WO2009030654A1 - Mikrobiologische herstellung von isoprenoiden - Google Patents

Mikrobiologische herstellung von isoprenoiden Download PDF

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WO2009030654A1
WO2009030654A1 PCT/EP2008/061471 EP2008061471W WO2009030654A1 WO 2009030654 A1 WO2009030654 A1 WO 2009030654A1 EP 2008061471 W EP2008061471 W EP 2008061471W WO 2009030654 A1 WO2009030654 A1 WO 2009030654A1
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seq
cell
enzyme
sequence
isoprenoids
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PCT/EP2008/061471
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German (de)
English (en)
French (fr)
Inventor
Volker Sieber
Johannes Kaiser
Broder RÜHMANN
Nicole Potgrave
Eva-Maria Wittmann
Achim Marx
Norbert Windhab
Christian Ewering
Jens Kroll
Christian Schulze Gronover
Professor Dirk PRÜFER
Alexander Steinbüchel
David Wurbs
Adelbert Bacher
Wolfgang Eisenreich
Tobias Wilhelm GRÄWERT
Victoria Illarinova
Michael Groll
Felix Rohdich
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Evonik Degussa Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to a genetically modified cell as compared to its wild type, to a method for producing a genetically modified cell, to the genetically modified cell obtainable by this method, to a method for producing isoprenoids, to the isoprenoids obtainable by this method, to isolated nucleic acids and to isolated polypeptides ,
  • the terpenoids include compounds that are based on isoprene units.
  • the terpenoids include the subgroup of terpenes, whose carbon atoms can always be divided by 5, but also compounds derived from the terpenes, in which carbon atoms are later removed in the biosynthesis, and therefore their carbon atoms can not be divided by 5.
  • Terpenoids are important features for plant identification because a particular ingredient pattern is characteristic of a particular plant. There are more than 40,000 terpenoids known, about 8,000 of which belong to the subgroup of terpenes.
  • terpenoids are divided into the following subclasses: hemiterpenes (C5, such as isoprene), monoterpenes (Cio such as farnesyl), sesquiterpenes (C 5), diterpenes (C20, such as Taxol), triterpenes (C30, such as squalene) , Tetraterpenes (C 4 o, such as lycopene) and polyterpenes with more than 40 carbon atoms, such as rubber.
  • C5 hemiterpenes
  • monoterpenes Cao such as farnesyl
  • sesquiterpenes C 5
  • diterpenes C20, such as Taxol
  • triterpenes C30, such as squalene
  • Tetraterpenes C 4 o, such as lycopene
  • polyterpenes with more than 40 carbon atoms such as rubber.
  • Feed additives in skin or hair care products (QlO) or as resistant polymers (natural rubber).
  • cis-polyisoprene is found in rubber, while trans-polyisoprene is a major constituent of gutta-percha.
  • Gutta-percha is formed when the latex of the tropical tree Palaquium gutta dries.
  • Chicle obtained from the broadbush tree, represents a 1: 2 mixture of trans and cis polyisoprene.
  • terpenoids such as cis-polyisoprene based rubber
  • cis-polyisoprene based rubber are too complex to be prepared chemically, or their chemical preparation is cost-prohibitive over natural organism production.
  • the production of such complex isoprenoids by natural organisms, particularly by plants has the great disadvantage that the availability of these plants is often dependent on factors that can not be influenced by the manufacturer, such as drought and parasitic infestation or infection of the plants ,
  • the present invention has for its object to provide a method for providing terpenoids, with which it is possible to produce even complex terpenoids such as polyisoprenes, the economics of Production of terpenoids as far as possible does not depend on factors such as climatic conditions, parasite infestation or infections.
  • a further object of the present invention was to provide a process for the provision of terpenoids, with which it is possible to produce terpenoids with as constant a chemical quality as possible, in particular with a chemical composition that is as constant as possible.
  • a contribution to the solution of the abovementioned objects is made by a cell which has been genetically modified in comparison with its wild type in such a way that it is able to form isoprenoids enclosed in defined compartments.
  • cells in particular bacteria or yeast cells
  • Recombinant modification of the cell results in cells that were either unable to form or barely detectable levels of isoprenoids prior to recombinant modification or that were capable of forming isoprenoids prior to recombinant modification but were unable to express these isoprenoids
  • Include compartments in the cell, after performing the recombinant modification are now able to form isoprenoids and include them in defined compartments of the cell.
  • isoprenoid 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").
  • isoprenoids are the terpenoids, with the terpenoids again being the polyisoprene, in particular poly-cis-isoprene, poly-trans-isoprene or mixtures of polyisisoprene and poly-trans-isoprene, the most preferred isoprenoids.
  • 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.
  • wild-type cell is preferably referred to as a cell whose genome is in a state naturally established by evolution.The term will apply to both the entire cell and individual genes used. The term “wild-type” therefore does not include, in particular, those cells or genes whose gene sequences have been altered at least in part by humans by means of recombinant methods.
  • defined compartments of a cell preferably means certain organelles in the cytoplasm of a cell, which organelles may optionally be surrounded by a biomembrane
  • inclusion bodies which is usually a size in the range of 1 X 10 4 to 1 x 10 12 ⁇ 3 (cubic angstroms), more preferably in a range of 1 X 10 5 to 1 X. 10 10 3 and most preferably in a range from 1 ⁇ 10 6 to 1 ⁇ 10 8 ⁇ 3 and appear in electron micrographs as less electron-dense, milky-white appearing irregularly roundish shaped organelles in the cytoplasm.
  • inclusion bodies have a cylindrical structure.
  • the cells of the invention may be prokaryotes or eukaryotes. These may be mammalian cells (such as human cells), plant cells, or microorganisms such as yeasts, fungi or bacteria, with microorganisms being most preferred and bacteria and yeasts being most preferred.
  • mammalian cells such as human cells
  • plant cells such as plant cells
  • microorganisms such as yeasts, fungi or bacteria, with microorganisms being most preferred and bacteria and yeasts being most preferred.
  • yeasts or fungi especially those bacteria, yeasts or fungi are suitable, which in the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany, as bacterial, yeast or fungal strains are deposited.
  • DSMZ German Collection of Microorganisms and Cell Cultures GmbH
  • Bacteria suitable according to the invention belong to the genera under
  • Yeasts which are suitable according to the invention belong to those genera which are listed under
  • Particularly preferred cells according to the invention 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, Pseu
  • the genetically modified cell prefferably be genetically modified in such a way that it is particularly preferred within a defined time interval, preferably within 2 hours, more preferably within 8 hours, and most preferably within 24 hours, at least 2 times more preferably at least 100 times, more preferably at least 100 times, and most preferably at least 100,000 times more isoprenoids and more preferably in the defined compartments of the cell than the wild type of the cell.
  • the increase in the formation and the inclusion of the isoprenoids can be determined, for example, by cultivating the cell according to the invention and the wild-type cell separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specific time interval in a suitable nutrient medium and then determining the amount of isoprenoids contained in the inclusion bodies.
  • the wording "that the genetically engineered cell is genetically engineered to include more isoprenoids and to include in the defined compartments of the cell than the wild type of the cell” also applies to the case that the wild-type of the genetically engineered cell either does not have any isoprenoids at all forms detectable amounts of isoprenoids or indeed forms isoprenoids, but these are not in defined compartments, in particular in the inclusion bodies described above, and only after the genetic modification detectable amounts of these components can be formed and included in the defined compartments of the cell.
  • the expression, and thus also the activity, of at least one enzyme involved in the production of isoprenoids, preferably in the preparation of polyisoprenes having the abovementioned number of carbon atoms is in comparison therewith to the wild type, which enzyme is preferably an enzyme selected from the group consisting of a rubber binding protein, such as the srpp3-tk gene encoded small rubber binding protein Rubber elongation factor, such as the "rubber elongation factor" encoded by the ref2-hb gene, and a prenyltransferase, wherein increasing the activity of a prenyltransferase is particularly preferred.
  • a rubber binding protein such as the srpp3-tk gene encoded small rubber binding protein
  • Rubber elongation factor such as the "rubber elongation factor" encoded by the ref2-hb gene
  • prenyltransferase wherein increasing the activity of a prenyltransferase is particularly preferred.
  • 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 is described, 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
  • Another suitable gene for a gum-binding protein is the ghs gene described by In Jeong Kim et al. in "A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro", Journal of Experimental Botany, Vol. 55
  • 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 is described, inter alia, by 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).
  • 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.
  • the cell according to the invention has an increased activity of an enzyme Ei compared to 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 Ei and in the following statements in connection with the enzymes E 2 , etc., is preferably to be understood as an increased intracellular activity.
  • the following statements on increasing the enzyme activity in cells apply both to the increase in the activity of the enzyme Ei and to all the enzymes mentioned below, the activity of which may optionally be increased.
  • an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence (s) encoding the enzyme, using a strong promoter, or using a gene or allele encoding a corresponding enzyme with enhanced activity and, where appropriate, combining these measures.
  • Genetically engineered cells according to the invention are produced for example by transformation, transduction, conjugation or a combination of these methods with a vector which contains the desired gene, an allele of this gene or parts thereof and a vector which enables expression of the gene.
  • the heterologous expression is achieved in particular by the particularly preferred integration according to the invention of the gene or of the alleles into the chromosome of the cell or an extrachromosomally replicating vector.
  • the expression of the above and all of the following enzymes or genes is with the aid of 1- and 2-dimensional Protein gel separation and subsequent optical identification of the protein concentration with appropriate evaluation software detectable in the gel. 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 described by Hermann et al.
  • 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., CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY USA, 1989) and subsequent optical evaluation with appropriate software for
  • 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).
  • DNA band shift assays also called gel retardation
  • 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. CoId Spring Harbor Laboratory Press, CoId 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 specific methods for determining the activity of a specific enzyme are specified in the following, the determination of the increase in the enzyme activity and also the determination of the reduction of an enzyme activity are preferably carried out by means of the methods described in Hermann et al. , Electophoresis, 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).
  • mutations can be generated either undirected by classical methods, such as by UV irradiation or by mutagenic chemicals, or specifically by genetic engineering methods such as deletion (s), insertion (s) and / or nucleotide exchange (s). These mutations result in genetically engineered cells.
  • Particularly preferred mutants of enzymes are in particular those enzymes which are no longer or at least reduced in comparison to the wild-type enzyme reduced jfeecibacJc-inhibitable.
  • 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 act expression cassettes, the upstream of the structural gene. Inducible promoters also make it possible to increase expression at any time.
  • 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.
  • 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.
  • overexpression of the genes in question can be achieved by changing the composition of the medium and culture.
  • the skilled person will find, inter alia, in Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al.
  • episomal plasmids are used, for example. Suitable plasmids are in particular those which are replicated in coryneform bacteria.
  • plasmid vectors such as pZ1 (Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989)), pEKEx1 (Eikmanns et al., Gene 107: 69-74 (1991)) or pHS2-l (1989).
  • pZ1 Moskel et al., Applied and Environmental Microbiology 64: 549-554 (1989)
  • pEKEx1 (Eikmanns et al., Gene 107: 69-74 (1991)) or pHS2-l (1989).
  • Clar et al., Gene 107: 69-74 (1991) are based on the cryptic plasmids pHM1519, pBLI or pGAl.
  • Other plasmid vectors such as those on pCG4
  • plasmid vectors by means of which one can apply the method of gene amplification by integration into the chromosome, as described for example by Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) for duplication or amplification of the hom-thrB operon.
  • the complete gene is cloned into a plasmid vector which can be replicated in a host (typically Escherichia coli) but not in Corynebacterium glutamicum.
  • vectors examples include pSUP301 (Simon et al., Bio / Technology 1: 784-791 (1983)), pKl ⁇ mob or pK19mob (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 (shrink et al, Journal of.
  • the enzyme Ei is preferably a prenyltransferase (e.g., EC 2.5.1.29, 2.5.1.10 or 2.5.1.1), but more preferably a cis-1,4-prenyltransferase.
  • 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.
  • 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).
  • this has an increased activity of the enzyme egg, wherein this enzyme is encoded by a DNA selected from the group consisting of:
  • 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 encoding a protein or peptide comprising the amino acid sequence of 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.
  • a sequence having a sequence according to a) to c) of at least 80%, preferably at least 85%, particularly preferably at least 90%, moreover preferably at least 95% and most preferably at least 99% % is identical, this sequence preferably coding for an enzyme which catalyzes the transfer of an isopentenyl diphosphate unit to an allylic diphosphate initiator, e) a sequence which is linked to the complementary strand of a sequence according to one of groups a) to d) hybridized or hybridized in consideration of the degeneracy of the genetic code, this sequence preferably coding for an enzyme which inhibits the transfer of an isopentenyl catalysed diphosphate unit on an 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), said sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate
  • SEQ. ID No. Ol 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, and h) a sequence having neutral sense mutations of SEQ. -id-
  • 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.
  • sequences according to SEQ. ID No. 01 to SEQ. ID. -No. 07 are derived from the genus of Russian dandelion and encode proteins that are responsible for the terpenoid formation and have inter alia a prenyl transferase activity.
  • nucleotide identity and also the “amino acid identity” mentioned below within the meaning of the present invention are determined by means of known methods.
  • special computer programs with algorithms are under Consideration of special requirements used. 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
  • BLASTP BLASTN and FASTA (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).
  • Blossom 62 matrix is used using the default settings (gap weight: 12, length weight: 1).
  • An identity of 80% according to the above algorithm means 80% identity in the context of the present invention. The same applies to higher identities.
  • sequence which would hybridize with the opposite strand of a sequence according to one of groups a) to d), particularly preferably according to group a), or would hybridize in consideration of the degeneracy of the genetic code indicates a sequence, which hybridizes under preferably stringent conditions with the complementary strand of a sequence according to one of the groups a) to d), particularly preferably according to group a), or would hybridize taking into account the degeneracy of the genetic code
  • the hybridizations can be carried out at 68 ° C. in 2 ⁇ SSC or according to the protocol of the dioxygenin labeling kit from Boehringer (Mannheim) Preferred hybridization conditions are eg incubation at 65 ° C. overnight in 7% SDS, 1% BSA, 1 mM EDTA, 250 mM sodium phosphate buffer (pH 7 , 2) and subsequent washing at 65 ° C with 2 X 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 as exchange glycine for alanine or aspartic acid for 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.
  • this cell comprises a DNA sequence selected from the group consisting of:
  • this intron-free sequence preferably encodes an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator, c) a sequence encoding a protein or peptide having the amino acid sequence of 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.
  • a sequence having a sequence according to a) to c) of at least 80%, preferably at least 85%, particularly preferably at least 90%, moreover preferably at least 95% and most preferably at least 99% % is identical, this sequence preferably coding for an enzyme which catalyzes the transfer of an isopentenyl diphosphate unit to an allylic diphosphate initiator, e) a sequence which is linked to the complementary strand of a sequence according to one of groups a) to d) hybridized or hybridized in consideration of the degeneracy of the genetic code, which sequence preferably codes for an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator, f) by substitution, addition, inversion and / or Deletion of one or more bases obtained derivative of a sequence according to one of the groups a) to e), said sequence preferablyphil r encodes an enzyme which catalyzes the transfer of an isopentenyl
  • This code preferably corresponds to an enzyme which catalyzes the transfer of an isopentenyl diphosphate moiety to an allylic diphosphate initiator, and h) a sequence with neutral sense mutations of SEQ. -id-
  • 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.
  • Coenzyme A catalyzed 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 the conversion of mevalonate to
  • Mevalonate 5-phosphate catalyzes; an enzyme E 5 , which catalyzes the conversion of mevalonate-5-phosphate to mevalonate-5-diphosphate; an enzyme Ee, 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 Ei, besides increasing the activity of the enzyme Ei and a gum extension protein, besides increasing the activity of the enzyme Ei and a gum binding protein or besides increasing the activity of the enzyme Ei, a gum extender 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 , EiE 2 , EiE 3 , EiE 4 , E 1 E 5 , E 2 E 3 , E 2 E 4 , E 2 E 5 , E 2 Eg, E 3 E 4 , E 3 E 5 , E 3 Eg, E 4 E 5 , E 4 Eg, E 5 E 6 , EiE 2 E 3 , EiE 2 E 4 , EiE 2
  • E 2 is a hydroxymethylglutaryl-coenzyme A synthase
  • 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 Eg is a diphosphomevalonate decarboxylase (EC 4.1.1.33)
  • the enzyme E 2 is preferably encoded by a gene selected from the group consisting of hmgcsl, hmgcs2, hmgs, hcs, hgsA, pksG, mvaS, hmcM, mvaS.I, mvaS2, hmcS, mvaB, mvaBl and mvaB2.
  • the enzyme E 3 is preferably encoded by a gene selected from the group consisting of hmgcr, hmgl, hmg2, hmgA, hmgB, mvaA, mvaS.I, mvaAl and mvaA2.
  • the enzyme E 4 is preferably encoded by genes selected from the group consisting of mvk, lmbP, mvaKl 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, mvdl, mvaD and dmd.
  • the nucleotide sequences of the abovementioned genes and of further genes for the enzymes E2 to E 6 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 2 to E 6 is increased can be found, for example, in US 2007/0166782 A1.
  • 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's egg, the activity of the egg enzyme and a gum extension protein, the activity of the egg enzyme and a gum-binding protein or the activity of the egg enzyme, a gum elongation factor and a gum-binding protein are increased.
  • 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 Ei, besides increasing the activity of the enzyme Ei and a gum extension protein, besides increasing the activity of the enzyme Ei and a gum-binding protein or besides increasing the activity of the enzyme Ei, a rubber elongation 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 reaction of D-glyceraldehyde-3-phosphate and pyruvate to l-deoxy-D-xylulose-5-phosphate; an enzyme Eg, 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 conversion of 2-C-methyl-D-erythritol-4-phosphate to 4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol; an enzyme E10, which is the reaction of 4- (cytidine-5'-diphospho) -2-C-methyl-D-erythritol to 2-phospho-4- (cytidine-5'-diphospho) -2-C-methyl- D-erythritol catalyses; an En enzyme which catalyzes the conversion of 2-phospho-4- (cy
  • 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 production of polyisoprenes having the abovementioned number of carbon atoms, are particularly preferred
  • Increasing the activity of the enzyme Ei besides increasing the activity of the enzyme Ei and a gum extension protein, besides increasing the activity of the enzyme Ei and a gum binding protein or besides increasing the activity of the enzyme Ei, a gum elongation factor and a Gum binding protein the activity of the following enzymes or enzyme combinations is increased: E 7 , E 8 , E 9 , Ei 0 , En, E i2 , E i3 , E 7 E 8 , E 7 Eg, E 7 Ei O , E 7 En, E 7 Ei 2 , E 7 Ei 3 , E 8 Eg, E 8 Ei O , E 8 En, E 8 Ei 2 , E 8 Ei 3 , EgEio, EgEn, EgEi 2 , EgEi 3
  • E 7 is an l-deoxy-D-xylulose-5-phosphate synthase (EC 2.2.1.7)
  • E 8 is an l-deoxy-D-xylulose-5-phosphate reductoisomerase
  • E 10 is a 4- (cytidine-5 '-diphospho) -2-C-methyl-D-erythritol kinase
  • E 11 is a 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase
  • E 12 is a 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase
  • E 13 is a 4-hydroxy-3-methylbut-2-enyl diphosphate reductase
  • the enzyme E 7 is preferably selected from a gene selected from the group consisting of dxs, dxs-1, dxs-2, dxsA, dxsB or tktB coded.
  • the enzyme E 8 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 Ei 0 are selected from the group consisting of ispE, ychB, ipk, thrBl, thrB, yabH and cmeK.
  • Enzyme En 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, lytBl, 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 can be found, for example, in US 2004/0176570 A1.
  • 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 Ei, the activity of the enzyme Ei and a gum extension protein, the activity of the enzyme Ei and a gum-binding protein or the activity of the enzyme Ei, a gum elongation factor 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 elongation factor ("Rubber Enlongation Factor”) and a prenyl transferase in the cell.
  • an enzyme selected from the group consisting of a rubber binding protein, a gum elongation factor ("Rubber Enlongation Factor") and a prenyl transferase in the cell.
  • 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 prenyltransferase and a rubber elongation factor in the cell or the step of increasing the expression of a prenyltransferase, a gum extender and a gum-binding protein in the cell.
  • 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 i3 .
  • 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 introducing it into the cell in the form of an expression vector.
  • the cell is a microorganism, in particular a bacterial or yeast cell in which the cells preferred as bacterial or yeast cells are those which have already been mentioned at the beginning in connection with the cell according to the invention.
  • the cells according to the invention or the cells obtainable by the process according to the invention are first brought into contact with a culture medium containing at least one carbon source under conditions under which the cell can form isoprenoids from the carbon source, which in defined compartments in be injected into the cell.
  • 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 GB-A-1009370.
  • the culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington D.C, USA, 1981).
  • 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 are used. These substances can be used individually or as a mixture. Particularly preferred is the use of carbohydrates, in particular monosaccharides, oligosaccharides or polysaccharides, as described in US 6,01,494 and US 6,136,576, of Cs sugars or of glycerin.
  • the nitrogen source there may be used organic nitrogen-containing compounds such as peptones, 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.
  • organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea
  • inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.
  • the nitrogen sources can be used singly or as a mixture.
  • phosphorus source can phosphoric acid
  • the culture medium must further contain salts of metals, e.g. Magnesium sulfate or iron sulfate necessary for growth.
  • essential growth factors such as amino acids and vitamins can be used in addition to the above-mentioned substances.
  • suitable precursors can be added to the culture medium.
  • the said feedstocks may be added to the culture in the form of a one-time batch or fed in a suitable manner during the cultivation.
  • basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are suitably used.
  • acidic compounds such as phosphoric acid or sulfuric acid are suitably used.
  • anti-foaming agents such as fatty acid polyglycol esters can be used.
  • suitable selective substances may be added to the medium such as adding antibiotics.
  • oxygen or oxygen-containing gas mixtures such as air are introduced into the culture.
  • the temperature of the culture is usually more than 20 0 C, preferably more than 30 0 C, it may also be more than 40 0 C, wherein advantageously a cultivation temperature of 95 ° C, more preferably 90 0 C and most preferably 80 0 C is not exceeded.
  • the isoprenoids included in the defined compartments are isolated in process step ii) ,
  • this isolation of the encapsulated isoprenoids can be carried out in the manner in which recombinantly produced proteins usually enclosed in inclusion bodies are isolated from microorganisms such as, for example, E. coli.
  • the isolation of the isoprenoids included in the defined compartments comprises the following process steps:
  • the cells are first digested to obtain a cell lysate comprising the defined compartments of the cell, preferably the inclusion bodies, in which the isoprenoids are included.
  • This disruption can be carried out by all methods known to those skilled in the art, which are usually used for disrupting cells.
  • the mechanical cell termination such as the homogenization in a blender with rotating blades, for example by means of an Ultra-Turrax, mechanical separation by means of the Potter-Elvehjem method, in which a piston closely spaced from a stationary one
  • a mechanical disruption by means of ultrasound or, in the case of a continuous digestion process the mechanical disruption, for example by means of a Manton-Gaulin Homogenizer, in which the cells are forced through a tight valve at high pressure.
  • digestion is accomplished using a Manton-Gaulin homogenizer at a pressure in the range of 1,000 to 50,000 psi, more preferably in the range of 10,000 to 20,000 psi.
  • non-mechanical digestion methods are also possible, such as treatment of the cells by repeated freezing and thawing, treatment of the cells with hypotonic buffer solutions, autolysis with toluene, enzymatic lysis with enzymes such as zymolyase, treatment of the cells Cells with detergents such as Triton-X-100 or the treatment of the cells with complexing compounds, such as with EDTA solutions.
  • autolysis with organic solvents in particular with halogenated hydrocarbons such as, for example, toluene.
  • Such use of organic solvents is particularly advantageous because not only the cells can be digested, but also, depending on the organic solvent, at the same time the defined compartments of other cell components can be separated, for example by precipitation, so that the process steps iia) and üb ) can be carried out at the same time.
  • the defined compartments preferably the inclusion bodies in which the isoprenoids are enclosed, are separated from the cell lysate.
  • This one Inclusion bodies have a high density compared to other components of the cell lysate, they can be separated from the other components, for example by high-speed centrifugation, in which the inclusion bodies are obtained as a pellet.
  • the high-speed centrifugation is carried out at a centrifugal acceleration in a range of 1,000 to 20,000 xg, more preferably in a range of 5,000 to 15,000 x g.
  • the defined inclusion bodies separated in this manner are subsequently purified in process step iic), this purification serving in particular for separating proteins still contained in the inclusion bodies or adhering to the surface of the inclusion bodies or other impurities other than the isoprenoids.
  • This purification preferably takes place by washing the separated, defined compartments with suitable washing solutions.
  • suitable washing solutions are, in particular, water or saline buffer solutions. It can be advantageous, in particular, to treat the inclusion bodies separated in process step (iv) also with an enzyme-containing washing solution, for example an aqueous proteinase solution, in order to remove proteins which are enclosed in the inclusion bodies and / or adhere to the surface of the inclusion bodies.
  • 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 encoding a protein or peptide comprising the amino acid sequence of 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.
  • a sequence having a sequence according to a) to c) of at least 80%, preferably at least 85%, more preferably at least 90%, moreover preferably at least 95% and most preferably at least 99% % is identical, this sequence preferably coding for a protein which catalyzes the transfer of an isopentenyl diphosphate unit to an allylic diphosphate initiator, e) a sequence which would hybridize to the opposite strand of a sequence according to one of the groups a) to d) or would hybridize taking into account the degeneracy of the genetic code, this sequence preferably coding for a protein which comprises the transfer of an isopentenyl diphosphate unit catalysed on an allylic diphosphate initiator, f) a obtained 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), said sequence preferably encodes a protein which the Catalyzed transfer of an isopentenyl
  • SEQ. ID No. Ol 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 having 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.
  • this sequence preferably coding for a protein which catalyzes the transfer of an isopentenyl diphosphate unit to an allylic diphosphate initiator, and i) a complementary sequence to a sequence according to one of the groups a) to h).
  • RNA sequences were then amplified For details, see the examples.
  • a vector preferably an expression vector, comprising a DNA having a sequence according to one of the groups a) to h), as defined above.
  • Suitable vectors are all vectors known to those skilled in the art, which are usually used for introducing DNA into a host cell.
  • Preferred vectors are selected from the group comprising plasmids, such as the E.
  • viruses such as bacteriophages, adenoviruses, vaccinia viruses, baculoviruses, measles viruses and retroviruses, cosmids or YACs, wherein plasmids are vectors most preferred.
  • the DNA having a sequence according to one of the groups a) to h) is under the control of a regulatable promoter which is suitable for expression of the polypeptide encoded by these DNA sequences in the cell of a microorganism, preferably a bacterial , Yeast or Pilzelle, particularly preferably a bacteria of the yeast cell, is suitable.
  • a regulatable promoter which is suitable for expression of the polypeptide encoded by these DNA sequences in the cell of a microorganism, preferably a bacterial , Yeast or Pilzelle, particularly preferably a bacteria of the yeast cell.
  • promoters are, for example, the trp promoter or the tac promoter.
  • the vector according to the invention should preferably comprise a ribosome binding site as well as a terminator. It is particularly preferred that the DNA according to the invention is incorporated into an expression cassette of the vector comprising the promoter, the ribosome binding site and the terminator.
  • the vector may further comprise selection genes known to those skilled in the art.
  • the use of the above-described vector for the transformation of a cell as well as the cell obtained by transformation with this vector further contribute to the solution of the abovementioned objects.
  • the cells which can be transformed with the vector according to the invention are preferably those cells which have already been described at the outset as preferred cells according to the invention.
  • an isolated polypeptide comprising the amino acid sequence of 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 an 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.
  • Figure 1 shows the electron micrograph of an E. coli Rosetta gami B (DE3) plysS pET23a cell as a control.
  • FIG. 2 shows electron micrographs of an E. coli origami (DE3) plysS pET23a :: srpp3-tJc cell according to the invention, in which a Prenyltransferase from T. kok-saghyz was overexpressed.
  • FIG. 3 shows 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.
  • the Figure 4 shows 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 prenyltransferase from T. kok-saghyz were together overexpressed.
  • cytoplasm of the cell which is shown in Figures 2 to 4, defined compartments can be seen, in which isoprenoids are included.
  • Figure 5 shows the prenyl-transferase activity of the enzymes derived from the genes of SEQ. ID No. Ol and SEQ. ID No. 07 are coded. Furthermore, the prenyltransferase activity of prenyltransferase from Hevea brasiliensis is shown. The determination is carried out 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 :: cptl-tk ), "2" of the cell-free extract of E.
  • 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 :: cptl-tk
  • 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" a mixture of cell-free crude extract from E. coli Rosetta gami B (DE3) plysS (pET23a:: cptl-tk) and cell-free crude extract from E. coli Rosetta gami B (DE3) plysS (pEXP5CT: srpp3-tk).
  • FIG. 6 shows an electron micrograph of an E. coli HMS174 (DE3) pMR07-EK according to the invention
  • pCDFDuet-1 DXIPP cell in which the genes cptl-tk to cpt4-tk and srppl-tk to srpp3-tk were together overexpressed.
  • the bar corresponds to 0.5 ⁇ m.
  • Figure 7 shows an electron micrograph of an E. coli HMS174 (DE3) pACYC184; pCDFDuet-1 :: DXIPP control cell. The bar corresponds to 0.5 ⁇ m.
  • E. coli HMS174 DE3 pACYC184; pCDFDuet-1 :: DXIPP control cell.
  • the bar corresponds to 0.5 ⁇ m.
  • An analogous picture results for the cells analogous constructs in E. coli tuner (DE3), cf. below.
  • Figure 8 shows the numbering for NMR measurements of relevant carbon atoms in isoprene
  • RNA homogenization buffer 4 M guanidinium isothiocyanate, 100 mM Tris / HCl, pH 7
  • 800 ul TriFast ® 800 ul TriFast ® (PeqLab) and the procedure according to the manufacturer.
  • 2 ⁇ g of the total RNA were inserted into the RT and were purified by means of the reverse transcriptase "Super Scriptll” (Invitrogen) using the primers srpp-TKl-3_rev (SEQ ID No. 16, SEQ. ID.
  • srpp3-tk (comprises 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 X His fusion protein (Srpp3-tkCT 6 x 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.
  • 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).
  • the Taq DNA polymerase was used to generate 3 '-A overhangs.
  • the 5 'primer generated no further interfaces and was identical to the first sequence section.
  • 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 grown after transformation into E. coli TOP10 on LB ampicillin plates, plasmid DNA was isolated and probed with the primers TOPO_Fw and TOPO_Rev (SEQ. ID No. 33 and SEQ. ID 34). A positive E. coli TOP10 clone and its prepared pEXP5CT:: srpp3-tk plasmid was final for used further studies, transformed into various DE3 expression strains and preserved.
  • the codon usage of the cis-1, 4-prenyltransferase hrt2 from Hevea brasiliensis was 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).
  • common restriction sites for further cloning were deducted from the optimized nucleotide sequence to allow for easy subcloning.
  • flanking restriction sites Ndel and EcoRI were chosen. The Ndel and EcoRI sites allowed direct subcloning into the vector pET23a.
  • the synthetic gene hrt2-jk was delivered in the bacterial cloning vector pUC57.
  • a restriction digest was carried out with the enzymes Ndel and EcoRI and the structural gene was purified in an agarose gel for further use.
  • To prepare the target vector pET23a for ligation it was also completely digested with the enzymes Ndel and EcoRI and purified in an agarose gel. This was followed by a ligation of structural gene and target vector.
  • positive clones or their prepared pET23a:: hrt2-jk plasmids were completely sequenced, transformed into different expression strains and conserved for further investigations.
  • Preparation of the expression vector pET23a:: cptl-tk (comprises a cis-1,4-prenyltransferase from Taraxacum kok-saghyz)
  • PCR polymerase chain reaction
  • the 927 bp DNA fragment were amplified by means of the oligonucleotides Cpt Vspl 5 'and Cpt_EcoRI_3' (SEQ ID No. 35 and SEQ ID No. 36) by means of PCR.
  • the Pfx-DNAPolymerase was used, which has a proof reading function.
  • the 5 'primer generated the restriction site for Vspl before the start codon already contained in the gene.
  • the use of Vspl after digestion with the enzyme of the same name enabled the presence of a compatible overhang to ⁇ / del digested restriction sites in the target vector.
  • the use and generation of this interface became necessary because cptl-tk already had a Ndel recognition site.
  • TAA gene termination sequence
  • the 950 bp DNA fragments obtained after the PCR were separated in an agarose gel and purified for further use.
  • the purified PCR fragments digested by the restriction enzymes Vspl and EcoRI.
  • the target vector pET23a was previously isolated from E. coli cells, purified and digested by the restriction endonucleases Ndel and EcoRI overnight.
  • the linearized vector fragment became final still purified via an agarose gel and ligated into the linearized vector pET23a.
  • srpp3-tk The cleavage of srpp3-tk in the pET23a vector enabled subcloning of the entire gene into the MCS 2.
  • the purified expression plasmid pET23a :: srpp3-tk was digested with the restriction enzymes Ndel and Xhol and purified.
  • 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.
  • the vector pETDuet-1:: srpp3-tk restricted with the same restriction enzymes was subsequently ligated with the gene hrt2-jk to obtain the expression vector pETDuetl :: hrt2-jk :: srpp3-tk.
  • Another expression vector pMR07-EK, SEQ. ID No. 37, based on the low copy vector pACYC184, which contains the genes cptl-tk to cpt4-tk and srppl-tk to srpp3-tk in an optimized form for expression in E. coli.
  • the region responsible for the expression of these genes is flanked on both sides by manx sequences which allow Lamdba-mediated integration.
  • This vector was synthesized at the company Geneart optimized for E. coli codon usage.
  • Flavodoxin 1 amplified Beiude amplificates were fused by PCR (fusion PCR) and subsequently digested with the restriction enzymes SacI and SacII.
  • the cleaved fusion amplificate was cloned into the SacI and SacII digested vector pBScyclo (Hecht S. et al., 2001, Studies on the nonmevalonate pathway to terpenes: The role of the GcpE (IspG) protein, Proceedings of the National Academy of Sciences of the United States of America 98: 14837-14842).
  • the resulting vector is called pBSDXIPP, SEQ. ID No. 38.
  • This vector was digested with the restriction endonucleases SacI and KpnI.
  • the resulting fragment was ligated into the SacI and KpnI digested vector pCDF-Duet (Novagen), the resulting vector is called pCDF-Duet :: DXIPP
  • competent E. coli cells were prepared.
  • the E. coli strains to be transformed starting from a preculture in 50 ml of LB medium to an OD 6 oonm of 0.3 to 0.5 at 37 0 C on a rotary shaker (Controlled environment incubator shaker, New Brunswick Scientific Co. Inc., Edison, USA) and stored sterile harvested in 10 ml batches by centrifugation at 3,500 rpm for 10 min at 4 0 C.
  • the sedimented cells were dissolved in 5 ml of ice-cold 0.1 M CaCl 2. " Solution and incubated for 10 min on ice. 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.
  • the transfer of DNA into E. coli cells was carried out by transformation of the competent cells obtained above. 200 .mu.l of competent E. coli cells were thoroughly mixed with 50-250 .mu.g of the expression vectors. To adsorb the DNA on the surface, the cells were incubated on ice for 30 min. By a heat shock of the cells for exactly 90 seconds at 42 0 C and a short cooling on ice for 2-5 min, the DNA was taken up by the cells. For regeneration of cells and for expression of the plasmid-encoded antibiotic resistance to the transformation mixture was added 600 ul of LB medium was added sterile and the cells incubated for 60-90 min at 37 0 C. Finally, aliquots were plated on selective agar of 70-200 ul and cultured for the isolation of the recombinant clones overnight at 37 0 C. As a control, a batch without DNA was included.
  • E. coli origami (DE3) plysS pET23a :: srpp3-tk
  • E. coli HMS174 (DE3) pMR07-EK; pCDFDuet-1 :: DXIPP 7.
  • E. coli tuner (DE3) pMR07-EK; pCDFDuet-1 :: DXIPP
  • E. coli tuner (DE3) pACYC184; pCDFDuet-1 :: DXIPP
  • 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.
  • cells of E. coli in LB medium New Brunswick Scientific Co., Inc., Edison Fa., USA
  • 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 starting from a pure culture on solid medium in test tubes with 5 ml LB medium or Erlenmeyer flask 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.
  • a single colony of E. coli cells was used as Impfgut and usually cultured at 37 0 C until reaching an optical density (OD ⁇ OOnm) of 0.7.
  • Velcro piston allowed a comfortable checking of the optical density (OD).
  • Using a Klett Summerson Colorimeter Frter No.
  • the OD of the culture was monitored at 520-580 nm.
  • cell growth was measured by measuring the OD with a photometer (Ultrospec 2000 UV / Visible
  • the French-Press cell was disrupted by passing three times through an ice-cold French Press cell (Amicon, Silver Spring, Maryland, USA) at a pressure of 130 MPa.
  • Cell free crude extracts were xg by centrifugation for 20 min at 13,000 won in a cooling centrifuge at 4 0 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.
  • BSA bovine serum albumin
  • the solution was filtered before use.
  • 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.
  • the reaction mixture was pipetted into 1.5 ml reaction vessels, incubated for 4 h at 30 0 C and then extracted with 0.6 ml of 1-butanol.
  • the reaction mixture was incubated vigorously on a vortex shaker of the type VV3 (VWR International GmbH, Darmstadt, Germany) with a suitable reaction vessel attachment for 15 minutes at room temperature. This was followed by centrifugation at 14,000 x g.
  • the top 1-butanol phase was carefully removed without damage to the middle protein layer and directly into a 3 ml scintillation vial IRGASAFE plus scintillation cocktail (Zinsser Analytic GmbH, Frankfurt, Germany).
  • the remaining aqueous phase and the protein layer were extracted twice with 0.6 ml of toluene / hexane (mixing ratio 1: 1) in the same manner.
  • 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 shown by way of example for the enzymes encoded by the genes hrt2-jk, cptl-tk (SEQ ID No. 01) and srpp3-tk (SEQ ID No. 07) in FIG.
  • Step 2 Extraction and Detection of Polyisoprene Compounds.
  • the cells obtained in step 1 are lyophilized, suspended with 1 ml of deuterated methylene chloride (CD 2 Cl 2) or other deuterated apolar solvents and extracted with shaking for 1 h at room temperature. Subsequently, the sample is centrifuged at 13,000 rpm for 15 minutes, and the supernatant is subjected to 13 C-NMR spectroscopic analysis.
  • CD 2 Cl 2 deuterated methylene chloride
  • Table 1 NMR data of 10 mg trans- or cis-1, 4th polyisoprene (reference sample) dissolved in 0.5 mL deuterated solvent.
  • the measurement parameters were default values that were included in the
  • the chain length of formed products can be estimated on the basis of signals derived from carbon atoms at the end of the chain. It is important here that an OH or OPP is bonded to C-I at the chain end and the corresponding carbinol signal is observable at about 60 ppm.
  • the NMR intensities of the C-I signal of the terminal isoprene unit (60 ppm) with the signals for C-I from the chain (27 ppm) the number of isoprene units and thus the molecular weight can be estimated.
  • the assignment of the NMR signals to cis- or trans-polyisoprene compounds can be made on the basis of Table 1.
  • the signals for C-4 and C-5 can be used to distinguish between the two substance classes.
  • the used marking strategy can in the present Example, the signal from C-4 as a basis (see Table 2).
  • Table 2 13 C NMR data of [1, 2, 4- 13 C 3 ] trans-1,4-polyisoprene from a typical labeling experiment compared to 13 C NMR literature data for cis and trans polyisoprene.
  • the quantification of cis- or trans-polyisoprene can be carried out by dividing the SSttaannddaarrddiissiieerruunngg c on the 13 C-NMR signal of the solvent CD 2 Cl 2 .
  • Figure 9 shows relevant excerpts from the 13 C NMR spectra of the crude extracts of E. coli tuner (DE3) pACYC184; pCDFDuet-1 :: DXI PP and E. coli tuner (DE3) pMR07-EK; pCDFDuet-1 :: DXIPP.
  • the 13 C NMR spectra of the extracts showed clear NMR signals of 13 C-labeled apolar polyisoprene compounds. It was possible to detect three 13 C-coupled signals with high intensity. The chemical shifts and coupling constants were in good agreement with comparative data on polyisoprene compounds (see Table 1). For example, the signal observed at 39.5 ppm in Figure 9 is specific for C-4 of a trans-configured compound.
  • trans- or cis-polyisoprene was carried out by the specific signals of C atoms 4 at 39.5 ppm (trans), and 31.9 ppm (ice). In both spectra, the corresponding signals can be detected, but with different relative signal intensities, in particular for the signal at 31.9 ppm for cis-polyisoprene.
  • the quantification was carried out by integration of one (well-separated) signal component of the respective doublets for the signals of C-4 (caused by 13 C- 13 C coupling with 13 CI of the adjacent isoprene unit
  • the observed increase can be attributed in particular to the increased amount of cis-polyisoprene, in which the relative integral value increased from 0.06 to 0.10. This corresponds to an increase of 67% for the cis Polyisoprene compound by the plasmid pACYC184. In the case of trans-polyisoprene, the integral value increased from 0.71 to 0.84 (increase by 18%).

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10787688B2 (en) 2012-05-11 2020-09-29 Evonik Operations Gmbh Multi-stage synthesis method with synthesis gas
CN108715825A (zh) * 2018-06-01 2018-10-30 天津大学 基因过表达及获得的菌株、应用
CN108715825B (zh) * 2018-06-01 2021-03-09 天津大学 基因过表达及获得的菌株、应用

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