the enzymes E ₂ , etc., is preferably to be understood as increased intracellular activity.

The following statements on increasing the enzyme activity in cells apply both to the increase in the activity of the enzyme E ₁ and to all the enzymes mentioned below, the activity of which may optionally be increased.

in principle can be an increase in enzymatic activity Achieve by the copy number of the gene sequence or the Increases gene sequences encoding the enzyme, use a strong promoter or use a gene or allele, that for a corresponding enzyme with an increased activity coded and if necessary combined these measures. Genetically modified according to the invention Cells are transformed, for example, by transformation, transduction, Conjugation or a combination of these methods with a vector generates the desired gene, an allele of this gene or parts thereof and one that enables the expression of the gene Vector contains. The heterologous expression is particularly by the invention particularly preferred integration of the gene or alleles in the chromosome of the cell or an extrachromosomal scored replicating vector.

An overview of the possibilities for increasing the enzyme activity in cells using the example of pyruvate carboxylase gives DE-A-100 31 999 , which is hereby incorporated by reference, and the disclosure of which relates to the possibilities of increasing the enzyme activity in cells forms part of the disclosure of the present invention. Invention forms.

The expression of the above and all of the enzymes or genes mentioned below can be detected in the gel with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software. If the increase in enzyme activity is based solely on an increase in the expression of the corresponding gene, the quantification of the increase in enzyme activity can be determined in a simple manner by comparing the 1- or 2-dimensional protein separations between wild type and genetically modified cell. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that of Hermann et al. (Electrophoresis, 22: 1712.23 (2001) described procedure. The protein concentration can also be determined by Western blot hybridization with an antibody specific for the protein to be detected ( Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989 ) and subsequent optical evaluation with appropriate software for concentration determination ( Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647 ) to be analyzed. The activity of DNA-binding proteins can be measured by DNA band shift assays (also called gel retardation) (Wilson et al., (2001) Journal of Bacteriology, 183: 2151-2155 ). The effect of DNA-binding proteins on the expression of other genes can be demonstrated by various well-described methods of the reporter gene assay ( Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989 ). The intracellular enzymatic activities can be determined by various methods described ( Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627 ; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284 ; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823 ). Unless specified in the following statements concrete methods for determining the activity of a particular enzyme, the determination is carried out the increase in the enzyme activity and also the determination of the reduction of an enzyme activity, preferably by means of in Hermann et al., Electrophoresis, 22: 1712-23 (2001) . Lohaus et al., Biospektrum 5 32-39 (1998) . Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001) described methods.

Becomes the increase of enzyme activity by mutation of the endogenous gene, such Mutations generated either undirected by classical methods be such as by UV irradiation or by mutagenic Chemicals, or specifically by means of genetic engineering methods such as Deletion (s), insertion (s) and / or nucleotide exchange (s). By These mutations will be genetically engineered cells receive. Particularly preferred mutants of enzymes are in particular even those enzymes that are no longer or at least compared to Wild-type enzyme reduces feedback-inhibitable.

If the increase in enzyme activity is accomplished by increasing the expression of an enzyme, for example, one increases the copy number of the corresponding genes or mutates the promoter and regulatory region or the ribosome binding site, which is upstream of the structural gene. In the same way, expression cassettes act, which are installed upstream of the structural gene. Inducible promoters also make it possible to increase expression at any time. Furthermore, the enzyme gene can be assigned as regulatory sequences but also so-called "enhancers" which also cause an increased gene expression via an improved interaction between RNA polymerase and DNA. Measures to extend the lifetime of m-RNA also improve expression. Furthermore, by preventing degradation of the enzyme protein, enzyme activity is also enhanced. The genes or gene constructs are either present in plasmids with different copy numbers or are integrated and amplified in the chromosome, an integration in the genome being particularly preferred. Alternatively, overexpression of the genes in question can be achieved by changing the composition of the medium and culture. Instructions for this, the expert finds among others Martin et al. (Bio / Technology 5, 137-146 (1987)) , at Guerrero et al. (Gene 138, 35-41 (1994)) . Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)) , at Eikmanns et al. (Gene 102, 93-98 (1991)) , in EP-A-0 472 869 , in the US 4,601,893 , at Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991) , at Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) , at LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)) , in WO-A-96/15246 , at Malumbres et al. (Gene 134, 15-24 (1993) , in JP-A-10-229891 , at Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) and in known textbooks of genetics and molecular biology. The measures described above as well as the mutations lead to genetically modified cells.

To increase the expression of the respective genes, episomal plasmids are used, for example. Suitable plasmids are in particular those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as pZl ( Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989) ), pEKExl (Eikmanns et al., Gene 107: 69-74 (1991) ) or pHS2-1 ( Sonnen et al., Gene 107: 69-74 (1991) ) are based on the cryptic plasmids pHM1519, pBLI or pGAl. Other plasmid vectors, such as those based on pCG4 ( US 4,489,160 ) or pNG2 ( Serwold-Davis et al., FEMS Microbiology Letters 66: 119-124 (1990) ) or pAG1 ( US 5,158,891 ) can be used in the same way.

Also suitable are those plasmid vectors, by means of which one can apply the method of gene amplification by integration into the chromosome, as it is for example of Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) for duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned into a plasmid vector which can be replicated in a host (typically Escherichia coli) but not in Corynebacterium glutamicum. As vectors, for example, pSUP301 ( Simon et al., Bio / Technology 1: 784-791 (1983) ), pKI8mob or pKI9mob ( Schäfer et al., Gene 145: 69-73 (1994) ), pGEM-T (Promega Corporation, Madison, Wisconsin, USA), pCR2.1-TOPO ( Shuman, Journal of Biological Chemistry 269: 32678-84 (1994) (), PCR ^® Blunt (Invitrogen, Groningen, The Netherlands), pEMl Schrumpf et al., Journal of Bacteriology 173: 4510-4516) ) or pBGS8 ( Spratt et al., Gene 41: 337-342 (1986) ) in question. The plasmid vector containing the gene to be amplified is then converted by conjugation or transformation into the desired strain of Corynebacterium glutamicum. The method of conjugation is for example at Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994) described. Methods for transformation are for example Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988) . Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989) and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994) described. After homologous recombination by means of a cross-over event the resulting strain has at least two copies of the gene in question.

The enzyme E ₁ is preferably a prenyltransferase (for example EC 2.5.1.29, 2.5.1.10 or 2.5.1.1), but more preferably a cis-1,4-prenyltransferase. Examples of genes for a prenyltransferase are, for example, ggps1, qm, bts1, bet4, ram2, ctrE, ispA, idsA1, ptlB, fppS, sds, gds-1, gds, fdps, yqiD, fppS, ispAB, fps, ptlB, idsA, idsB or mutants of these genes, with ctrE, ispA, fps and idsA being particularly preferred. The nucleotide sequence of these genes as well as other suitable genes for a prenyltransferase can be found, for example, in the "Kyoto Encyclopedia of Genes and Genomes" (KEGG database), the National Library of Biotechnology Information (NCBI) databases of the National Library of Medicine (Bethesda, MD , USA) or the nucleotide sequence database of European Molecular Biology Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK, respectively).

• a) einer Sequenz nach SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07,
• b) einer intronfreien Sequenz, die von einer Sequenz nach a) abgeleitet ist und das gleiche Protein oder Peptid kodiert wie die Sequenz nach SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07, wobei diese intronfreie Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• c) einer Sequenz, die ein Protein oder Peptid kodiert, das die Aminosäure-Sequenz nach SEQ.-ID-Nr. 08, SEQ.-ID-Nr. 09, SEQ.-ID-Nr. 10, SEQ.-ID-Nr. 11, SEQ.-ID-Nr. 12, SEQ.-ID-Nr. 13 oder SEQ.-ID-Nr. 14 umfasst,
• d) einer Sequenz, die mit einer Sequenz nach a) bis c) zu mindestens 80%, vorzugsweise zu mindestens 85%, besonders bevorzugt zu mindestens 90%, darüber hinaus bevorzugt zu mindestens 95% und am meisten bevorzugt zu mindestens 99% identisch ist, wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyldiphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• e) einer Sequenz, die mit dem Gegenstrang einer Sequenz nach einer der Gruppen a) bis d) hybridisiert oder unter Berücksichtigung der Degeneration des genetischen Codes hybridisieren würde, wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• f) einem durch Substitution, Addition, Inversion und/oder Deletion einer oder mehrerer Basen erhaltenen Derivat einer Sequenz nach einer der Gruppen a) bis e), wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• g) einer Sequenz, die der SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07 innerhalb der Degeneration des genetischen Codes entspricht, wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert, sowie
• h) einer Sequenz mit neutralen Sinnmutationen der SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07, wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert.

According to a particular embodiment of the cell according to the invention, it has an increased activity of the enzyme E ₁ , this enzyme being encoded by a DNA selected from the group consisting of:

• a) a sequence according to SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07,
• b) an intron-free sequence which is derived from a sequence according to a) and encodes the same protein or peptide as the sequence according to SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, wherein said intron-free sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator,
• c) a sequence which encodes a protein or peptide comprising the amino acid sequence according to SEQ.ID-No. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ. ID. NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 comprises
• d) a sequence which is identical to a sequence according to a) to c) at least 80%, preferably at least 85%, particularly preferably at least 90%, moreover preferably at least 95% and most preferably at least 99% identical which sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator,
• e) a sequence which would hybridize to the opposite strand of a sequence according to any one of groups a) to d) or would hybridize in consideration of the degeneracy of the genetic code, this sequence preferably coding for an enzyme which comprises the transfer of an isopentenyl diphosphate unit catalyzes an allylic diphosphate initiator,
• f) a derivative of a sequence according to one of the groups a) to e) obtained by substitution, addition, inversion and / or deletion of one or more bases, this sequence preferably coding for an enzyme which comprises the transfer of an isopentenyl diphosphate unit catalyzes an allylic diphosphate initiator,
• g) a sequence corresponding to SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07 within the degeneracy of the genetic code, which sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator, as well as
• h) a sequence with neutral sense mutations of SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, which sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator.

The Sequences according to SEQ.-ID-No. 01 to SEQ. 07 are from the genome of the Russian dandelion and encode for proteins responsible for terpenoid formation and, among other things, a prenyltransferase activity exhibit.

• – GAP (Deveroy, J. et al., Nucleic Acid Research 12 (1984), Seite 387 , Genetics Computer Group University of Wisconsin, Medicine (Wi), und
• – BLASTP, BLASTN und FASIA ( Altschul, S. et al., Journal of Molecular Biology 215 (1990), Seiten 403–410 . Das BLAST-Programm kann erhalten werden vom National Center For Biotechnology Information (NCBI) und aus weiteren Quellen ( BLAST Handbuch, Altschul S. et al., NCBI NLM NIH Bethesda ND 22894; Altschul S. et al., vorstehend).

The "nucleotide identity" and also the "amino acid identity" mentioned below within the meaning of the present invention are determined by means of known methods. In general, special computer programs are used with algorithms taking into account special requirements. Preferred methods for determining identity initially produce the greatest match between the sequences to be compared. Computer identity determination programs include, but are not limited to, the GCG program package, including

• - CAP (Deveroy, J. et al., Nucleic Acid Research 12 (1984), page 387 , Genetics Computer Group University of Wisconsin, Medicine (Wi), and
• - BLASTP, BLASTN and FASIA ( Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410 , The BLAST program can be obtained from the National Center For Biotechnology Information (NCBI) and from other sources ( BLAST Handbook, Altschul S. et al., NCBI NLM NIH Bethesda ND 22894; Altschul S. et al. supra).

Those skilled in the art will be aware that various computer programs are available for the calculation of similarity or identity between two nucleotide or amino acid sequences. Thus, the percentage identity between two amino acid sequences z. B. by the Needleman and desire ( Biol. (48): 444-453 (1970). ) Algorithm, which can be found in the GAP program in the GCG software package (available via http://www.gcg.com ), using either a Blossom 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5 , or 6. Those skilled in the art will recognize that the use of different parameters will lead to slightly different results, but that the percent identity between two amino acid sequences in total will not be significantly different. Typically, the Blossom 62 matrix is used using the default settings (gap weight: 12, length weight: 1).

A Identity of 80% according to the above Algorithm means in the context of the present invention 80% identity. The same applies to higher ones Identities.

The Feature "sequence that coincides with the opposite strand of a sequence according to one of the groups a) to d), more preferably according to group a), hybridized or taking into account the degeneration of the genetic code would hybridize "according to alternative e) indicates a sequence which is preferably stringency Conditions with the opposite strand of a sequence after one of the groups a) to d), particularly preferably according to group a), hybridized or considering the degeneration of the genetic Codes would hybridize. For example, you can hybridizations at 68 ° C in 2X SSC or according to the protocol of the dioxygenin labeling kit from Boehringer (Mannheim). Preferred hybridization conditions are z. B. Incubation at 65 ° C overnight in 7% SDS, 1% BSA, 1mM EDTA, 250mM sodium phosphate buffer (pH 7.2) and subsequent washing at 65 ° C with 2X SSC; 0.1% SDS.

Derivatives of the isolated DNA according to the invention, which according to alternative f) can be obtained by substitution, addition, inversion and / or deletion of one or more bases of a sequence according to one of groups a) to e), include in particular those sequences which are described in US Pat Protein which they encode to conservative amino acid substitutions, such. B. the exchange of glycine for alanine or aspartic acid against glutamic acid. Such functionally neutral mutations are referred to as sense mutations and do not lead to any fundamental change in the activity of the polypeptide. Furthermore, it is known that changes to the N- and / or C-terminus of a polypeptide can not significantly affect its function or even stabilize it, so that correspondingly also DNA sequences in which at the 3 'end or at the 5' end of the Sequence with the SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07 bases are included, are encompassed by the present invention. Information on this is the expert, among others Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)) , at O'Regan et al. (Gene 77: 237-251 (1989)) , at Sahin-Toth et al. (Protein Sciences 3: 240-247 (1994)) , at Hochuli et al. (Bio / Technology 6: 1321-1325 (1988) ) and in well-known textbooks of genetics and molecular biology.

• a) einer Sequenz nach SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07,
• b) einer intronfreien Sequenz, die von einer Sequenz nach a) abgeleitet ist und das gleiche Protein oder Peptid kodiert wie die Sequenz nach SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07, wobei diese intronfreie Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• c) einer Sequenz, die ein Protein oder Peptid kodiert, das die Aminosäure-Sequenz nach SEQ.-ID-Nr. 08, SEQ.-ID-Nr. 09, SEQ.-ID-Nr. 10, SEQ.-ID-Nr. 11, SEQ.-ID-Nr. 12, SEQ.-ID-Nr. 13 oder SEQ.-ID-Nr. 14 umfasst,
• d) einer Sequenz, die mit einer Sequenz nach a) bis c) zu mindestens 80%, vorzugsweise zu mindestens 85%, besonders bevorzugt zu mindestens 90%, darüber hinaus bevorzugt zu mindestens 95% und am meisten bevorzugt zu mindestens 99% identisch ist, wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyldiphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• e) einer Sequenz, die mit dem Gegenstrang einer Sequenz nach einer der Gruppen a) bis d) hybridisiert oder unter Berücksichtigung der Degeneration des genetischen Codes hybridisieren würde, wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyldiphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• f) einem durch Substitution, Addition, Inversion und/oder Deletion einer oder mehrerer Basen erhaltenen Derivat einer Sequenz nach einer der Gruppen a) bis e), wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• g) einer Sequenz, die der SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07 innerhalb der Degeneration des genetischen Codes entspricht, wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert, sowie
• h) einer Sequenz mit neutralen Sinnmutationen der SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07, wobei diese Sequenz vorzugsweise für ein Enzym kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,

wobei diese Sequenz entweder in das Genom der Zelle integriert ist oder aber auf einem Expressionsvektor lokalisiert ist.According to a particularly preferred embodiment of the cell according to the invention, this cell comprises a DNA sequence selected from the group consisting of:

• a) a sequence according to SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07,
• b) an intron-free sequence which is derived from a sequence according to a) and encodes the same protein or peptide as the sequence according to SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, wherein said intron-free sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator,
• c) a sequence which encodes a protein or peptide comprising the amino acid sequence according to SEQ.ID-No. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ. ID. NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 comprises
• d) a sequence which is identical to a sequence according to a) to c) at least 80%, preferably at least 85%, particularly preferably at least 90%, moreover preferably at least 95% and most preferably at least 99% identical which sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator,
• e) a sequence which would hybridise to the opposite strand of a sequence according to one of the groups a) to d) or would hybridize in consideration of the degeneracy of the genetic code, this sequence preferably coding for an enzyme which comprises the transfer of an isopentenyl diphosphate unit to a catalyses allylic diphosphate initiator,
• f) a derivative of a sequence according to one of the groups a) to e) obtained by substitution, addition, inversion and / or deletion of one or more bases, this sequence preferably coding for an enzyme which comprises the transfer of an isopentenyl diphosphate unit catalyzes an allylic diphosphate initiator,
• g) a sequence corresponding to SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07 within the degeneracy of the genetic code, which sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator, as well as
• h) a sequence with neutral sense mutations of SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, which sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator,

this sequence being either integrated into the genome of the cell or localized on an expression vector.

In addition to the increase in the activity E ₁ in a cell according to the invention or in addition to the expression of one or more of the proteins which are represented by the gene sequences according to SEQ ID NO. 01 to SEQ ID NO. 07, especially in a microorganism such as a bacterial or yeast cell, it may also be preferred according to the invention to increase the activity of one or more enzymes responsible for the formation of the C ₅ precursor compounds necessary for isoprenoid formation.

• – eines Enzyms E₂, welches die Umsetzung von Acetoacetyl-Coenzym A zu 3-Hydroxy-3-methylglutaryl-Coenzym A katalysiert;
• – eines Enzyms E₃, welches die Umsetzung von 3-Hydroxy-3-methylglutaryl-Coenzym A zu Mevalonat katalysiert;
• – eines Enzyms E₄, welches die Umsetzung von Mevalonat zu Mevalonat-5-phosphat katalysiert;
• – eines Enzyms E₅, welches die Umsetzung von Mevalonat-5-phosphat zu Mevalonat-5-diphosphat katalysiert;
• – eines Enzyms E₆, welches die Umsetzung von Mevalonat-5-diphosphat zu Isopentenyl-diphosphat katalysiert.

In this connection, according to a first variant of the cell according to the invention, it is preferred that these are particularly preferred in addition to increasing the activity of an enzyme involved in the production of isoprenoids, preferably in the preparation of polyisoprenes having the aforementioned number of carbon atoms besides increasing the activity of the enzyme E ₁ , besides increasing the activity of the enzyme E ₁ and a gum extension protein, besides increasing the activity of the enzyme E ₁ and a gum-binding protein or in addition to increasing the activity of the enzyme E ₁ , a gum extension factor and a gum-binding protein also has an increased activity of at least one of the enzymes E ₂ to E ₆ :

• An enzyme E ₂ which catalyzes the conversion of acetoacetyl-coenzyme A to 3-hydroxy-3-methylglutaryl-coenzyme A;
• An enzyme E ₃ which catalyzes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate;
• An enzyme E ₄ which catalyzes the conversion of mevalonate to mevalonate 5-phosphate;
• An enzyme E ₅ which catalyzes the conversion of mevalonate 5-phosphate to mevalonate 5-diphosphate;
• - An enzyme E ₆ , which catalyzes the conversion of mevalonate-5-diphosphate to isopentenyl diphosphate.

Particularly preferred cells according to the invention are those cells which, in addition to increasing the activity of an enzyme involved in the production of isoprenoids, preferably in the preparation of polyisoprenes having the aforementioned number of carbon atoms, are particularly preferred besides increasing the activity of the enzyme E ₁ , in addition to increasing the activity of the enzyme E ₁ and a gum extension protein, in addition to increasing the activity of the enzyme E ₁ and a gum-binding protein or in addition to increasing the activity of the enzyme E ₁ , a gum Extension factor and a gum-binding protein, the activity of the following enzymes or enzyme combinations is increased: E ₂ , E ₃ , E ₄ , E ₅ , E ₆ , E ₁ E ₂ , E ₁ E ₃ , E ₁ E ₄ , E ₁ E ₅ , E ₂ E ₃ , E ₂ E ₄ , E ₂ E ₅ , E ₂ E ₆ , E ₃ E ₄ , E ₃ E ₅ , E ₃ E ₆ , E ₄ E ₅ , E ₄ E ₆ , E ₅ E ₆ , E ₁ E ₂ E ₃ , E ₁ E ₂ E ₄ , E ₁ E ₂ E ₅ , E ₁ E ₂ E ₆ , E ₁ E ₃ E ₄ , E ₁ E ₃ E ₅ , E ₁ E ₃ E ₆ , E ₁ E ₄ E ₅ , E ₁ E ₄ E ₆ , E ₁ E ₅ E ₆ , E ₂ E ₃ E ₄ , E ₂ E ₃ E ₅ , E ₂ E ₃ E ₆ , E ₂ E ₄ E ₅ , E ₂ E ₄ E ₆ , E ₂ E ₅ E ₆ , E ₃ E ₄ E ₅ , E ₃ E ₄ E ₆ , E ₃ E ₅ E ₆ , E ₄ E ₅ E ₆ , E ₁ E ₂ E ₃ E ₄ , E ₁ E ₂ E ₃ E ₅ , E ₁ E ₂ E ₃ E ₆ , E ₁ E ₂ E ₄ E ₅ , E ₁ E ₂ E ₄ E ₆ , E ₁ E ₂ E ₅ E ₆ , E ₁ E ₃ E ₄ E ₅ , E ₁ E ₃ E ₄ E ₆ , E ₁ E ₃ E ₅ E ₆ , E ₁ E ₄ E ₅ E ₆ , E ₂ E ₃ E ₄ E ₅ , E ₂ E ₃ E ₄ E ₆ , E ₂ E ₃ E ₅ E ₆ , E ₂ E ₄ E ₅ E ₆ , E ₃ E ₄ E ₅ E ₆ , E ₁ E ₂ E ₃ E ₄ E ₅ , E ₁ E ₂ E ₃ E ₄ E ₆ , E ₁ E ₂ E ₃ E ₅ E ₆ , E ₁ E ₂ E ₄ E ₅ E ₆ , E ₁ E ₃ E ₄ E ₅ E ₆ , E ₂ E ₃ E ₄ E ₅ E ₆ and E ₁ E ₂ E ₃ E ₄ E ₅ E ₆ , wherein E ₁ E ₂ E ₃ E ₄ E ₅ E _{6 is} most preferred.

In this context, it is further preferred that the enzyme
E _{2 is} a hydroxymethylglutaryl-coenzyme A synthase (EC 2.3.3.1.10),
E _{3 is} a hydroxymethylglutaryl-coenzyme A reductase (EC 1.1.1.34),
E ₄ a mevalonate kinase (EC 2.7.1.36),
E _{5 is} a phosphomevalonate kinase (EC 2.7.4.1), and
E _{6 is} a diphosphome valonate decarboxylase (EC 4.1.1.33).

The enzyme E ₂ is preferably encoded by a gene selected from the group consisting of hmgcs1, hmgcs2, hmgs, hcs, hgsA, pksG, mvaS, hmcM, mvaS.1, mvaS2, hmcS, mvaB, mvaB1 and mvaB2. The enzyme E ₃ is preferably from a gene selected from the group consisting of HMGCR, HMG1, HMG2, HMGA, HMGB, mvaA, mvaS.1 encoded mvaA1 and mvaA2. The enzyme E ₄ is preferably encoded by genes selected from the group consisting of mvk, lmbP, mvaK1 and yeaG. Suitable genes for the enzyme E ₅ are selected from the group consisting of pmvk and mvaK2. The enzyme E ₆ is preferably encoded by a gene selected from the group consisting of mvd, mvd1, mvaD and dmd. The nucleotide sequences of the abovementioned genes and of further genes for the enzymes E ₂ to E ₆ can be taken from among others the KEGG database, the NCBI database or the EMBL database.

in the Connection with the above-described, first variant of Cell according to the invention, in which the provision of isopentenyl diphosphate via the mevalonate pathway If desired, it may further be preferred to those enzyme activities in the cell increase, an increase in acetoacetyl coenzyme A deployment. In this context, it may be particular be beneficial to the activity of acetyl coenzyme A-C acetyltransferase (EC 2.3.1.9).

Examples of a cell in which the activity of one or more of the enzymes E ₂ to E _{6 is} increased, for example, the US 2007/0166782 A1 be removed. The recombinant cells described in this patent application can be used as cells in which additionally the activity of an enzyme involved in the production of isoprenoids, preferably in the preparation of polyisoprenes having the aforementioned number of carbon atoms, in particular the activity of the Enzyme E ₁ , the activity of the enzyme Ei and a gum extension protein, the activity of the enzyme E ₁ and a gum-binding protein or the activity of the enzyme E ₁ , a gum elongation factor and a gum-binding protein is increased.

• – eines Enzyms E₇, welches die Umsetzung von D-Glyceraldehyd-3-phosphat und Pyruvat zu 1-Deoxy-D-xylulose-5-phosphat katalysiert;
• – eines Enzyms E₈, welches die Umsetzung von 1-Deoxy-D-Xylulose-5-Phosphat zu 2-C-Methyl-D-erythritol-4-phosphat katalysiert;
• – eines Enzyms E₉, welches die Umsetzung von 2-C-Methyl-D-erythritol-4-phosphat zu 4-(Cytidin-5'-diphospho)-2-C-methyl-D-erythritol katalysiert;
• – eines Enzyms E₁₀, welches die Umsetzung von 4-(Cytidin-5'-diphospho)-2-C-methyl-D-erythritol zu 2-Phospho-4-(cytidin-5'-diphospho)-2-C-methyl-D-erythritol katalysiert;
• – eines Enzyms E₁₁, welches die Umsetzung von 2-Phospho-4-(cytidin-5'-diphospho)-2-C-methyl-D-erythritol zu 2-C-Methyl-D-erythritol-2,4-cyclodiphosphat katalysiert;
• – eines Enzyms E₁₂, welches die Umsetzung von 2-C-Methyl-D-erythritol-2,4-cyclodiphosphat zu 1-Hydroxy-2-methyl-2-butenyl-4-diphosphat katalysiert;
• – eines Enzyms E₁₃, welches die Umsetzung von 1-Hydroxy-2-methyl-2-butenyl-4-diphosphat zu Isopentenyl-diphosphat katalysiert.

According to another variant of the cell according to the invention, it is preferable that besides the increase in the activity of an enzyme involved in the production of isoprenoids, preferably in the production of polyisoprenes having the above-mentioned number of carbon atoms, it is more preferable besides increasing the activity of the enzyme E ₁ , in addition to increasing the activity of the enzyme E ₁ and a gum extension protein, in addition to increasing the activity of the enzyme E ₁ and a gum-binding protein or in addition to increasing the activity of the enzyme E ₁ , a gum Extension factor and a gum-binding protein also has an increased activity of at least one of the enzymes E ₇ to E ₁₃ :

• An enzyme E ₇ which catalyzes the conversion of D-glyceraldehyde-3-phosphate and pyruvate to 1-deoxy-D-xylulose-5-phosphate;
• An enzyme E ₈ which catalyzes the conversion of 1-deoxy-D-xylulose-5-phosphate to 2-C-methyl-D-erythritol-4-phosphate;
• An enzyme E ₉ which catalyzes the reaction of 2-C-methyl-D-erythritol-4-phosphate into 4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol;
• An enzyme E ₁₀ which inhibits the conversion of 4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol to 2-phospho-4- (cytidine-5'-diphospho) -2-C- catalyzes methyl-D-erythritol;
• - An enzyme E ₁₁ , which is the reaction of 2-phospho-4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate catalyzes;
• An enzyme E ₁₂ which catalyzes the conversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl-4-diphosphate;
• - An enzyme E ₁₃ , which catalyzes the reaction of 1-hydroxy-2-methyl-2-butenyl-4-diphosphate to isopentenyl diphosphate.

Particularly preferred cells according to the invention are those cells which, in addition to increasing the activity of an enzyme involved in the production of isoprenoids, preferably in the preparation of polyisoprenes having the aforementioned number of carbon atoms, are particularly preferred besides increasing the activity of the enzyme E ₁ , in addition to increasing the activity of the enzyme E ₁ and a gum extension protein, in addition to increasing the activity of the enzyme E ₁ and a gum-binding protein or in addition to increasing the activity of the enzyme E ₁ , a gum Extension factor and a gum-binding protein, the activity of the following enzymes or enzyme combination is increased: E ₇ , E _8i E ₉ , E ₁₀ , E ₁₁ , E ₁₂ , E ₁₃ , E ₇ E ₈ , E ₇ E ₉ , E ₇ E ₁₀ , E ₇ E ₁₁ , E ₇ E ₁₂ , E ₇ E ₁₃ , E ₈ E ₉ , E ₈ E ₁₀ , E ₈ E ₁₁ , E ₈ E ₁₂ , E ₈ E ₁₃ , E ₉ E ₁₀ , E ₉ E ₁₁ , E ₉ E ₁₂ , E ₉ E ₁₃ , E ₁₀ E ₁₁ , E ₁₀ E ₁₂ , E ₁₀ E ₁₃ , E ₁₁ E ₁₂ , E ₁₁ E ₁₃ , E ₁₂ E ₁₃ , E ₇ E ₈ E ₉ , E ₇ E ₈ E ₁₀ , E ₇ E ₈ E ₁₁ , E ₇ E ₈ E ₁₂ , E ₇ E ₈ E ₁₃ , E ₇ E ₉ E ₁₀ , E ₇ E ₉ E ₁₁ , E ₇ E ₉ E ₁₂ , E ₇ E ₉ E ₁₃ , E ₇ E ₁₀ E ₁₁ , E ₇ E ₁₀ E ₁₂ , E ₇ E ₁₀ E ₁₃ , E ₇ E ₁₁ E ₁₂ , E ₇ E ₁₁ E ₁₃ , E ₇ E ₁₂ E ₁₃ , E ₈ E ₉ E ₁₀ , E ₈ E ₉ E ₁₁ , E ₈ E ₉ E ₁₂ , E ₈ E ₉ E ₁₃ , E ₈ E ₁₀ E ₁₁ , E ₈ E ₁₀ E ₁₂ , E ₈ E ₁₀ E ₁₃ , E ₈ E ₁₁ E ₁₂ , E ₈ E ₁₁ E ₁₃ , E ₈ E ₁₂ E ₁₃ , E ₉ E ₁₀ E ₁₁ , E ₉ E ₁₀ E ₁₂ , E ₉ E ₁₀ E ₁₃ , E ₉ E ₁₁ E ₁₂ , E ₉ E ₁₁ E ₁₃ , E ₉ E ₁₂ E ₁₃ , E ₁₀ E ₁₁ E ₁₂ , E ₁₀ E ₁₁ E ₁₃ , E ₁₀ E ₁₂ E ₁₃ , E ₁₁ E ₁₂ E ₁₃ , E ₇ E ₈ E ₉ E ₁₀ , E ₇ E ₈ E ₉ E ₁₁ , E ₇ E ₈ E ₉ E ₁₂ , E ₇ E ₈ E ₉ E ₁₃ , E ₇ E ₈ E ₁₀ E ₁₁ , E ₇ E ₈ E ₁₀ E ₁₂ , E ₇ E ₈ E ₁₀ E ₁₃ , E ₇ E ₈ E ₁₁ E ₁₂ , E ₇ E ₈ E ₁₁ E ₁₃ , E ₇ E ₈ E ₁₂ E ₁₃ , E ₇ E ₉ E ₁₀ E ₁₁ , E ₇ E ₉ E ₁₀ E ₁₂ , E ₇ E ₉ E ₁₀ E ₁₃ , E ₇ E ₉ E ₁₁ E ₁₂ , E ₇ E ₉ E ₁₁ E ₁₃ , E ₇ E ₉ E ₁₂ E ₁₃ , E ₇ E ₁₀ E ₁₁ E ₁₂ , E ₇ E ₁₀ E ₁₁ E ₁₃ , E ₇ E ₁₀ E ₁₂ E ₁₃ , E ₇ E ₁₁ E ₁₂ E ₁₃ , E ₈ E ₉ E ₁₀ E ₁₁ , E ₈ E ₉ E ₁₀ E ₁₂ , E ₈ E ₉ E ₁₀ E ₁₃ , E ₈ E ₉ E ₁₁ E ₁₂ , E ₈ E ₉ E ₁₀ E ₁₃ , E ₈ E ₉ E ₁₁ E ₁₂ , E ₈ E ₉ E ₁₁ E ₁₃ , E ₈ E ₉ E ₁₂ E ₁₃ , E ₈ E ₁₀ E ₁₁ E ₁₂ , E ₈ E ₁₀ E ₁₁ E ₁₃ , E ₈ E ₁₀ E ₁₂ E ₁₃ , E ₈ E ₁₁ E ₁₂ E ₁₃ , E ₉ E ₁₀ E ₁₁ E ₁₂ , E ₉ E ₁₀ E ₁₁ E ₁₃ , E ₉ E ₁₀ E ₁₂ E ₁₃ , E ₉ E ₁₁ E ₁₂ E ₁₃ , E ₁₀ E ₁₁ E ₁₂ E ₁₃ , E ₇ E ₈ E ₉ E ₁₀ E ₁₁ , E ₇ E ₈ E ₉ E ₁₀ E ₁₂ , E ₇ E ₈ E ₉ E ₁₀ E ₁₃ , E ₇ E ₈ E ₉ E ₁₁ E ₁₂ , E ₇ E ₈ E ₉ E ₁₁ E ₁₃ , E ₇ E ₈ E ₉ E ₁₂ E ₁₃ , E ₇ E ₈ E ₁₀ E ₁₁ E ₁₂ , E ₇ E ₈ E ₁₀ E ₁₁ E ₁₃ , E ₇ E ₈ E ₁₀ E ₁₂ E ₁₃ , E ₇ E ₈ E ₁₁ E ₁₂ E ₁₃ E ₇ E ₉ E ₁₀ E ₁₁ E ₁₂ , E ₇ E ₉ E ₁₀ E ₁₁ E ₁₃ , E ₇ E ₉ E ₁₀ E ₁₂ E ₁₃ , E ₇ E ₉ E ₁₁ E ₁₂ E ₁₃ , E ₇ E ₁₀ E ₁₁ E ₁₂ E ₁₃ E ₈ E ₉ E ₁₀ E ₁₁ E ₁₂ , E ₈ E ₉ E ₁₀ E ₁₁ E ₁₃ , E ₈ E ₉ E ₁₀ E ₁₂ E ₁₃ , E ₉ E ₁₀ E ₁₁ E ₁₂ E ₁₃ , E ₇ E ₈ E ₉ E ₁₀ E ₁₁ E ₁₂ , E ₇ E ₈ E ₉ E ₁₀ E ₁₁ E ₁₃ , E ₇ E ₈ E ₉ E ₁₀ E ₁₂ E ₁₃ , E ₇ E ₈ E ₉ E ₁₁ E ₁₂ E ₁₃ , E ₇ E ₈ E ₁₀ E ₁₁ E ₁₂ E ₁₃ , E ₇ E ₉ E ₁₀ E ₁₁ E ₁₂ E ₁₃ , E ₈ E ₉ E ₁₀ E ₁₁ E ₁₂ E ₁₃ and E ₇ E ₈ E ₉ E ₁₀ E ₁₁ E ₁₂ E ₁₃ , where E ₇ E ₈ E ₉ E ₁₀ E ₁₁ E ₁₂ E ₁₃ most preferred.

In this context, it is further preferred that the enzyme
E _{7 is} 1-deoxy-D-xylulose-5-phosphate synthase (EC 2.2.1.7),
E _{8 is} a 1-deoxy-D-xylulose-5-phosphate reductoisomerase (EC 1.1.1.267),
E _{9 is} a 2-C-methyl-D-erythritol-4-phosphate-cytidylyl transferase (EC 2.7.7.60),
E _{10 is} a 4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol kinase (EC 2.7.1.148),
E _{11 is} a 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (EC 4.6.1.12),
E _{12 is} a 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC 1.17.4.3), and
E _{13 is} a 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2)
is.

The enzyme E ₇ is preferably encoded by a gene selected from the group consisting of dxs, dxs-1, dxs-2, dxsA, dxsB or tktB. The enzyme E ₉ is preferably encoded by a gene selected from the group consisting of ispC, dxr, yaeM, dxr-1, dxr-2 and dxrA. The enzyme E ₉ is preferably encoded by genes selected from the group consisting of ispD, ygbP, ispF, ispDF, yacM and mecT. Suitable genes for the enzyme E ₁₀ are selected from the group consisting of ispF, ychB, ipk, thrB1, thrB, yabH and cmeK. The enzyme E ₁₁ is preferably encoded by a gene selected from the group consisting of ispF, ygbB, ispD, ispDF, yacN, mecS and trmD. Suitable genes for the enzyme E ₁₂ are selected from the group consisting of ispG, gcpE, aarC and yqfY. The enzyme E ₁₃ is preferably encoded by a gene selected from the group consisting of ispH, lytB, ispH-1, ispH-2, lytB1, lytB2 and yqfP. The nucleotide sequences of the abovementioned genes and of other genes for the enzymes E ₇ to E ₁₃ can be taken from among others the KEGG database, the NCBI database or the EMBL database.

Examples of a cell in which the activity of one or more of the enzymes E ₇ to E ₁₃ , but in particular the activity of the enzyme E ₁₂ , is increased, may, for example, be US 2004/0176570 A1 be removed. The recombinant cells described in this patent application can also be used as cells in which additionally the activity of an enzyme involved in the production of isoprenoids, preferably in the preparation of polyisoprenes having the abovementioned number of carbon atoms, in particular the activity of the enzyme E ₁ , the activity of the enzyme E ₁ and a gum extension protein, the activity of the enzyme E ₁ and a gum-binding protein or the activity of the enzyme E ₁ , a gum extender and a gum-binding protein are increased.

A further contribution to achieving the abovementioned objects is made by a method for producing a genetically modified cell comprising the method step of increasing the expression of an enzyme selected from the group consisting of a rubber binding protein, a gum extension factor ("Rubber Enlongation Factor") and a prenyl transferase in the cell. According to a particularly preferred embodiment of this method according to the invention, the method comprises the step of increasing the expression of a prenyl-transferase in the cell, the step of increasing the expression of a prenyl-transferase and a gum-binding protein in the cell, the step of increasing the expression a prenyl-transferase and a gum extension factor in the cell or the step of increasing the expression of a prenyl-transferase, a gum extender and a gum-binding protein in the cell. In addition, the method may additionally comprise the step of increasing one or more of the activities of the enzymes E ₂ to E ₆ or E ₇ to E ₁₃ . In this context, it is furthermore preferred that the increase in the expression of the corresponding enzyme activities takes place by integrating a gene coding for the corresponding enzyme into the genome of the cell or else in the form of an expression vector into the cell is introduced.

Farther it is in connection with the method described above for producing a genetically modified cell, that the cell is a microorganism, in particular is a bacterial or yeast cell, with bacterial or yeast or yeast cells those cells are preferred that already at the beginning in connection with the cell according to the invention have been called.

a further contribution to the solution of the aforementioned tasks continues to perform by the method described above available, genetically modified cell.

• i) in Kontakt bringen einer erfindungsgemäßen Zelle oder einer durch das erfindungsgemäße Verfahren erhältlichen Zelle mit einem Kulturmedium beinhaltend mindestens eine Kohlenstoffquelle unter Bedingungen, unter denen die Zelle aus der Kohlenstoffquelle Isoprenoide zu bilden vermag, welche in definierten Kompartimenten in der Zelle eingeschossen werden;
• ii) Isolierung der in den definierten Kompartimenten eingeschlossenen Isoprenoide.

In particular, a method for the production of isoprenoids, comprising the following method steps, also makes a contribution to the solution of the abovementioned objects:

• i) contacting a cell according to the invention or a cell obtainable by the process according to the invention with a culture medium containing at least one carbon source under conditions in which the cell from the carbon source is capable of forming isoprenoids injected into defined compartments in the cell;
• ii) isolation of the isoprenoids included in the defined compartments.

in the Step i) of the method according to the invention first the cells of the invention or by the method according to the invention containing cells containing a culture medium at least one carbon source is brought into contact under conditions under which the cell from the carbon source to form isoprenoids capable of injecting into defined compartments in the cell become.

The genetically modified cells according to the invention can be brought into contact with the nutrient medium continuously or discontinuously in the batch process (batch culturing) or in the fed-batch process (feed process) or repeated-fed-batch process (repetitive feed process) for the purpose of producing the isoprenoids brought and thus cultivated. Also conceivable is a semi-continuous process, as described in the GB-A-1009370 is described. A summary of known cultivation methods can be found in the textbook by Chmiel ( "Bioprocessing Technology 1. Introduction to Bioprocess Engineering" (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook of Storhas ( "Bioreactors and Peripheral Facilities", Vieweg Verlag, Braunschweig / Wiesbaden, 1994 ).

The culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981) contain.

As a carbon source carbohydrates such. As glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such. As soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such. As palmitic acid, stearic acid and linoleic acid, alcohols such. As glycerol and methanol, hydrocarbons such as methane, amino acids such as L-glutamate or L-valine or organic acids such. As acetic acid can be used. These substances can be used individually or as a mixture. Particular preference is given to the use of carbohydrates, in particular monosaccharides, oligosaccharides or polysaccharides, as described in US Pat US 6,01,494 and US 6,136,576 of C ₅ sugars or glycerol.

When Nitrogen source can be organic nitrogen containing compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, Soybean meal and urea or inorganic compounds such as Ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate are used. The nitrogen sources can used singly or as a mixture.

When Phosphorus source can phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing ones Salts are used. The culture medium must continue to salts of Metals include such. As magnesium sulfate or iron sulfate, the necessary for growth. Finally, you can essential growth factors such as amino acids and vitamins in addition used for the above-mentioned substances. The culture medium In addition, suitable precursors may be added become. The stated feedstocks can be used to culture in Formed as a one-off approach or as appropriate be fed during cultivation.

For pH control of the culture, basic compounds such as sodium hydroxide, potassium hydroxide, Am ammonia or ammonia or acidic compounds such as phosphoric acid or sulfuric acid used in a suitable manner. To control the foam development antifoams such. B. fatty acid polyglycol esters are used. To maintain the stability of plasmids, the medium suitable selective substances such. B. antibiotics are added. In order to maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such. For example, air is introduced into the culture.

The Temperature of the culture is usually more than 20 ° C, preferably at more than 30 ° C, it can also more than 40 ° C, wherein advantageously a cultivation temperature of 95 ° C, more preferably 90 ° C and most preferably 80 ° C is not exceeded.

After this in process step i) a sufficient amount of defined in Compartments including isoprenoids have been formed or after a sufficient amount of cell division, Biomass based on recombinant microorganisms has been obtained is in process step ii) in the defined compartments enclosed isoprenoids isolated. This isolation can the trapped isoprenoids basically on the Manner, usually also in inclusion bodies enclosed, recombinantly produced proteins from microorganisms such as For example, E. coli can be isolated.

• iia) Aufschließen der Zellen unter Erhalt eines Zelllysates, welches die definierten Kompartimente der Zelle umfasst, in denen die Isoprenoide eingeschlossen sind;
• iib) Abtrennen der definierten Kompartimente, in denen die Isoprenoide eingeschlossen sind, aus dem Zelllysat;
• iic) gegebenenfalls Reinigung der abgetrennten, definierten Kompartimente.

According to a preferred embodiment of the process according to the invention, the isolation of the isoprenoids included in the defined compartments comprises the following process steps:

• iia) digesting the cells to obtain a cell lysate comprising the defined compartments of the cell in which the isoprenoids are entrapped;
• iib) separating the defined compartments, in which the isoprenoids are included, from the cell lysate;
• iic) optionally purification of the separated, defined compartments.

In Step iia), the cells are initially under Obtaining a cell lysate containing the defined compartments the cell, preferably the inclusion bodies, comprises in which the isoprenoids are included, open-minded. This Aufschließ can by all the skilled person known methods usually used to unlock Cells are used.

In Consideration is given in particular to the mechanical cell disruption, such as the homogenization in a blender with rotating Knives, for example by means of an Ultra-Turrax, a mechanical digestion middle of the Potter-Elvehjem process, in which a piston, which is tightly encased by a stationary vessel is, moved and by the shear forces generated during this movement a destruction of the cells is effected, a mechanical one Digestion by means of a mortar or in a glass bead mill, one mechanical disruption by means of ultrasound or, in the case of a continuous digestion process, the mechanical digestion for example by means of a Manton-Gaulin homogenizer, in which the cells are pressed under great pressure through a tight valve become. The digestion preferably takes place when using a Manton-Gaulin Homogenizer at a pressure in a range of 1,000 to 50,000 psi, more preferably in a range of 10,000 up to 20,000 psi.

Next A mechanical breakdown of the cells are also non-mechanical Digestion methods, such as the treatment of the cells by repeated freezing and thawing, the treatment of the cells with hypotonic buffer solutions, autolysis with toluene, enzymatic lysis with enzymes such as zymolyase, the treatment of the cells with detergents such as Triton-X-100 or the treatment of cells with complexing compounds, such as with EDTA solutions. Particularly according to the invention preferred is autolysis with organic solvents, especially with halogenated hydrocarbons such as toluene. Such use of organic solvents is especially advantageous because not only the cells are digested can be, depending on the organic solvent, at the same time also the defined compartments of other cell components for example, can be separated by precipitation, so that the method steps iia) and iib) are carried out at the same time can be.

From the aforementioned types of digestion, those are preferred which leads to a disruption of the cells while preserving the cell organelles lead, in particular, a mechanical digestion proved by the Potter Elvehjem method to be advantageous Has. It may be advantageous, the cells initially mittles an Ultra-Turrax and the thus obtained Subsequently, digestion by means of a Manton-Gaulin homogenizer continue to catch up.

in the Process step iib) are the defined compartments, preferably the inclusion bodies in which the isoprenoids are included are separated from the cell lysate. Because these inclusion bodies a great compared to other components of the cell lysate Have density, they can, for example, by high-speed centrifugation, in which the inclusion bodies are obtained as a pellet, be separated from the other components. advantageously, the high-speed centrifugation takes place at a centrifugal acceleration in a range of 1,000 to 20,000 × g, more preferably in a range of 5,000 to 15,000 × g.

The in this way separated defined inclusion bodies are then cleaned in process step iic), wherein this cleaning in particular for the separation of even in the Inclusion bodies contained or from on the surface the inclusion body adhering proteins or other, from the isoprenoids different impurities serves. Preferably This cleaning is done by washing the separated defined Compartments with suitable. Washing solutions. Come as washing solutions especially water or saline buffer solutions in Consideration. It may be advantageous in particular, in the process step iib) separated inclusion bodies also with an enzyme-containing Washing solution, for example an aqueous proteinase solution, to treat proteins present in the inclusion bodies enclosed and / or on the surface of the inclusion bodies stick to remove.

a Contribute to the solution of the problem mentioned above furthermore the isoprenoids obtained by this process, in particular however, the cis-polyisoprenes, trans-polyisoprenes obtainable by this process or mixtures of cis-polyisoprenes and trans-polyisoprenes.

• a) eine Sequenz nach SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07,
• b) eine intronfreie Sequenz, die von einer Sequenz nach a) abgeleitet ist und das gleiche Protein oder Peptid kodiert wie die Sequenz nach SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07, wobei diese intronfreie Sequenz vorzugsweise für ein Protein kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• c) eine Sequenz, die ein Protein oder Peptid kodiert, das die Aminosäure-Sequenz nach SEQ.-ID-Nr. 08, SEQ.-ID-Nr. 09, SEQ.-ID-Nr. 10, SEQ.-ID-Nr. 11, SEQ.-ID-Nr. 12, SEQ.-ID-Nr. 13 oder SEQ.-ID-Nr. 14 umfasst,
• d) eine Sequenz, die mit einer Sequenz nach a) bis c) zu mindestens 80%, vorzugsweise zu mindestens 85%, besonders bevorzugt zu mindestens 90%, darüber hinaus bevorzugt zu mindestens 95% und am meisten bevorzugt zu mindestens 99% identisch ist, wobei diese Sequenz vorzugsweise für ein Protein kodiert, welches die Übertragung einer Isopentenyldiphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• e) eine Sequenz, die mit dem Gegenstrang einer Sequenz nach einer der Gruppen a) bis d) hybridisiert oder unter Berücksichtigung der Degeneration des genetischen Codes hybridisieren würde, wobei diese Sequenz vorzugsweise für ein Protein kodiert, welches die Übertragung einer Isopentenyldiphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• f) ein durch Substitution, Addition, Inversion und/oder Deletion einer oder mehrerer Basen erhaltenes Derivat einer Sequenz nach einer der Gruppen a) bis e), wobei diese Sequenz vorzugsweise für ein Protein kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• g) eine Sequenz, die der SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07 innerhalb der Degeneration des genetischen Codes entspricht, wobei diese Sequenz vorzugsweise für ein Protein kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert,
• h) eine Sequenz mit neutralen Sinnmutationen der SEQ.-ID-Nr. 01, SEQ.-ID-Nr. 02, SEQ.-ID-Nr. 03, SEQ.-ID-Nr. 04, SEQ.-ID-Nr. 05, SEQ.-ID-Nr. 06 oder SEQ.-ID-Nr. 07, wobei diese Sequenz vorzugsweise für ein Protein kodiert, welches die Übertragung einer Isopentenyl-diphosphat-Einheit auf einen allylischen Diphosphat-Initiator katalysiert, sowie
• i) eine komplementäre Sequenz zu einer Sequenz nach einer der Gruppen a) bis h).

A contribution to the solution of the problem mentioned at the outset continues to be provided by an isolated nucleic acid whose sequence is selected from the following sequences:

• a) a sequence according to SEQ.-ID-Nr. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07,
• b) an intron-free sequence which is derived from a sequence according to a) and encodes the same protein or peptide as the sequence according to SEQ.ID-No. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, wherein said intron-free sequence preferably encodes a protein which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator,
• c) a sequence which encodes a protein or peptide having the amino acid sequence according to SEQ.ID-No. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ. ID. NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 comprises
• d) a sequence which is identical to a sequence according to a) to c) to at least 80%, preferably to at least 85%, particularly preferably to at least 90%, moreover preferably to at least 95% and most preferably at least 99% identical this sequence preferably encodes a protein which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator,
• e) a sequence which would hybridise with the opposite strand of a sequence according to any one of groups a) to d) or would hybridize in consideration of the degeneracy of the genetic code, this sequence preferably coding for a protein which comprises the transfer of an isopentenyl diphosphate unit to one catalyses allylic diphosphate initiator,
• f) a derivative obtained by substitution, addition, inversion and / or deletion of one or more bases of a sequence according to one of the groups a) to e), this sequence preferably coding for a protein which comprises the transfer of an isopentenyl diphosphate unit catalyzes an allylic diphosphate initiator,
• g) a sequence corresponding to SEQ.ID.NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07 within the degeneracy of the genetic code, which sequence preferably encodes a protein which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator,
• h) a sequence with neutral sense mutations of SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, wherein said sequence preferably encodes a protein which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator, and
• i) a complementary sequence to a sequence according to one of the groups a) to h).

Surprisingly, it was found that overexpression of one or more of these genes with SEQ. ID. NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07 from the Russian dandelion in microorganisms, such as E. coli cells, to results in these cells now being able to form isoprenoids included in inclusion bodies. These genes can therefore be used individually or in combination with one another in order to genetically modify cells, in particular microorganisms, in such a way that these cells can be used for the targeted production of isoprenoids.

The Isolation of these genes was done by first RNA for RT-PCR (reverse transcription polymerase Chain Reaction ") from the latex of T. kok-saghyz has been. Using appropriate primers, the DNA sequences were then amplified. Details can be found in the examples.

a Contribute to the solution of the above-mentioned tasks furthermore a vector, preferably an expression vector, comprising a DNA having a sequence according to any one of groups a) to h), such as defined above. As vectors all known to the expert Vectors are usually considered for introduction be used by DNA in a host cell. Preferred vectors are selected from the group comprising plasmids, such as such as the E. coli plasmids pTE13, pTrc99A, pBR345 and pBR322, viruses, such as bacteriophages, adenoviruses, vaccinia viruses, baculoviruses, Measles viruses and retroviruses, cosmids or YACs, with plasmids as Vectors are most preferred.

According to one preferred embodiment of the invention Vector is the DNA with a sequence according to one of the groups a) to h) under the control of a regulatable promoter, which for expression of the polypeptide encoded by these DNA sequences in the cell of a microorganism, preferably a bacterial, Yeast or Pil cell, more preferably a yeast cell, suitable is. Examples of such promoters are about the trp promoter or the tac promoter.

Of the Vector of the invention should be in addition to a promoter preferably a ribosome binding site and a terminator include. It is particularly preferred that the inventive DNA into an expression cassette of the vector comprising the promoter, the ribosome binding site and the terminator is incorporated. Next The above-mentioned structural elements may be the vector furthermore comprise selection genes known to the person skilled in the art.

a Contribute to the solution of the tasks mentioned above the use of the vector described above for Transformation of a cell as well as transformation by this vector obtained cell. The cells which with the inventive Vector can be transformed are preferably those Cells, which are already considered at the beginning as preferred according to the invention Cells have been described.

a Contribute to the solution of the above-mentioned tasks furthermore, an isolated polypeptide containing the amino acid sequence with the SEQ ID NO. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ. ID. NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14, or one Amino acid sequence that has an identity of at least 50%, preferably at least 55%, moreover preferred at least 60%, moreover at least 65% and most preferably at least 70% to the amino acid sequence according to SEQ.-ID-No. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ. ID. NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 has.

The The invention will now be described by way of non-limiting examples and drawings explained in more detail.

1 the electron micrograph of an E. coli Rosetta gami B (DE3) plysS pET23a cell as control.

2 Electron micrographs of an E. coli origami (DE3) plysS pET23a :: srpp3-tk cell according to the invention, in which a Prenyl-transferase from T. kok-saghyz was overexpressed.

3 Electron micrographs of an E. coli Rosetta gami B (DE3) plysS pET23a :: hrt2-jk cell according to the invention, in which the activity of a cis-1,4-prenyltransferase from Hevea brasiliensis was overexpressed.

4 Electron micrographs of an E. coli Rosetta gami B (DE3) plysS pETdUET-1 :: hrt2-jk :: srpp3-tk cell according to the invention, in which the activity of a cis-1,4-prenyltransferase from Hevea brasiliensis and a T. kok-saghyz prenyltransferase were overexpressed together.

In the cytoplasm of the cell, which in the 2 to 4 is reproduced, defined compartments can be seen in which isoprenoids are included.

5 the prenyl-transferase activity of the enzymes derived from the genes of SEQ. ID. NO. 01 and SEQ ID NO. 07 are coded. Also shown is the prenyl-transferase activity of prenyltransferase from Hevea brasiliensis. The determination takes place in a cell-free extract, where "K" is the cell-free crude extract from Rosettagami B (DE3) plysSpET23a, "1" is the cell-free crude extract from E. coli Rosetta-gami B (DE3) plysS (pET23a :: cpt1-tk), "2" of the cell-free extract of E. coli origami (DE3) pLysS (pET23a :: hrt2-jk), "3" of the cell-free extract of E. coli Rosetta-gami B (DE3) plysS (pEXP5CT :: srpp3-tk) and "4" is a mixture of cell-free crude extract from E. coli Rosetta gami B (DE3) plysS (pET23a :: cpt1-tk) and cell-free crude extract from E. coli Rosetta gami B (DE3) plysS (pEXP5CT :: srpp3- tk) is.

Examples

Isolation of the genes with the sequences according to SEQ.-ID-No. 01 to 07

For the isolation of the genes with the sequences according to SEQ.-ID-No. 05, SEQ ID NO. 06 and SEQ ID NO. 07 (hereafter referred to as srpp1-tk, srpp2-tk and srpp3-tk) have each been specific forward and reverse primers of sequences of a cDNA library which is now publicly available ( http://plantta.tigr.org/ ) (SEQ ID NO: 15 to SEQ ID NO: 20). For the isolation of the genes with the sequences according to SEQ.-ID-No. 01, SEQ ID NO. 02, SEQ ID NO. 03 and SEQ ID NO. 04 (hereinafter called cpt1-tk, cpt2-tk, cpt3-tk and cpt4-tk), a degenerate primer (SEQ.ID.NO.:21) was derived from other organisms using a conserved region of cpts.

to Amplification of the genes was RNA for RT-PCR ("Reverse Transcription Polymerase Chain Reaction ") from the Milk Juice (Latex) isolated from T. kok-saghyz.

For this purpose, 100 .mu.l of latex in 100 .mu.l RNA homogenization buffer (4 M guanidinium isothiocyanate, 100 mM Tris / HCl, pH 7) were added, mixed with 800 .mu.l TriFast ^® (company PegLab) and according to the manufacturer. 2 μg of the total RNA were inserted into the RT and were purified by means of the reverse transcriptase "Super Script II" (Invitrogen) using the primers srpp-TK1-3 rev (SEQ.-ID No. 16, SEQ. ID 18 and SEQ.ID.NO.:20 or the primer "adapter-dT" (SEQ ID NO: 22) were transcribed into single-stranded cDNA. No. 15, SEQ ID No. 17, SEQ ID No. 19, and SEQ ID No. 21) with the primer "adapter" (SEQ ID No. 23), so full length cDNAs were obtained for the srpp genes and partial cDNA fragments for the cpt genes. "To complete the cpt sequences, the" Genome Walker Universal Kit (Clontech) "with the primer" cpt-revGW "( SEQ.ID.NO.:24), the sequence of which was derived from the partial cDNA fragment, performed an extension in the 5 'direction to give a total of four different cpt genes, the full length of which were cDNAs containing the specific primers (SEQ. ID No. 25 to S EQ.-ID No. 30) was amplified.

Production of expression vectors

a) Preparation of the expression vector pEXP5CT :: srpp3-tk (includes the small gum-binding protein from Taraxacum kok-saghyz (Russian dandelion)

The cloning of the gene srpp3-tk into the bacterial T7 expression vector pEXP5CT was used for the future expression of a C-terminal 6 × His fusion protein (Srpp3-tkCT 6 × His). This fusion protein should facilitate or facilitate future purification and immunodetection of the small rubber particle protein. The principle of the TOPO vector pEXP5CT is based on the covalent linkage of a Taq polymerase amplified PCR product with the linearized target vector by a vector-bound topoisomerase. For this purpose, the 717 bp fragment was amplified by PCR using the oligonucleotides Srpp3_TOPO_start_5 'and Srpp3_TOPO_nostop_3' (SEQ ID NO: 31 and SEQ ID NO: 32). Here, the Taq DNA polymerase was used to generate 3'-A overhangs. The 5 'primer did not generate any further interfaces and was identical to the first sequence segment. The 3 'primer used instead removed the stop codon (TGA) to allow complete reading of the vectorially encoded hexahistidine fusion pot. The 714 bp truncated srpp3-tk fragments obtained after the PCR were separated in an agarose gel, excised, and purified for further use. This was followed by ligation into the topo vector pEXP5CT. Due to the high ligation efficiency of the topoisomerase vector system, a ligation efficiency of 90% could be observed. From selected colonies following transformation into E. coli TOP10 on LB ampicillin plates plasmid DNA was isolated and sequenced with the primers TOPO Fw and TOPORev (SEQ ID NO: 33 and SEQ ID NO: 34). A positive E. coli TOP10 clone and its prepared pEXP5CT :: srpp3-tk plasmids were finally used for further investigations, transformed into various DE3 expression strains and preserved.

b) Preparation of the expression vector pET23a :: hrt2-jk (includes the cis-1,4-prenyltransferase from Hevea brasiliensis)

to Production of the expression vector pET23a :: hrt2-jk became the codon usage of cis-1,4-prenyltransferase hrt2 from Hevea brasiliensis optimized for heterologous expression in E. coli on the computer and a new gene (hrt2-jk) with identical amino acid sequence to hrt2 synthesized (Genscript Corp., USA). additionally were common restriction sites for further cloning from the optimized nucleotide sequence calculated out to allow a simple sub-cloning. As flanking restriction sites were NdeI and EcoRI selected. The interfaces NdeI and EcoRI enabled a direct subcloning into the vector pET23a. The synthetic one Gene hrt2-jk was delivered in the bacterial cloning vector pUC57. Starting from the plasmid pUC57 :: hrt2-jk was a restriction digest carried out with the enzymes NdeI and EcoRI and the structural gene in an agarose gel for further use. To the Target vector pET23a was prepared for ligation also this with the enzymes NdeI and EcoRI completely digested and purified in an agarose gel. There followed a ligation of structural gene and target vector. Positive clones, or its prepared pET23a :: hrt2-jk plasmids were finally complete sequenced, for further investigations transformed into different expression strains and preserved.

c) Preparation of the expression vector pET23a :: cpt1-tk (comprises a taraxacum cis-1,4-prenyltransferase kok-saghyz)

The Cloning of the gene for the cis-1,4-prenyltransferase cpt1-tk in the vector pET23a was used to express the gene in the native Protein used. Starting from a bacterial intermediate cloning vector (pCRII-TOPO :: cpt1-tk) with the gene cpt1-tk, was the one for this coding gene section for the generation of suitable restriction sites amplified by polymerase chain reaction (PCR). For this purpose were the 927 bp DNA fragment using the oligonucleotides Cpt VspI 5 'and Cpt EcoRI 3' (SEQ.ID.NO.:35 and SEQ.ID.NO.:36) amplified by PCR. Here, the Pfx-DNAPolymerase was used, which has a proof reading function. The 5 'primer generated before the start codon already contained in the gene, the restriction site for VspI. The use of VspI enabled after digestion with the enzyme of the same name presence of one compatible overhang to NdeI-digested restriction sites in the destination vector. The use and generation of this interface became necessary because cpt1-tk already had an NdeI recognition site. The used 3 'primer added to the gene termination sequence (TAA) a restriction site for EcoRI. The after the PCR obtained. DNA fragments of one size of 950 bp were separated in an agarose gel and used for the further use purified. The purified PCR fragments digested by the restriction enzymes VspI and EcoRI. The target vector pET23a was previously isolated, purified and purified from E. coli cells the restriction endonucleases NdeI and EcoRI overnight digested. The linearized vector fragment became final still on an agarose gel purified and linearized in the Vector pET23a ligated. By using two different Interfaces for the restriction endonucleases VspI and EcoRI became the desired orientation of the fragment inevitably ensured in the vector. After the ligation were permanently competent E. coli TOP10 cells with the ligation approaches transformed and streaked on ampicillin-containing LB agar plates. A check of the receiving clones on carriers of the cpt1-tk gene was carried out by a plasmid isolation and a subsequent digestion with the enzymes XbaI and EcoRI. Of the Use of XbaI as the 5 'control site in front of the gene in the vector pET23a became necessary because of the ligation of the vectorially digested Interface NdeI with the compatible VspI overhang lost the digestion of the cpt1-tk insert. Positive clones, or its prepared pET23a :: cpt1-tk plasmids were final completely sequenced, for continuing Studies in expression strains transformed and conserved.

d) Preparation of the expression vector pETDuet1 :: hrt2-jk :: srpp3-tk (comprises a cis-1,4-prenyltransferase Hevea brasiliensis and the small gum-binding protein from Taraxacum kok-saghyz)

The cleavage of srpp3-tk in the pET23a vector enabled subcloning of the entire gene into the MCS 2. For this, the purified expression plasmid pET23a :: srpp3-tk was digested with the restriction enzymes NdeI and XhoI and purified. With the restriction enzymes of the same name and the identical procedure, the vector pETDuet-1 was treated and subsequently ligated together with the previously restricted by restriction structural gene srpp3-tk to obtain the vector pETDuet-1 :: srpp3-tk.

By those already chosen in the construction of the plasmid pET23a :: hrt2-jk Interfaces, it was also possible to complete the Gene hrt2-jk with ribosomal binding site in the multiple cloning site 1 (MCS1) of the vector pETDuet-1 :: srpp3-tk. Therefor was the purified plasmid pET23a :: hrt2-jk with XbaI and EcoRI digested and the gene fragment purified. The one with the same restriction enzymes restricted vector pETDuet-1 :: srpp3-tk was subsequently added with the gene hrt2-jk to obtain the expression vector pETDuet1 :: hrt2-jk :: srpp3-tk ligated.

Transformation of cells

First, competent E. coli cells were prepared. For this purpose, the E. coli strains to be transformed, starting from a preculture in 50 ml of LB medium to an OD _{600 nm} of 0.3 to 0.5 at 37 ° C on a rotary shaker (Controlled environment incubator shaker, New Brunswick Scientific Co. Inc., Edison, USA) and harvested sterile in 10 ml batches by centrifugation at 3500 rpm for 10 minutes at 4 ° C. The sedimented cells were taken up in 5 ml of an ice-cold 0.1 M CaCl ₂ solution and incubated on ice for 10 min. The centrifugation was repeated under the same conditions. The pellet was then resuspended in 0.5 to 1 ml of 0.1 M CaCl ₂ solution and the cells were stored on ice until transformation, but for a maximum of 24 hours.

Of the Transfer of DNA into E. coli cells was by transformation the competent cells obtained above. 200 μl each competent E. coli cells were given 50-250 μg the expression vectors well mixed. For adsorption of the DNA on the surface, the cells were incubated for 30 min incubated on ice. Heat shocked the cells for exactly 90 seconds at 42 ° C and a short cooling on ice for 2-5 min was the DNA from the cells added. For the regeneration of cells and expression the plasmid-encoded antibiotic resistance were the transformation approach 600 .mu.l LB medium added sterile and the cells for Incubated at 37 ° C for 60-90 min. Finally aliquots of 70-200 μl were plated on selective agar and to isolate the recombinant clones overnight Cultured at 37 ° C. The control was a batch without DNA carried.

• 1. E. coli Origami (DE3)plysS pET23a::srpp3-tk
• 2. E. coli Rosetta-gami B(DE3)plusS pEP23a::hrt2-jk
• 3. E. coli Rosetta-gami B(DE3)plysS pETdUET-1::hrt2-jk::srpp3-tk
• 4. E. coli Rosetta-gami B(DE3)plysS pET23a::cpt1-tk
• 5. E. coli Rosetta-gamiB(DE3)plysS pEXP5CT::srpp3-tk

By the procedure described above, the following recombinant cells were obtained:

• 1. E. coli origami (DE3) plysS pET23a :: srpp3-tk
• 2. E. coli Rosetta gami B (DE3) plus S pEP23a :: hrt2-jk
• 3. E. coli Rosetta gami B (DE3) plysS pETdUET-1 :: hrt2-jk :: srpp3-tk
• 4. E. coli Rosetta gami B (DE3) plysS pET23a :: cpt1-tk
• 5. E. coli Rosetta gamiB (DE3) plysS pEXP5CT :: srpp3-tk

Electron microscopic analyzes

For the electron microscopic examinations of the produced recombinant E. coli cells with respect to intracellular occurring Inclusion bodies and morphological changes these were, unless otherwise specified, cultivated in LB medium, washed and harvested by centrifugation. A fixation of Cells were both treated / resuspended at 0.5%. (w / v) glutaraldehyde in Sörensen phosphate buffer as well by 1% (w / v) osmium tetroxide. Following were the preparations drained, embedded in spurr resin and by means of a Cut diamond cutters into approx. 70 nm thick sections. The at the preparation performed embedding and Cutting steps were carried out in the working group of Prof. R. Reichelt, Institute of Medical Physics and Biophysics of the Westphalian Wilhelms University Münster. The transmission electronic Recordings on a Hitachi H500 (Hitachi Ltd. Corporation, Tokyo, Japan) became self-sufficient at an acceleration voltage of 75 kV carried out.

Assay for determination of prenyltransferase activity in cell-free crude extracts

To assess the functional activity of prenyltransferases, a latex particle-based enzyme assay was established and performed. Cell-free crude extracts of recombinant E. coli cells were used in this enzyme assay. For this purpose, cells of E. coli were grown in LB medium, at 37 ° C. in a rotary shaker (New Brunswick Scientific Co. Inc., Edison, USA) and cultivated at 37 ° C. overnight. A suitable antibiotic was also added to the media. Erlenmeyer flasks were used for cultivation in liquid media, the volume ratio of vessel to liquid being 10: 1 to 5: 1. Precultures were prepared from a pure culture on solid medium in test tubes with 5 ml LB medium or Erlenmeyer flask each with 30 ml LB liquid medium. Well-grown pre-cultures were used for inoculation of the main cultures, with the main culture inoculum corresponding to 0.1-5% of the pre-culture volume. For this purpose, a single colony of E. coli cells was used as Impfgut and usually cultured at 37 ° C until reaching an optical density (OD600 nm) of 0.7. Subsequently, the cultures were induced with 1 mM IPTG for 3 h at 37 ° C. The use of Velcro piston allowed a comfortable checking of the optical density (OD). Using a Klett-Summerson colorimeter (Filter No. 54, Manostat Corporation Cat. No. 76-550-220, USA), the OD of the culture was monitored at 520-580 nm. Alternatively, cell growth was determined by measuring the OD with a photometer (Ultrospec 2000 UV / Visible Spectrophotometer, Pharmacia Biotech, Freiburg, Germany) at a wavelength of 600 nm. The reference was sterile medium. Samples with an OD600 nm> 0.3 were appropriately diluted with medium and measured again. Subsequently, the cell harvest took place. For cell harvesting, up to 50 ml of cell suspension were each aliquoted into 50 ml Falcontubes and harvested by centrifugation for 15-30 min at 3500 × g and 4 ° C. From larger culture volumes, the cells were harvested by centrifugation in a Sorvall RC-5B centrifuge (Du Pont de Nemours, Newton, Conn., USA) at 6,000 rpm (Rotor GS3) at 4 ° C for 20 min. The cell disruption by French-Press was carried out by passing three times through an ice-cooled French Press cell (Amicon, Silver Spring, Maryland, USA) at a pressure of 130 MPa. Cell-free crude extracts were recovered by centrifugation for 20 min at 13,000 x g in a refrigerated centrifuge at 4 ° C. The crude extract thus obtained was stored on ice until use. The determination of the protein concentration in aqueous solution was based on the colorimetric protein determination according to BRADFORD 1976. The determination of the protein concentration in aqueous solution based on the formation of a dye-protein complex in which the absorption maximum of the dye is shifted from 465 nm to 595 nm. For the test, 20 μl sample were mixed with 980 μl Bradford reagent (see below) and incubated for 10 min. protected from light at room temperature. Subsequently, the absorbance at a wavelength of 595 nm was measured with the Ultraspec III® photometer (Pharmacia LKB, Uppsala, Sweden) against the blank value of the corresponding buffer used. As a reference, a measurement with bovine serum albumin (BSA) in the range of 100-900 μg protein ml-1 was performed and a calibration line was prepared. If the absorbance of a sample was above the calibration range, it was diluted with the appropriate buffer. In this case, the buffer components in the concentration used have no negative influence on the test. Bradfordreagenz Serva Blue G 70 mg Ethanol (96%, v / v) 50 ml Phosphoric acid (85%, v / v) 100 ml H2O _bidist . ad. 1,000 ml The solution was filtered before use.

By determining the protein concentrations, defined protein concentrations could be used for the subsequent enzyme test. The latex particle-based enzyme assay used here is for the radiometric measurement of prenyltransferase activity. The ¹⁴ C-IPP monomers are successively added to existing polyisoprene units of the added purified latex particles from T. kok-saghyz by a condensation reaction in the form of a chain extension reaction. Subsequent to the reaction, extraction of both short- and long-chain polyisoprenes covers the radiolabeled polyisoprene. The radioactivity of these fractions can be quantified by scintillation counting. The following is the approach for the radiometric determination of prenyltransferase activity. 20 μg crude extracts of the following E. coli strains were always used for the enzyme test: K, control batch without addition of external prenyltransferases, 1 20 μg cell-free crude extract from E. coli Rosetta gami B (DE3) plysS (pET23a :: cpt1-tk) , 2.20 μg cell-free crude extract from E. coli origami (DE3) pLysS (pET23a :: hrt2-jk), 3.20 μg cell-free crude extract from E. coli Rosetta gami B (DE3) plysS (pEXP5CT :: srpp3-tk ) 4 10 μg cell-free crude extract from E. coli Rosetta gami B (DE3) plysS (pET23a :: cpt1-tk) and 10 μg cell-free crude extract from E. coli Rosetta gami B (DE3) plysS (pEXP5CT :: srpp3- tk). Test for the radiometric measurement of prenyltransferase activity Reaction mixture (see below) 100 μl Farnesyl diphosphate 0.6 (mM) 5 μl ¹⁴ C-isopentenyl pyrophosphate 1.85 GBq mmol ^-1, 1.85 MBq ml ^-1 6 μl

cell-free crude extract × μl T. kok saghyz latex particles (washed) 20 mg H2O _bidist . ad 200 μl reaction Tris 100 mM KCl 60 mM MgCl ₂ 4 mM ZnCl ₂ 10 mM dithiothreitol 10 mM KF 40 mM Desoxychelat 0.2 mM, pH 7.4

Of the Reaction batch was in 1.5 ml reaction vessels pipetted, incubated for 4 h at 30 ° C and then extracted with 0.6 ml of 1-butanol. To this was added after addition of 1-butanol the reaction mixture on a vortex shaker of the type VV3 (VWR International GmbH, Darmstadt, Germany) with matching Reaction vessel attachment for 15 minutes shaking vigorously at room temperature. Subsequently centrifugation was carried out at 14,000 × g. They are now The top 1-butanol phase became cautious without damage taken from the middle protein layer and directly into a scintillation vial with 3 ml IRGASAFE plus scintillation cocktail (Zinsser Analytic GmbH, Frankfurt, Germany). The rest aqueous phase and the protein layer were washed 2 times 0.6 ml of toluene / hexane (mixing ratio 1: 1) to the same Way extracted.

Here, the combined toluene / hexane extracts were also fed to a scintillation vial with 3 ml IRGASAFE plus scintillation cocktail and finally the decay rate (decays per minute, DPM) was measured in a scintillation counter (LS 6500). The result of this enzyme text is illustrative of the enzymes encoded by the genes hrt2-jk, cpt1-tk (SEQ ID NO: 01) and srpp3-tk (SEQ ID NO: 07) in the 5 shown.

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

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://plantta.tigr.org/ [0090]

Claims (21)

A cell opposite to its wild type was genetically engineered to be in defined compartments enclosed isoprenoids can form. The cell of claim 1, wherein the cell is compared to their wild type more included in the defined compartments Isoprenoids can form. The cell of claim 1 or 2, wherein the cell is selected from the group consisting of bacterial cells and yeast cells. The cell according to any one of the preceding claims, wherein the isoprenoid is a polyisoprene. The cell of claim 4, wherein the polyisoprene Poly-cis-isoprene, poly-trans-isoprene or a mixture of poly-cis-isoprene and poly-trans isoprene. Cell according to one of the preceding claims, wherein in the cell, the expression of at least one enzyme which in the preparation of polyisoprenes is particularly preferred the expression of an enzyme selected from the group consisting from a rubber binding protein, a Rubber elongation factor ("rubber elongation factor") and a prenyltransferase compared to the wild type is. The cell of any one of the preceding claims, wherein the cell has an increased activity of an enzyme E ₁ , enhanced compared to its wild type, which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator. A cell according to claim 7, wherein the enzyme E _{1 is} a prenyltransferase, preferably a cis-1,4-prenyltransferase. The cell of claim 8, wherein the prenyltransferase is encoded by a DNA selected from the following sequences: a) a sequence according to SEQ.-ID-Nr. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, b) an intron-free sequence generated by derived from a sequence according to a) and the same protein or Peptide encoded as the sequence of SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07 c) a sequence containing a protein or Coded peptide containing the amino acid sequence according to SEQ.-ID-No. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ. ID. NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 comprises d) a sequence, those with a sequence according to a) to c) at least 80% identical is e) a sequence associated with the opposite strand of a sequence hybridized according to one of the groups a) to d) or taking into account would hybridize to the degeneration of the genetic code, f) by substitution, addition, inversion and / or deletion of a or more bases derived derivative of a sequence according to a of groups a) to e), g) a sequence corresponding to SEQ.ID.NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07 within the degeneration corresponds to the genetic code, h) a sequence with neutral Sense mutations of SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, as well i) a complementary sequence to a sequence after one the groups a) to h). The cell according to any one of claims 7 to 9, wherein in addition to the activity of the enzyme E ₁ in the cell, the activity of at least one of the enzymes E ₂ to E _{6 is} increased: - an enzyme E ₂ , which is the reaction of acetoacetyl-coenzyme A catalysed to 3-hydroxy-3-methylglutaryl-coenzyme A; An enzyme E ₃ which catalyzes the conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate; An enzyme E ₄ which catalyzes the conversion of mevalonate to mevalonate 5-phosphate; An enzyme E ₅ which catalyzes the conversion of mevalonate 5-phosphate to mevalonate 5-diphosphate; An enzyme E ₆ which catalyzes the conversion of mevalonate 5-diphosphate to isopentenyl diphosphate Siert. The cell according to claim 10, wherein the enzyme E _{2 is} a hydroxymethylglutaryl-coenzyme A synthase (EC 2.3.3.1.10), E _{3 is} a hydroxymethylglutaryl-coenzyme A reductase (EC 1.1.1.34), E _{4 is} a mevalonate kinase ( EC 2.7.1.36), E _{5 is} a phosphomevalonate kinase (EC 2.7.4.1), and E _{6 is} a diphosphomevalonate decarboxylase (EC 4.1.1.33). The cell according to any one of claims 7 to 9, wherein in addition to the activity of the enzyme E ₁ in the cell, the activity of at least one of the enzymes E ₇ to E _{13 is} increased: an enzyme E ₇ , which is the reaction of D-glyceraldehyde 3-phosphate and pyruvate catalyzed to 1-deoxy-D-xylulose-5-phosphate; An enzyme E ₈ which catalyzes the conversion of 1-deoxy-D-xylulose-5-phosphate to 2-C-methyl-D-erythritol-4-phosphate; An enzyme E ₉ which catalyzes the reaction of 2-C-methyl-D-erythritol-4-phosphate into 4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol; An enzyme E ₁₀ which inhibits the conversion of 4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol to 2-phospho-4- (cytidine-5'-diphospho) -2-C- catalyzes methyl-D-erythritol; - An enzyme E ₁₁ , which is the reaction of 2-phospho-4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate catalyzes; An enzyme E ₁₂ which catalyzes the conversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl-4-diphosphate; - An enzyme E ₁₃ , which catalyzes the reaction of 1-hydroxy-2-methyl-2-butenyl-4-diphosphate to isopentenyl diphosphate. The cell according to claim 12, wherein the enzyme E _{7 is} a 1-deoxy-D-xylulose-5-phosphate synthase (EC 2.2.1.7), E _{8 is} a 1-deoxy-D-xylulose-5-phosphate reductoisomerase (EC 1.1.1.267), E _{9 is} a 2-C-methyl-D-erythritol-4-phosphate cytidylyl transferase (EC 2.7.7.60), E _{10 is} a 4- (cytidine-5'-diphospho) -2-C-methyl- D-erythritol kinase (EC 2.7.1.148), E ₁₁ a 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (EC 4.6.1.12), E ₁₂ a 4-hydroxy-3-methylbutyl 2-en-1-yl diphosphate synthase (EC 1.17.4.3), and E _{13 is} a 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2). A process for producing a genetically engineered modified cell, comprising the step of the Increasing the expression of an enzyme selected from the group consisting of a gum-binding protein ("rubber binding protein "), a rubber extension factor (" rubber Enlongation factor ") and a prenyl transferase in the cell. The method of claim 14, wherein the cell is selected from the group consisting of bacterial cells and yeast cells. A cell, obtainable by a process after 14 or 15. A process for the preparation of isoprenoids, comprising the following method steps: i) bring into contact A cell according to any one of claims 1 to 13 or 16 a culture medium containing at least one carbon source under conditions in which the cell from the carbon source isoprenoids which can form in defined compartments in the cell be shot; ii) isolation of those defined in Compartments include isoprenoids. The method of claim 17, wherein the method step ii) comprises the following method steps; iia) unlock the cells to obtain a cell lysate, which defined Compartments of the cell includes, in which the isoprenoids included are; iib) separating the defined compartments in which the isoprenoids are included in the cell lysate; iic) if appropriate, purification of the separated, defined compartments. Isoprenoids, obtainable by a process according to claim 17 or 18. An isolated nucleic acid, its sequence is selected from the following sequences: a) one Sequence according to SEQ.-ID-Nr. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, b) an intron-free sequence derived from a sequence according to a) is and encodes the same protein or peptide as the sequence according to SEQ.-ID-No. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, c) a sequence encoding a protein or peptide encoding the amino acid sequence according to SEQ.-ID-No. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14 comprises d) a sequence having a sequence according to a) to c) at least 80% identical, e) a sequence with the opposite strand a sequence according to one of the groups a) to d) hybridized or under Consideration of the degeneration of the genetic code would hybridise, f) a by substitution, addition, Inversion and / or deletion of one or more bases Derivative of a sequence according to one of the groups a) to e), G) a sequence corresponding to SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07 within the degeneracy of the genetic code, H) a sequence with neutral sense mutations of SEQ ID NO. 01, SEQ ID NO. 02, SEQ ID NO. 03, SEQ ID NO. 04, SEQ ID NO. 05, SEQ ID NO. 06 or SEQ ID NO. 07, as well i) a complementary one Sequence to a sequence according to one of the groups a) to h). An isolated polypeptide containing the amino acid sequence with the SEQ ID NO. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ. ID. NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 or SEQ ID NO. 14, or one Amino acid sequence that has an identity of at least 50%, preferably at least 55%, moreover preferably at least 60%, moreover, preferably at least 65% and most preferably at least 70% to the amino acid sequence according to SEQ ID NO. 08, SEQ ID NO. 09, SEQ ID NO. 10, SEQ. ID. NO. 11, SEQ ID NO. 12 SEQ ID no. 13 or SEQ ID NO. 14 has.