EP1179187A1 - Use of genes of the deoxy-d-xylulose phosphate biosynthetic pathway for altering the concentration of isoprenoid - Google Patents

Use of genes of the deoxy-d-xylulose phosphate biosynthetic pathway for altering the concentration of isoprenoid

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Publication number
EP1179187A1
EP1179187A1 EP00935082A EP00935082A EP1179187A1 EP 1179187 A1 EP1179187 A1 EP 1179187A1 EP 00935082 A EP00935082 A EP 00935082A EP 00935082 A EP00935082 A EP 00935082A EP 1179187 A1 EP1179187 A1 EP 1179187A1
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Prior art keywords
gcpe
yfgb
protein
parasites
bacteria
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EP00935082A
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German (de)
French (fr)
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Hassan Jomaa
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Jomaa Pharmaka GmbH
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Jomaa Pharmaka GmbH
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Priority claimed from DE19923568A external-priority patent/DE19923568A1/en
Priority claimed from DE19923567A external-priority patent/DE19923567A1/en
Application filed by Jomaa Pharmaka GmbH filed Critical Jomaa Pharmaka GmbH
Publication of EP1179187A1 publication Critical patent/EP1179187A1/en
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation

Definitions

  • the present invention relates to the use of D ⁇ A sequences (SEQ: 1,3,5,7) which code for the gcpE or yfgB protein from bacteria or parasites and which are integrated into the genome of viruses, eukaryotes and prokaryotes change the isoprenoid content and methods for measuring the activity of the gcpE gene in relation to isoprenoid synthesis. It also relates to methods for identifying substances with herbicidal, antiparasitic, antiviral, fungicidal activity in plants and antiparasitic, antifungal and antiviral activity in humans and animals.
  • CH 2 C (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2
  • CH 2 C (CH 3 ) -CH (OH) -CH 2 -OH
  • CH (OH) C (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2
  • CH (OH) C (CH 3 ) -CH (OH) -CH 2 -OH, CH 3 -
  • the invention therefore relates to the use of DNA sequences which encode the gcpE or yfgB protein from bacteria or parasites from bacteria or the gcpE or yfgB protein from parasites or DNA sequences which are analogous or derivatives thereof Encode proteins in which one or more amino acids have been deleted, added or substituted by other amino acids without significantly reducing the enzymatic action of the polypeptide. It relates in particular to the use of the SEQ 1,3,5,7 DNA sequences.
  • the original origin of the specified sequences SEQ 1 and 5 and of proteins 2 and 6 is the organism Escherichia coli, strain K12.
  • the original origin of the specified sequences SEQ 3 and 7 and of proteins 4 and 8 is the organism Plasmodium Falciparum, strain 3D7.
  • sequences according to the invention are suitable for the expression of genes in viruses, eukaryytes and prokaryotes which are responsible for the isoprenoid biosynthesis of the 1-deoxy-D-xylulose pathway.
  • the eukaryotes or eukaryotic cells include animal cells, plant cells, algae, yeasts, fungi and the prokaryotes or prokaryotic bacteria are archaebacteria and eubacteria.
  • viruses, eukaryotes and prokaryotes When a DNA sequence is integrated into a genome on which one of the above-mentioned DNA sequences is located, the expression of the above-described genes in viruses, eukaryotes and prokaryotes is made possible.
  • the viruses, eukaryotes and prokaryotes transformed according to the invention are grown in a manner known per se and the isoprenoid formed in the process is isolated and, if appropriate, purified. Not all isoprenoids need to be isolated because in some cases the isoprenoids are released directly into the air.
  • the transgenic viruses, eukaryotes and prokaryotes used to change the isoprenoid content can be produced in the following steps: a) Production of a DNA sequence with the following partial sequences i) promoter which is active in viruses, eukaryotes and prokaryotes and ensures the formation of an RNA in the intended target tissue or the target cells, ii) DNA sequence which is suitable for a polypeptide with the amino acid sequence of Code gcpE or the yfgB protein from bacteria or parasites or for an analog or derivative of this polypeptide, üi) 3 'untranslated sequence which is used in viruses, eukaryotes and prokaryotes to add poly-A residues to the 3 ' end of the RNA leads, b) transfer and incorporation of the DNA sequence into the genome of viruses, prokaryotic or eukaryotic cells with or without the use of a vector (eg plasmid, viral DNA).
  • a vector eg plasmid,
  • the intact whole plants can be regenerated from the transformed plant cells.
  • sequences coding for the gcpE or the yfgB proteins or their analogs or derivatives can be provided with a promoter which ensures transcription in certain organs or cells and which is in sense orientation (3 'end of the promoter to the 5' end of the coding sequence) is coupled to the sequence encoding the protein to be formed.
  • a termination signal determining the termination of the mRNA synthesis is appended to the 3 end of the coding sequence.
  • a sequence coding for a so-called signal sequence or a transit peptide can be placed between the promoter and the coding sequence.
  • the sequence must be in the same reading frame as the coding sequence of the protein.
  • cloning vectors which contain a replication signal for E. coli and a marker which permits selection of the transformed cells. Examples of vectors are pBR 322, pUC series, M13mp series, pACYC 184, EMBL 3 etc. Depending on the method of introducing desired genes into the plant, further DNA sequences may be required.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least one right boundary, but often the right and left boundary of the Ti and Ri plasmid T-DNA, must be inserted as the flank region of the genes to be introduced become.
  • T-DNA for the transformation of plant cells has been intensively investigated and is sufficient in EP 120516; Hoekama, in: The Binary Plant Vector System, Offset-drukkerij Kanters BV Alblasserdam (1985), Chapter V; Fraley et al., Crit.Rev.Plant Sei. 4,1-46 and An et al. (1985) EMBO J. 4, 277-287 have been described.
  • the inserted DNA is integrated in the genome, it is usually stable and is also retained in the offspring of the originally transformed cells. It normally receives a selection marker which imparts resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin and the like, to the transformed plant cells.
  • the individually used marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA.
  • agrobacteria e.g. Agrobacterium tu- mefaciens
  • the fusion of protoplasts the microinjection of DNA
  • electroporation as well as ballistic methods and virus infection.
  • Whole plants can then be regenerated from transformed plant material in a suitable medium, which may contain antibiotics or biocides for selection. When it comes to injection and electroporation, there are no special requirements for the plasmids. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary.
  • the transformed cells grow within the plants in the usual way (McCormick et al. (1986), Plant Cell Reports 5, 81-84).
  • the plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup. The resulting individuals have the corresponding phenotypic properties.
  • Expression vectors which contain one or more of the DNA sequences according to the invention are suitable for introducing the DNA into the host organisms.
  • Such expression vectors are obtained by providing the DNA sequences according to the invention with suitable functional regulatory signals.
  • Such regulatory signals are DNA sequences which are responsible for expression, for example promoters, operators, enhancers, ribosomal binding sites and which are recognized by the host organism.
  • further regulation signals which control, for example, replication or recombination of the recombinant DNA in the host organism, can be part of the expression vector.
  • Particularly suitable for the expression of the enzymes according to the invention are host cells and organisms which have no intrinsic enzymes with the function of DOXP synthase, DOXP reductoisomerase or gcpE kinase. This applies to archaebacteria, animals, fungi, slime molds and some eubacteria. The lack of these intrinsic enzyme activities makes the detection and purification of the recombinant enzymes essential facilitated. This also makes it possible to measure the activity and in particular the inhibition of the activity of the recombinant enzymes according to the invention by various chemicals and pharmaceuticals in crude extracts from the host cells with little effort.
  • the enzymes according to the invention are advantageously expressed in eukaryotic cells if post-translational modifications and a native folding of the polypeptide chain are to be achieved.
  • introns are eliminated by splicing the DNA and the enzymes are produced in the polypeptide sequence characteristic of the parasites.
  • Sequences coding for introns can also be removed by recombinant DNA technology from the DNA sequences to be expressed or inserted experimentally.
  • the protein can be isolated from the host cell or the culture supernatant of the host cell by methods known to the person skilled in the art. In vitro reactivation of the enzymes may also be required.
  • the enzymes according to the invention or partial sequences of the enzymes can be expressed as a fusion protein with various peptide chains.
  • Oligo-histidine sequences and sequences derived from glutathione-S-transferase, thioredoxin or calmodulin-binding peptides are particularly suitable for this purpose. Fusions with thioredoxin-derived sequences are particularly suitable for prokaryotic expression, since this increases the solubility of the recombinant enzymes.
  • the enzymes according to the invention or partial sequences of the enzymes can be expressed as fusion proteins with peptide chains known to those skilled in the art that the recombinant enzymes are transported into the extracellular environment or into certain compartments of the host cells. This enables both the purification and the investigation of the biological activity of the enzymes to be facilitated.
  • the enzymes according to the invention When expressing the enzymes according to the invention, it may prove expedient to change individual codons.
  • the targeted exchange of bases in the coding region also makes sense if the codons used in the parasites differ from the codon use in the heterologous expression system in order to ensure optimal synthesis of the protein.
  • deletions of untranslated 5 'or 3 'sections make sense, for example if there are several destabilizing sequence motifs ATTTA in the 3' region of the DNA. Then these should be deleted in the preferred expression in eukaryotes. Changes of this type are deletions, additions or exchange of bases and are also the subject of the present invention.
  • the enzymes according to the invention can be obtained under standardized conditions by techniques known to the person skilled in the art by in vitro translation. Suitable systems are rabbit reticulocyte and wheat germ extracts and bacterial lysates. In vitro transcribed mRNA can also be translated into Xenopus oocytes.
  • Oligo- and polypeptides can be produced by chemical synthesis, the sequences being derived from the peptide sequence of the enzymes according to the invention. With a suitable choice of the sequences, such peptides have properties which are characteristic of the complete enzymes according to the invention. Such peptides can be produced in large quantities and are particularly suitable for studies on the kinetics of enzyme activity, the regulation of enzyme activity, the three-dimensional structure of the enzymes, the inhibition of enzyme activity by different chemicals and pharmaceuticals and the binding geometry and binding affinity of different ligands.
  • Another object of this invention are methods for determining the enzymatic activity of gcpE kinase. This can be determined according to the known instructions.
  • CH 2 C (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2
  • CH 2 (OH) -C ( CH 2 ) -C (OH) -CH 2 -OH
  • CH (OH) C (CH 3 ) -CH (OH) -CH2-O-PO (OH) 2
  • CH (OH) C (CH 3 ) -CH (OH) -CH 2 -OH
  • CH 3 - C (CH 3 ) CH-CH 2 -O-PO (OH) 2
  • CH 3 -C (CH 3 ) CH-CH 2 -OH
  • CH 2 C (CH 3 ) -CH 2 -CH 2 - O-PO (OH) 2 , detected.
  • Another object of this invention is the use of these measuring methods for the determination of substances which inhibit the activity of the respective enzymes. It has been found that the deoxy-D-xylulose-phosphate pathway is also present in many parasites, viruses and fungi.
  • the invention therefore also includes a method for screening a compound.
  • a host organism which contains a recombinant expression vector, the vector having at least a part of the oligonucleotide sequence which codes for the gcpE or the yfgB protein, or variants or homologues thereof, and also a compound which is suspected that it has an antimicrobial, antiparasitic, antiviral and antifungal activity in humans and animals or a bactericidal, antimicrobial, herbicidal or fungicidal activity in plants.
  • the host organism is then brought into contact with the compound and the effectiveness of the compound is determined.
  • the plasmid pAC-LYC was constructed according to published protocols (Cunningham, FX Jr et al., 1996, Plant Cell 8: 1613-1626).
  • the plasmid carries the genes that are required for the synthesis of the carotenoid lycopene from IPP and DMAPP.
  • E. coli cells transformed with pAC-LYC therefore form pink colonies. If the availability of the starting substances for carotenoid synthesis is increased, carotenoids are increasingly enriched and the colonies appear deep pink. An increased formation of starting substances can be achieved by overexpressing genes of the DOXP pathway.
  • the gcpe and yfgB genes from E. coli were cloned into suitable expression vectors.
  • the gcpe gene was PCR by primers 5'-CCA TGG GCC ATA ACC AGG CTC CAA TCC AA-3 'and 5'-GGA TCC TTT TTC AAC CTG CTG AAC GTC AAT-3' of genomic E. coli DNA amplified and cloned into the pCR2.1-TOPO vector.
  • the insert was cloned into the expression vector pQE60 via the restriction sites Nco I and Bam HI.
  • the yfgB gene was amplified with the primers 5'-GGA TCC ATG TCT GAA CAA TTA GTC ACA-3 'and 5'-AAG CTT TCA GAC CGC TTT AAT GTC GAT GGC-3' and in the pCRT7 / NT TOPO - vector cloned.
  • the insert was cloned into the expression vector pQE30 via the restriction cleavage Bam HI and Hind III.
  • Bacteria which had been transformed with pAC-LYC and one of the two expression constructs showed a significantly deeper staining than bacteria which, as a control, had additionally been transformed only with the empty pQE30 vector.
  • Photometric quantification of the carotenoid enrichment gave 210% for gcpe and 173% for yfgB based on the control.
  • falciparum strain 3D7 was used as a template and thermostable Pwo DNA polymerase.
  • the PCR products were phosphorylated with T4 polynucleotide kinase and cloned into pQE32 vectors that had been linearized with Sma I and dephosphorylated with alkaline phosphatase.
  • the orientation of the inserts was verified by restriction analysis.
  • the bacterial colonies obtained with these constructs showed no clear color changes.
  • photometric evaluation showed a carotenoid enrichment of 117% (gcpe) and 113% (yfgB).
  • the relatively low carotenoid accumulation with the P. falciparum genes is apparently due to the frequently observed low expression of P. falciparum genes in E. coli as a result of the high A / T content.

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Abstract

The invention relates to the use of DNA sequences from bacteria and parasites, namely the genes gcpE and yfgB for integration in the genome of viruses, eukaryotes, and prokaryotes and thereby altering the concentration of isoprenoid. The invention also relates to methods for identifying substances that exhibit a herbicidal, antiparasitic, antiviral, and fungicidal activity in plants and an antimycotic, antiparasitic and antiviral activity in humans and animals.

Description

Nerwendung von Genen des Desoxy-D-xylulose-Phosphatbiosynthesewegs zur Veränderung der Isoprenoidkonzentration Use of genes of the deoxy-D-xylulose-phosphate biosynthetic pathway to change the isoprenoid concentration
Die vorliegende Erfindung betrifft die Nerwendung von DΝA-Sequenzen (SEQ: 1,3,5,7), die für das gcpE- oder das yfgB-Protein aus Bakterien oder Parasiten kodieren und die bei Integration in das Genom von Viren, Eukaryonten und Prokaryonten den Isoprenoid-Gehalt verändern sowie Verfahren zur Messung der Aktivität des gcpE-Gens in bezug auf die Isoprenoid- Synthese. Außerdem betrifft sie Verfahren zur Identifizierung von Stoffen mit herbizider, anti- parasitärer, antiviraler, fungizider Wirkung bei Pflanzen und antiparasitärer, antimykotischer und antiviraler Wirkung bei Mensch und Tier.The present invention relates to the use of DΝA sequences (SEQ: 1,3,5,7) which code for the gcpE or yfgB protein from bacteria or parasites and which are integrated into the genome of viruses, eukaryotes and prokaryotes change the isoprenoid content and methods for measuring the activity of the gcpE gene in relation to isoprenoid synthesis. It also relates to methods for identifying substances with herbicidal, antiparasitic, antiviral, fungicidal activity in plants and antiparasitic, antifungal and antiviral activity in humans and animals.
Der Biosyntheseweg zur Bildung von Isoprenoiden über den klassischen Acetat/Mevalonat- Weg und einen alternativen, Mevalonat-unabhängigen Biosyntheseweg, den Desoxy-D- xylulose-Phosphat-Weg, ist bereits bekannt (Rohmer, M., Knani, M., Simonin, P.~ Sutter, B., and Sahm, H. (1993): Biochem. J. 295: 517-524).The biosynthetic pathway for the formation of isoprenoids via the classic acetate / mevalonate route and an alternative, mevalonate-independent biosynthetic route, the deoxy-D-xylulose-phosphate route, is already known (Rohmer, M., Knani, M., Simonin, P. ~ Sutter, B., and Sahm, H. (1993): Biochem. J. 295: 517-524).
In der US 5 858 367 ist die Verwendung von aarC-Oligonukleotiden zum Auffinden von antibakteriellen Substanzen beschrieben.US Pat. No. 5,858,367 describes the use of aarC oligonucleotides for the detection of antibacterial substances.
Überraschend hat sich jedoch nun herausgestellt, daß das gcpE-Protein zusätzlich noch eine Kinasefünktion im alternativen Stoffwechselweg für die Isoprenoidbiosynthese hat und die Phosphorylierung eines Zuckers oder eines Phosphorzuckers oder einer Vorstufe der Isoprenoidbiosynthese, insbesondere die Phosphorylierung von 2-C-Methyl-D-erythritol, 2-C- Methyl-D-erythritol-phosphat, insbesondere 2-C-Methyl-D-erythritol-4-phosphat, 2-C-Methyl- D-erythrose, 2-C-Methyl-D-erythrose-phosphat, insbesondere 2-C-Methyl-D-erythrose-4- phosphat, CH2(OH)-C(CH3)=C(OH)-CH2-O-PO(OH)2,Surprisingly, however, it has now been found that the gcpE protein additionally has a kinase function in the alternative metabolic pathway for isoprenoid biosynthesis and the phosphorylation of a sugar or a phosphorus sugar or a precursor of isoprenoid biosynthesis, in particular the phosphorylation of 2-C-methyl-D-erythritol , 2-C-methyl-D-erythritol-phosphate, in particular 2-C-methyl-D-erythritol-4-phosphate, 2-C-methyl-D-erythrose, 2-C-methyl-D-erythrose-phosphate, in particular 2-C-methyl-D-erythrose-4-phosphate, CH 2 (OH) -C (CH 3 ) = C (OH) -CH 2 -O-PO (OH) 2 ,
CH2(OH)-C(CH3)=C(OH)-CH2-OH,CH 2 (OH) -C (CH 3 ) = C (OH) -CH 2 -OH,
CH2(OH)-CH(CH3)-CO-CH2-O-PO(OH)2, CH2(OH)-CH(CH3)-CO-CH2-OH CH2=C(CH3)-CO-CH2-O-PO(OH)2, CH2=C(CH3)-CO-CH2-OH,CH 2 (OH) -CH (CH 3 ) -CO-CH 2 -O-PO (OH) 2 , CH 2 (OH) -CH (CH 3 ) -CO-CH 2 -OH CH 2 = C (CH 3 ) -CO-CH 2 -O-PO (OH) 2, CH 2 = C (CH 3 ) -CO-CH 2 -OH,
CH2=C(CH3)-CH(OH)-CH2-O-PO(OH)2, CH2=C(CH3)-CH(OH)-CH2-OH,CH 2 = C (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2 , CH 2 = C (CH 3 ) -CH (OH) -CH 2 -OH,
CH2(OH)-C(=CH2)-C(OH)-CH2-O-PO(OH)2,CH 2 (OH) -C (= CH 2 ) -C (OH) -CH 2 -O-PO (OH) 2 ,
CH2(OH)-C(=CH2)-C(OH)-CH2-OHCH 2 (OH) -C (= CH 2 ) -C (OH) -CH 2 -OH
CHO-CH(CH3)-CH(OH)-CH2-O-PO(OH)2, CHO-CH(CH3)-CH(OH)-CH2-OH, CH2(OH)-C(OH)(CH3)-CH=CH-O-PO(OH)2,CHO-CH (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2 , CHO-CH (CH 3 ) -CH (OH) -CH 2 -OH, CH 2 (OH) -C ( OH) (CH 3 ) -CH = CH-O-PO (OH) 2 ,
CH2(OH)-C(OH)(CH3)-CH=CH-OHCH 2 (OH) -C (OH) (CH 3 ) -CH = CH-OH
CH(OH)=C(CH3)-CH(OH)-CH2-O-PO(OH)2, CH(OH)=C(CH3)-CH(OH)-CH2-OH, CH3-CH (OH) = C (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2 , CH (OH) = C (CH 3 ) -CH (OH) -CH 2 -OH, CH 3 -
C(CH3)=CH-CH2-O-PO(OH)2, CH3-C(CH3)=CH-CH2-OH, CH2=C(CH3)-CH2-CH2-O-PO(OH)2, CH2=C(CH3)-CH2-CH2-OH katalysiert.C (CH 3 ) = CH-CH 2 -O-PO (OH) 2 , CH 3 -C (CH 3 ) = CH-CH 2 -OH, CH2 = C (CH 3 ) -CH 2 -CH 2 -O-PO (OH) 2, CH 2 = C (CH 3 ) -CH 2 -CH 2 -OH catalyzed.
Die Erfindung betrifft daher die Verwendung von DNA-Sequenzen, die das gcpE- oder das yfgB-Protein aus Bakterien oder Parasiten aus Bakterien oder das gcpE- oder das yfgB-Protein aus Parasiten kodieren oder DNA-Sequenzen, die für ein Analoges oder Derivat dieses Proteins kodieren, worin eine oder mehrere Aminosäuren deletiert, hinzugefügt oder durch andere Aminosäuren substituiert worden sind, ohne die enzymatische Wirkung des Polypeptids wesentlich zu reduzieren. Sie betrifft insbesondere die Verwendung der DNA-Sequenzen SEQ 1,3,5,7. Die ursprüngliche Herkunft der angegebenen Sequenzen SEQ 1 und 5 sowie der Proteine 2 und 6 ist der Organismus Escherichia coli, Stamm K12. Die ursprüngliche Herkunft der angegebenen Sequenzen SEQ 3 und 7 sowie der Proteine 4 und 8 ist der Organismus Plasmodium Falci- parum, Stamm 3D7.The invention therefore relates to the use of DNA sequences which encode the gcpE or yfgB protein from bacteria or parasites from bacteria or the gcpE or yfgB protein from parasites or DNA sequences which are analogous or derivatives thereof Encode proteins in which one or more amino acids have been deleted, added or substituted by other amino acids without significantly reducing the enzymatic action of the polypeptide. It relates in particular to the use of the SEQ 1,3,5,7 DNA sequences. The original origin of the specified sequences SEQ 1 and 5 and of proteins 2 and 6 is the organism Escherichia coli, strain K12. The original origin of the specified sequences SEQ 3 and 7 and of proteins 4 and 8 is the organism Plasmodium Falciparum, strain 3D7.
Derartige DNA-Sequenzen sind in der US-5 858 367 offenbart sowie unter den folgenden Ac- cession-Nummern über die Internet- Adresse: http://www3.ncbi.nlm.nih.gov Entrez/protein.html zu finden: AAD07695, AAD18517, AAC75568, AAC67648, AAC65433, P36979, CAA15530, CAA98356, CAA98355, AAC24056, AAC07467, P54482, P44667, P27434, P27433, BAA17717, BAA20919, BAA16402, S23058, AAB51469, 1819264A, CAA45783, CAA45782, AAA21360, AAA21359, BAA02549, 139486, 2113330A.Such DNA sequences are disclosed in US Pat. No. 5,858,367 and can be found under the following access numbers via the Internet address: http://www3.ncbi.nlm.nih.gov Entrez / protein.html: AAD07695 , AAD18517, AAC75568, AAC67648, AAC65433, P36979, CAA15530, CAA98356, CAA98355, AAC24056, AAC07467, P54482, P44667, P27434, P27433, BAA17717, BAA20919, BA3058, A4306A4A2A4A2A4A2A4A2A4A5A4B5A5A4B5A5A4B5A5A05 , 139486, 2113330A.
Die erfindungsgemäßen Sequenzen eignen sich für die Expression von Genen in Viren, Euka- ryonten und Prokaryonten, die für die Isoprenoid-Biosynthese des 1-Desoxy-D-xylulose-Wegs verantwortlich sind.The sequences according to the invention are suitable for the expression of genes in viruses, eukaryytes and prokaryotes which are responsible for the isoprenoid biosynthesis of the 1-deoxy-D-xylulose pathway.
Erfindungsgemäß gehören zu den Eukaryonten oder eukaryontischen Zellen tierischen Zellen, Pflanzenzellen, Algen, Hefen, Pilze und zu den Prokaryonten oder prokaryontischen Bakterien Archaebakterien und Eubakterien.According to the invention, the eukaryotes or eukaryotic cells include animal cells, plant cells, algae, yeasts, fungi and the prokaryotes or prokaryotic bacteria are archaebacteria and eubacteria.
Bei Integration einer DNA-Sequenz in ein Genom, auf der eine der oben angegebenen DNA- Sequenzen lokalisiert ist, wird die Expression der oben beschriebenen Gene in Viren, Eukaryonten und Prokaryonten ermöglicht. Die erfindungsgemäß transformierten Viren, Eukaryonten und Prokaryonten werden in an sich bekannter Weise gezüchtet und das währenddessen gebildete Isoprenoid isoliert und gegebenenfalls gereinigt. Nicht alle Isoprenoide müssen iso- liert werden, da die Isoprenoide in einigen Fällen direkt in die Raumluft abgegeben werden.When a DNA sequence is integrated into a genome on which one of the above-mentioned DNA sequences is located, the expression of the above-described genes in viruses, eukaryotes and prokaryotes is made possible. The viruses, eukaryotes and prokaryotes transformed according to the invention are grown in a manner known per se and the isoprenoid formed in the process is isolated and, if appropriate, purified. Not all isoprenoids need to be isolated because in some cases the isoprenoids are released directly into the air.
Die Herstellung der verwendeten transgenen Viren, Eukaryonten und Prokaryonten zur Veränderung des Isoprenoid-Gehaltes, kann nach folgenden Schritten erfolgen: a) Herstellung einer DNA-Sequenz mit folgenden Teilsequenzen i) Promotor, der in Viren, Eukaryonten und Prokaryonten aktiv ist und die Bildung einer RNA im vorgesehenen Zielgewebe oder den Zielzellen sicherstellt, ii) DNA-Sequenz, die für ein Polypeptid mit der Aminosäuresequenz des gcpE- oder des yfgB-Proteins aus Bakterien oder Parasiten oder für ein Analoges oder Derivat dieses Polypeptids kodieren, üi) 3' -nichttranslatierte Sequenz, die in Viren, Eukaryonten und Prokaryonten zur Addition von Poly-A Resten an das 3' -Ende der RNA führt, b) Transfer und Einbau der DNA-Sequenz in das Genom von Viren, prokaryontischen oder eukaryontischen Zellen mit oder ohne Verwendung eines Vektors (z.B. Plasmid, virale DNA).The transgenic viruses, eukaryotes and prokaryotes used to change the isoprenoid content can be produced in the following steps: a) Production of a DNA sequence with the following partial sequences i) promoter which is active in viruses, eukaryotes and prokaryotes and ensures the formation of an RNA in the intended target tissue or the target cells, ii) DNA sequence which is suitable for a polypeptide with the amino acid sequence of Code gcpE or the yfgB protein from bacteria or parasites or for an analog or derivative of this polypeptide, üi) 3 'untranslated sequence which is used in viruses, eukaryotes and prokaryotes to add poly-A residues to the 3 ' end of the RNA leads, b) transfer and incorporation of the DNA sequence into the genome of viruses, prokaryotic or eukaryotic cells with or without the use of a vector (eg plasmid, viral DNA).
Aus den transformierten Pflanzenzellen können die intakten ganzen Pflanzen regeneriert wer- den.The intact whole plants can be regenerated from the transformed plant cells.
Die für die gcpE- oder die yfgB-Proteine oder ihre Analoga oder Derivate kodierenden Sequenzen können mit einem die Transkription in bestimmten Organen oder Zellen sicherstellenden Promotor versehen werden, der in sense-Orientierung (3' -Ende des Promotors zum 5' -Ende der kodierenden Sequenz) an die Sequenz, die das zu bildende Protein kodiert, gekoppelt ist. An das 3 -Ende der kodierenden Sequenz wird ein die Termination der mRNA-Synthese bestimmendes Terminationssignal angehängt. Um das zu exprimierende Protein in bestimmte subzelluläre Kompartimente, wie Chloroplasten, Amyloplasten, Mitochondrien, Vakuole, Cytosol oder Interzellularräume zu dirigieren, kann zwischen den Promotor und die kodierende Sequenz noch eine für eine sogenannte Signalsequenz oder ein Transitpeptid kodierende Sequenz gesetzt werden. Die Sequenz muß im gleichen Leserahmen wie die kodierende Sequenz des Proteins liegen. Zur Vorbereitung der Einführung der erfindungsgemäßen DNA-Sequenzen in höhere Pflanzen sind eine große Anzahl von Klonierungsvektoren verfügbar, die ein Repli- kationssignal für E.coli und einen Marker beinhalten, der eine Selektion der transformierten Zellen erlaubt. Beispiele für Vektoren sind pBR 322, pUC-Serien, M13mp-Serien, pACYC 184, EMBL 3 usw. Je nach Einführungsmethode gewünschter Gene in die Pflanze können weitere DNA-Sequenzen erforderlich sein. Werden zum Beispiel für die Transformation der Pflanzenzelle das Ti- oder Ri-Plasmid verwendet, so muß mindestens eine rechte Begrenzung, häufig jedoch die rechte und die linke Begrenzung der Ti- und Ri-Plasmid T-DNA als Flanken- bereich den einzuführenden Genen eingefügt werden. Die Verwendung von T-DNA für die Transformation von Pflanzenzellen ist intensiv untersucht und ausreichend in EP 120516; Hoe- kama, in: The Binary Plant Vector System, Offset-drukkerij Kanters B.V. Alblasserdam (1985), Chapter V; Fraley et al., Crit.Rev.Plant Sei. 4,1-46 und An et al. (1985) EMBO J. 4, 277-287 beschrieben worden. Ist die eingeführte DNA einmal im Genom integriert, so ist sie in der Regel stabil und bleibt auch in den Nachkommen der ursprünglich transformierten Zellen erhalten. Sie erhält normalerweise einen Selektionsmarker, der den transformierten Pflanzenzellen Resistenz gegenüber einem Biozid oder einem Antibiotikum, wie Kanamycin, G 418, Bleomycin, Hygromycin oder Phosphinotricin u.a. vermittelt. Der individuell verwendete Marker sollte daher die Selektion transformierter Zellen gegenüber Zellen, denen die eingefügte DNA fehlt, gestatten.The sequences coding for the gcpE or the yfgB proteins or their analogs or derivatives can be provided with a promoter which ensures transcription in certain organs or cells and which is in sense orientation (3 'end of the promoter to the 5' end of the coding sequence) is coupled to the sequence encoding the protein to be formed. A termination signal determining the termination of the mRNA synthesis is appended to the 3 end of the coding sequence. In order to direct the protein to be expressed into certain subcellular compartments, such as chloroplasts, amyloplasts, mitochondria, vacuoles, cytosols or intercellular spaces, a sequence coding for a so-called signal sequence or a transit peptide can be placed between the promoter and the coding sequence. The sequence must be in the same reading frame as the coding sequence of the protein. To prepare for the introduction of the DNA sequences according to the invention into higher plants, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker which permits selection of the transformed cells. Examples of vectors are pBR 322, pUC series, M13mp series, pACYC 184, EMBL 3 etc. Depending on the method of introducing desired genes into the plant, further DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, at least one right boundary, but often the right and left boundary of the Ti and Ri plasmid T-DNA, must be inserted as the flank region of the genes to be introduced become. The use of T-DNA for the transformation of plant cells has been intensively investigated and is sufficient in EP 120516; Hoekama, in: The Binary Plant Vector System, Offset-drukkerij Kanters BV Alblasserdam (1985), Chapter V; Fraley et al., Crit.Rev.Plant Sei. 4,1-46 and An et al. (1985) EMBO J. 4, 277-287 have been described. Once the inserted DNA is integrated in the genome, it is usually stable and is also retained in the offspring of the originally transformed cells. It normally receives a selection marker which imparts resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin and the like, to the transformed plant cells. The individually used marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA.
Für die Einführung von DNA in eine Pflanze stehen viele Techniken zur Verfügung. Diese Techniken umfassen die Transformation mit Hilfe von Agrobakterien, z.B. Agrobacterium tu- mefaciens , die Fusion von Protoplasten, die Mikroinjektion von DNA, die Elekroporation, sowie ballistische Methoden und die Virusinfektion. Aus transformiertem Pflanzenmaterial können dann im geeigneten Medium, welches Antibiotika oder Biozide zur Selektion enthalten kann, wieder ganze Pflanzen regeneriert werden. Bei der Injektion und Elektroporation sind an sich keine speziellen Anforderungen an die Plasmide gestellt. Sollen aber aus derartig transformierten Zellen ganze Pflanzen regeneriert werden, ist die Anwesenheit eines selektierbaren Markergens notwendig. Die transformierten Zellen wachsen innerhalb der Pflanzen in der üblichen Weise (McCormick et al. (1986), Plant Cell Reports 5, 81-84). Die Pflanzen können normal angezogen werden und mit Pflanzen, die die gleiche transformierte Erbanlage oder andere Erbanlagen haben, gekreuzt werden. Die daraus entstehenden Individuen haben die entsprechenden phänotypi sehen Eigenschaften.Many techniques are available for introducing DNA into a plant. These techniques include transformation using agrobacteria, e.g. Agrobacterium tu- mefaciens, the fusion of protoplasts, the microinjection of DNA, electroporation, as well as ballistic methods and virus infection. Whole plants can then be regenerated from transformed plant material in a suitable medium, which may contain antibiotics or biocides for selection. When it comes to injection and electroporation, there are no special requirements for the plasmids. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary. The transformed cells grow within the plants in the usual way (McCormick et al. (1986), Plant Cell Reports 5, 81-84). The plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup. The resulting individuals have the corresponding phenotypic properties.
Für die Einführung der DNA in die Wirtsorganismen eignen sich Expressionsvektoren, die eine oder mehrere der erfindungsgemäßen DNA-Sequenzen enthalten. Solche Expressionsvektoren erhält man, indem man die erfindungsgemäßen DNA-Sequenzen mit geeigneten fünktionellen Regulationssignalen versieht. Solche Regulationssignale sind DNA-Sequenzen, die für die Expression verantwortlich sind, beispielsweise Promotoren, Operatoren, Enhancer, ribosomale Bindungsstellen, und die vom Wirtsorganismus erkannt werden.Expression vectors which contain one or more of the DNA sequences according to the invention are suitable for introducing the DNA into the host organisms. Such expression vectors are obtained by providing the DNA sequences according to the invention with suitable functional regulatory signals. Such regulatory signals are DNA sequences which are responsible for expression, for example promoters, operators, enhancers, ribosomal binding sites and which are recognized by the host organism.
Gegebenenfalls können noch weitere Regulationssignale, die beispielsweise Replikation oder Rekombination der rekombinanten DNA im Wirtsorganismus steuern, Bestandteil des Expressionsvektors sein.If appropriate, further regulation signals, which control, for example, replication or recombination of the recombinant DNA in the host organism, can be part of the expression vector.
Für die Expression der erfindungsgemäßen Enzyme eignen sich besonders solche Wirtszellen und Organismen, die keine intrinsischen Enzyme mit der Funktion der DOXP-Synthase, der DOXP-Reduktoisomerase oder der gcpE-Kinase aufweisen. Dies trifft für Archaebacterien, Tiere, Pilze, Schleimpilze und einige Eubakterien zu. Durch das Fehlen dieser intrinsischen Enzymaktivitäten wird die Detektion und Aufreinigung der rekombinanten Enzyme wesentlich erleichtert. Auch wird es erst dadurch möglich, mit geringem Aufwand die Aktivität und insbesondere die Hemmung der Aktivität der erfindungsgemäßen rekombinanten Enzyme durch verschiedenen Chemikalien und Pharmaka in Rohextrakten aus den Wirtszellen zu messen.Particularly suitable for the expression of the enzymes according to the invention are host cells and organisms which have no intrinsic enzymes with the function of DOXP synthase, DOXP reductoisomerase or gcpE kinase. This applies to archaebacteria, animals, fungi, slime molds and some eubacteria. The lack of these intrinsic enzyme activities makes the detection and purification of the recombinant enzymes essential facilitated. This also makes it possible to measure the activity and in particular the inhibition of the activity of the recombinant enzymes according to the invention by various chemicals and pharmaceuticals in crude extracts from the host cells with little effort.
Die Expression der erfindungsgemäßen Enzyme erfolgt vorteilhaft dann in eukaryontischen Zellen, wenn posttranslatorische Modifikationen und eine native Faltung der Polypeptidkette erreicht werden soll. Außerdem wird in Abhängigkeit vom Expressionssystem bei der Expression genomischer DNA-Sequenzen erreicht, daß Introns durch Spleißen der DNA beseitigt und die Enzyme in der für die Parasiten charakteristischen Polypeptidsequenz produziert werden. Für Introns codierende Sequenzen können auch durch rekombinante DNA-Technologie aus den zu exprimierenden DNA-Sequenzen beseitigt oder experimentell eingefügt werden.The enzymes according to the invention are advantageously expressed in eukaryotic cells if post-translational modifications and a native folding of the polypeptide chain are to be achieved. In addition, depending on the expression system in the expression of genomic DNA sequences, introns are eliminated by splicing the DNA and the enzymes are produced in the polypeptide sequence characteristic of the parasites. Sequences coding for introns can also be removed by recombinant DNA technology from the DNA sequences to be expressed or inserted experimentally.
Die Isolierung des Proteins kann aus der Wirtszelle oder dem Kulturüberstand der Wirtszelle nach dem Fachmann bekannten Verfahren erfolgen. Es kann auch eine in vitro Reaktivierung der Enzyme erforderlich sein.The protein can be isolated from the host cell or the culture supernatant of the host cell by methods known to the person skilled in the art. In vitro reactivation of the enzymes may also be required.
Zur Erleichterung der Aufreinigung können die erfindungsgemäßen Enzyme oder Teilsequenzen der Enzyme als Fusionsprotein mit verschiedenen Peptidketten exprimiert werden. Dazu eigenen sich besonders Oligo-Histidin-Sequenzen und Sequenzen, die von der Glutathion-S- Transferase, Thioredoxin oder Calmodulin-bindenden Peptiden abgeleitet sind. Fusionen mit Thioredoxin-abgeleiteten Sequenzen eignen sich besonders für prokaryontische Expression, da dadurch die Löslichkeit der rekombinanten Enzyme erhöht wird.To facilitate the purification, the enzymes according to the invention or partial sequences of the enzymes can be expressed as a fusion protein with various peptide chains. Oligo-histidine sequences and sequences derived from glutathione-S-transferase, thioredoxin or calmodulin-binding peptides are particularly suitable for this purpose. Fusions with thioredoxin-derived sequences are particularly suitable for prokaryotic expression, since this increases the solubility of the recombinant enzymes.
Weiterhin können die erfindungsgemäßen Enzyme oder Teilsequenzen der Enzyme als Fusi- onsprotein mit solchen, dem Fachmann bekannten, Peptidketten exprimiert werden, daß die rekombinanten Enzyme in das extrazelluläre Millieu oder in bestimmte Kompartimente der Wirtszellen transportiert werden. Dadurch kann sowohl die Aufreinigung, als auch die Untersuchung der biologischen Aktivität der Enzyme erleichtert werden.Furthermore, the enzymes according to the invention or partial sequences of the enzymes can be expressed as fusion proteins with peptide chains known to those skilled in the art that the recombinant enzymes are transported into the extracellular environment or into certain compartments of the host cells. This enables both the purification and the investigation of the biological activity of the enzymes to be facilitated.
Bei der Expression der erfindungsgemäßen Enzyme kann es sich als zweckmäßig erweisen, einzelne Codone zu verändern. Dabei ist der gezielte Austausch von Basen in der kodierenden Region auch sinnvoll, wenn die genutzten Codone in den Parasiten abweichend sind von der Codonnutzung im heterologen Expressionssystem, um eine optimale Synthese des Proteins zu gewährleisten. Zudem sind oft Deletionen von nicht-translatierten 5 'bzw. 3 '-Abschnitten sinn- voll, beispielsweise wenn mehrere destabilisierende Sequenzmotive ATTTA im 3'-Bereich der DNA vorliegen. Dann sollten diese bei der bevorzugen Expression in Eukaryonten deletiert werden. Veränderungen dieser Art sind Deletionen, Additionen oder Austausch von Basen und ebenfalls Gegenstand der vorliegenden Erfindung. Weiterhin können die erfindungsgemäßen Enzyme unter standardisierten Bedingungen durch dem Fachmann bekannte Techniken durch in vitro-Translation gewonnen werden. Dafür geeignete Systeme sind Kaninchen-Reticulozyten- und Weizenkeimextrakte und Bakterienlysate. Auch kann in vitro transskribierte mRNA in Xenopus-Oocyten translatiert werden.When expressing the enzymes according to the invention, it may prove expedient to change individual codons. The targeted exchange of bases in the coding region also makes sense if the codons used in the parasites differ from the codon use in the heterologous expression system in order to ensure optimal synthesis of the protein. In addition, deletions of untranslated 5 'or 3 'sections make sense, for example if there are several destabilizing sequence motifs ATTTA in the 3' region of the DNA. Then these should be deleted in the preferred expression in eukaryotes. Changes of this type are deletions, additions or exchange of bases and are also the subject of the present invention. Furthermore, the enzymes according to the invention can be obtained under standardized conditions by techniques known to the person skilled in the art by in vitro translation. Suitable systems are rabbit reticulocyte and wheat germ extracts and bacterial lysates. In vitro transcribed mRNA can also be translated into Xenopus oocytes.
Durch chemische Synthese können Oligo- und Polypeptide hergestellt werden, der Sequenzen aus der Peptidsequenz der erfindungsgemäßen Enzyme abgeleitet sind. Bei geeigneter Wahl der Sequenzen besitzen derartige Peptide Eigenschaften, die für die vollständigen erfindungsgemä- ßen Enzyme charakteristisch sind. Derartige Peptide können in großen Mengen hergestellt werden und eignen sich besonders für Studien über die Kinetik der Enzymaktivität, die Regulation der Enzymaktivität, die dreidimensionale Struktur der Enzyme, die Hemmung der Enzymakti- vität durch verschiedenen Chemikalien und Pharmaka und die Bindungsgeometrie und Bindungsaffinität verschiedener Liganden.Oligo- and polypeptides can be produced by chemical synthesis, the sequences being derived from the peptide sequence of the enzymes according to the invention. With a suitable choice of the sequences, such peptides have properties which are characteristic of the complete enzymes according to the invention. Such peptides can be produced in large quantities and are particularly suitable for studies on the kinetics of enzyme activity, the regulation of enzyme activity, the three-dimensional structure of the enzymes, the inhibition of enzyme activity by different chemicals and pharmaceuticals and the binding geometry and binding affinity of different ligands.
Ein weiterer Gegenstand dieser Erfindung sind Methoden zur Bestimmung der enzymatische Aktivität der gcpE-Kinase. Diese kann nach den bekannten Anleitungen bestimmt werden. Hierbei wird die Phosphorylierung eines Zuckers oder eines Phosphorzuckers oder einer Vorstufe der Isoprenoidbiosynthese, insbesondere die Phosphorylierung von 2-C-Methyl-D- erythritol, 2-C-Methyl-D-erythritol-phosphat, insbesondere 2-C-Methyl-D-erythritol-4- phosphat, 2-C-Methyl-D-erythrose, 2-C-Methyl-D-erythrose-phosphat, insbesondere 2-C- Methyl-D-erythrose-4-phosphat, CH2(OH)-C(CH3)=C(OH)-CH2-O-PO(OH)2, CH2(OH)-C(CH3)=C(OH)-CH2-OH, CH2(OH)-CH(CH3)-CO-CH2-O-PO(OH)2, CH2(OH)-CH(CH3)-CO-CH2-OH CH2=C(CH3)-CO-CH2-OH,Another object of this invention are methods for determining the enzymatic activity of gcpE kinase. This can be determined according to the known instructions. The phosphorylation of a sugar or a phosphorus sugar or a precursor of isoprenoid biosynthesis, in particular the phosphorylation of 2-C-methyl-D-erythritol, 2-C-methyl-D-erythritol-phosphate, in particular 2-C-methyl-D- erythritol-4-phosphate, 2-C-methyl-D-erythrose, 2-C-methyl-D-erythrose-phosphate, especially 2-C-methyl-D-erythrose-4-phosphate, CH2 (OH) -C ( CH 3 ) = C (OH) -CH 2 -O-PO (OH) 2, CH 2 (OH) -C (CH 3 ) = C (OH) -CH 2 -OH, CH 2 (OH) -CH ( CH 3 ) -CO-CH 2 -O-PO (OH) 2 , CH 2 (OH) -CH (CH 3 ) -CO-CH2-OH CH 2 = C (CH 3 ) -CO-CH 2 -OH,
CH2=C(CH3)-CH(OH)-CH2-O-PO(OH)2, CH2=C(CH3)-CH(OH)-CH2-OH, CH2(OH)-C(=CH2)-C(OH)-CH2-O-PO(OH)2, CH2(OH)-C(=CH2)-C(OH)-CH2-OH CHO-CH(CH3)-CH(OH)-CH2-O-PO(OH)2, CHO-CH(CH3)-CH(OH)-CH2-OH, CH2(OH)-C(OH)(CH3)-CH*=CH-O-PO(OH)2, CH2(OH)-C(OH)(CH3)-CH=CH-OHCH 2 = C (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2 , CH 2 = C (CH 3 ) -CH (OH) -CH 2 -OH, CH2 (OH) -C (= CH 2 ) -C (OH) -CH 2 -O-PO (OH) 2, CH 2 (OH) -C (= CH 2 ) -C (OH) -CH 2 -OH CHO-CH (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2 , CHO-CH (CH 3 ) -CH (OH) -CH 2 -OH, CH 2 (OH) -C (OH) (CH 3 ) -CH * = CH-O-PO (OH) 2 , CH 2 (OH) -C (OH) (CH 3 ) -CH = CH-OH
CH(OH)=C(CH3)-CH(OH)-CH2-O-PO(OH)2, CH(OH)=C(CH3)-CH(OH)-CH2-OH, CH3- C(CH3)=CH-CH2-O-PO(OH)2, CH3-C(CH3)=CH-CH2-OH, CH2=C(CH3)-CH2-CH2-O-PO(OH)2, delektiert. Ein weiterer Ge- genstand dieser Erfindung ist die Verwendung dieser Meßverfahren zur Ermittlung von Stoffen, die die Aktivität der jeweiligen Enzyme inhibieren. Es hat sich herausgestellt, daß auch in vielen Parasiten, Viren und Pilzen dieser der Desoxy-D- xylulose-Phosphat-Stofϊwechselweg ebenfalls vorliegt.CH (OH) = C (CH 3 ) -CH (OH) -CH2-O-PO (OH) 2, CH (OH) = C (CH 3 ) -CH (OH) -CH 2 -OH, CH 3 - C (CH 3 ) = CH-CH 2 -O-PO (OH) 2 , CH 3 -C (CH 3 ) = CH-CH 2 -OH, CH 2 = C (CH 3 ) -CH 2 -CH 2 - O-PO (OH) 2 , detected. Another object of this invention is the use of these measuring methods for the determination of substances which inhibit the activity of the respective enzymes. It has been found that the deoxy-D-xylulose-phosphate pathway is also present in many parasites, viruses and fungi.
Die Erfindung umfaßt daher außerdem ein Verfahren zum Screening einer Verbindung. Gemäß diesem Verfahren wird ein Wirtsorganismus, der einen rekombinanten Expressionsvektor enthält, wobei der Vektor zumindest einen Teil der Olignukleotidsequenz, die für das gcpE- oder das yfgB-Protein kodiert, oder Varianten oder Homologe dieser aufweist, und außerdem eine Verbindung, von der vermutet wird, daß sie eine antimikrobielle, antiparasitäre, antivirale und antimykotische Wirkung bei Mensch und Tier oder eine bakterizide, antimikrobielle, herbizide oder füngizide Wirkung bei Pflanzen hat, bereitgestellt. Anschließend wird der Wirtsorganismus mit der Verbindung in Kontakt gebracht und die Wirksamkeit der Verbindung bestimmt.The invention therefore also includes a method for screening a compound. According to this method, a host organism which contains a recombinant expression vector, the vector having at least a part of the oligonucleotide sequence which codes for the gcpE or the yfgB protein, or variants or homologues thereof, and also a compound which is suspected that it has an antimicrobial, antiparasitic, antiviral and antifungal activity in humans and animals or a bactericidal, antimicrobial, herbicidal or fungicidal activity in plants. The host organism is then brought into contact with the compound and the effectiveness of the compound is determined.
Beispiel 1example 1
Erhöhte Carotinoid-Akkumulation in E. coli durch Überexpression von gcpe und yfgB.Increased carotenoid accumulation in E. coli due to overexpression of gcpe and yfgB.
Zunächst wurde das Plasmid pAC-LYC nach publizierten Protokollen konstruiert (Cunning- ham, F X Jr et al., 1996, Plant Cell 8: 1613-1626). Das Plasmid trägt die Gene, die für die Synthese des Carotinoids Lycopen aus IPP und DMAPP benötigt werden. E. coli-Zellen, die mit pAC-LYC transformiert wurden, bilden deshalb rosafarbene Kolonien. Wird die Verfüg- barkeit der Ausgangssubstanzen für die Carotinoidsynthese erhöht, werden verstärkt Carotinoi- de angereichert und die Kolonien erscheinen tief rosafarben. Eine verstärkte Bildung von Ausgangssubstanzen kann dadurch erreicht werden, daß Gene des DOXP-Wegs überexprimiert werden. Dazu wurden das gcpe- und yfgB- Gen von E. coli in geeignete Expressionsvektoren kloniert. Das gcpe-Gen wurde durch PCR mit den Primern 5'-CCA TGG GCC ATA ACC AGG CTC CAA TCC AA-3 ' und 5'-GGA TCC TTT TTC AAC CTG CTG AAC GTC AAT- 3' von genomischer E. coli-DNA amplifiziert und in den pCR2.1-TOPO- Vektor kloniert. Das Insert wurde über die Restriktionsschnittstellen Nco I und Bam H I in den Expressionsvektor pQE60 umkloniert. Das yfgB-Gen wurde mit den Primern 5'-GGA TCC ATG TCT GAA CAA TTA GTC ACA-3' und 5'-AAG CTT TCA GAC CGC TTT AAT GTC GAT GGC-3' amplifi- ziert und in den pCRT7/NT TOPO- Vektor kloniert. Das Insert wurde über die Restriktions- schnittsteüen Bam H I und Hind III in den Expressionsvektor pQE30 umkloniert. Bakterien, die mit pAC-LYC und einem der beiden Expressionskonstrukte transformiert worden waren, zeigten eine deutlich tiefere Färbung als Bakterien, die als Kontrolle zusätzlich nur mit dem leeren pQE30 Vektor transformiert worden waren. Photometrische Quantifizierung der Caroti- noid- Anreicherung ergab 210 % für gcpe und 173 % für yfgB bezogen auf die Kontrolle. Beispiel 2First, the plasmid pAC-LYC was constructed according to published protocols (Cunningham, FX Jr et al., 1996, Plant Cell 8: 1613-1626). The plasmid carries the genes that are required for the synthesis of the carotenoid lycopene from IPP and DMAPP. E. coli cells transformed with pAC-LYC therefore form pink colonies. If the availability of the starting substances for carotenoid synthesis is increased, carotenoids are increasingly enriched and the colonies appear deep pink. An increased formation of starting substances can be achieved by overexpressing genes of the DOXP pathway. For this purpose, the gcpe and yfgB genes from E. coli were cloned into suitable expression vectors. The gcpe gene was PCR by primers 5'-CCA TGG GCC ATA ACC AGG CTC CAA TCC AA-3 'and 5'-GGA TCC TTT TTC AAC CTG CTG AAC GTC AAT-3' of genomic E. coli DNA amplified and cloned into the pCR2.1-TOPO vector. The insert was cloned into the expression vector pQE60 via the restriction sites Nco I and Bam HI. The yfgB gene was amplified with the primers 5'-GGA TCC ATG TCT GAA CAA TTA GTC ACA-3 'and 5'-AAG CTT TCA GAC CGC TTT AAT GTC GAT GGC-3' and in the pCRT7 / NT TOPO - vector cloned. The insert was cloned into the expression vector pQE30 via the restriction cleavage Bam HI and Hind III. Bacteria which had been transformed with pAC-LYC and one of the two expression constructs showed a significantly deeper staining than bacteria which, as a control, had additionally been transformed only with the empty pQE30 vector. Photometric quantification of the carotenoid enrichment gave 210% for gcpe and 173% for yfgB based on the control. Example 2
Erhöhte Carotinoid-Akkumulation in P. falciparum durch Überexpression von gcpe und yfgBIncreased carotenoid accumulation in P. falciparum due to overexpression of gcpe and yfgB
Analoge Versuche wurden mit dem gcpe- und yfgB- Gen von P. falciparum durchgeführt. Das gcpe- Gen wurde mit den Primern 5'- CTG ATT CTT TTT ATG TTA CTG TTT TAT TCT CAT GTA-3' und 5'-CTA CCC TTT TTT ATT TGT AAG AAC ATC ATT AGT TAC GTT AAC-3' und das yfgB- Gen mit den Primern GAA AAG TCA AAA AGG TAC ATA AGC CTG ATT AAG und 5'-TAA CAT GTC CTC TAT GCC TAT ATA TTT ATA TAA TG-3' amplifiziert. Für die PCR wurde genomische DNA des P. falciparum- Stamms 3D7 als Template und thermostabile Pwo-DNA-Polymerase verwendet. Die PCR-Produkte wurden mit T4- Polynukleotidkinase phosphoryliert und in pQE32- Vektoren kloniert, die mit Sma I linearisiert und mit alkalischer Phosphatase dephosphoryliert worden waren. Die Orientierung der Inserts wurde durch Restriktionsanalyse verifiziert. Die Bakterienkolonien, die mit diesen Konstrukten erhalten wurden, zeigten optisch keine deutlichen Farbveränderungen. Photometrische Auswertung ergab jedoch eine Carotinoid- Anreicherung von 117 % (gcpe) und 113 % (yfgB). Die relativ geringe Carotinoid- Anreicherung mit den P. falciparum- Genen ist offensichtlich auf die häufig zu beobachtende geringe Expression von P. falciparum-Genen in E. coli in Folge des hohen A/T-Gehalts zurückzuführen. Analogous experiments were carried out with the gcpe and yfgB gene from P. falciparum. The gcpe gene was primed with 5'-CTG ATT CTT TTT ATG TTA CTG TTT TAT TCT CAT GTA-3 'and 5'-CTA CCC TTT TTT ATT TGT AAG AAC ATC ATT AGT TAC GTT AAC-3' and the yfgB - Gen amplified with the primers GAA AAG TCA AAA AGG TAC ATA AGC CTG ATT AAG and 5'-TAA CAT GTC CTC TAT GCC TAT ATA TTT ATA TAA TG-3 '. For the PCR, genomic DNA of the P. falciparum strain 3D7 was used as a template and thermostable Pwo DNA polymerase. The PCR products were phosphorylated with T4 polynucleotide kinase and cloned into pQE32 vectors that had been linearized with Sma I and dephosphorylated with alkaline phosphatase. The orientation of the inserts was verified by restriction analysis. The bacterial colonies obtained with these constructs showed no clear color changes. However, photometric evaluation showed a carotenoid enrichment of 117% (gcpe) and 113% (yfgB). The relatively low carotenoid accumulation with the P. falciparum genes is apparently due to the frequently observed low expression of P. falciparum genes in E. coli as a result of the high A / T content.

Claims

Patentansprüche claims
1. Verwendung der DNA-Sequenz des gcpE- oder des yfgB-Gens aus Bakterien oder Pa- rasiten zur Einführung in das Genom von Viren, eukaryontischen und prokaryantischen1. Use of the DNA sequence of the gcpE or yfgB gene from bacteria or parasites for introduction into the genome of viruses, eukaryotic and prokaryantic
Zellen.Cells.
2. Verwendung von DNA-Sequenzen, die mit der DNA-Sequenz des gcpE- oder des yfgB- Proteins aus Bakterien oder Parasiten oder deren Analoga oder deren Derivaten, die durch Insertion, Deletion oder Substitution von dieser Sequenz abgeleitet sind, hybridisieren und für ein plastidiäres Protein kodieren, das die biologischen Aktivität des gcpE- oder des yfgB-Gens besitzt, zur Einführung in das Genom von Viren, eukaryontischen und prokaryantischen Zellen.2. Use of DNA sequences which hybridize with the DNA sequence of the gcpE or yfgB protein from bacteria or parasites or their analogs or their derivatives, which are derived from this sequence by insertion, deletion or substitution, and for a Encode plastid protein, which has the biological activity of the gcpE or yfgB gene, for introduction into the genome of viruses, eukaryotic and prokaryantic cells.
3. Verwendung der DNA- Sequenz SEQ 1, 3, 5 oder 7 nach einem der Ansprüche 1 oder 2.3. Use of the DNA sequence SEQ 1, 3, 5 or 7 according to one of claims 1 or 2.
4. Verwendung nach Anspruch 1, 2 oder 3, dadurch gekennzeichnet, daß diese Sequenzen mit Steuerelementen, die die Transkription und Translation in den Zellen gewährleisten, verknüpft sind und zur Expression einer translatierbaren mRNA, die die Synthese eines gcpE- oder das yfgB-Gens bewirkt, führen.4. Use according to claim 1, 2 or 3, characterized in that these sequences are linked to control elements which ensure transcription and translation in the cells and for the expression of a translatable mRNA which the synthesis of a gcpE or the yfgB gene causes lead.
5. Pflanzenzellen, enthaltend die DNA-Sequenz oder DNA-Sequenzen, die mit der DNA- Sequenz des gcpE- oder des yfgB-Gens aus Bakterien oder Parasiten oder deren Analoga oder deren Derivaten, die durch Insertion, Deletion oder Substitution von dieser Se- quenz abgeleitet sind, hybridisieren und für ein plastidiäres Protein kodieren, das die biologischen Aktivität des gcpE- oder des yfgB-Proteins besitzt.5. Plant cells containing the DNA sequence or DNA sequences which are linked to the DNA sequence of the gcpE or yfgB gene from bacteria or parasites or their analogs or their derivatives by insertion, deletion or substitution of this sequence. are derived, hybridize and code for a plastid protein that has the biological activity of the gcpE or yfgB protein.
6. Transformierte Pflanzenzellen und aus diesen regenerierte transgene Pflanzen, enthaltend die DNA-Sequenz oder DNA-Sequenzen, die mit der DNA-Sequenz des gcpE- oder des yfgB-Gens aus Bakterien oder Parasiten oder deren Analoga oder deren Derivaten, die durch Insertion, Deletion oder Substitution von dieser Sequenz abgeleitet sind, hybridisieren und für ein plastidiäres Protein kodieren, das die biologischen Aktivität des gcpE- oder des yfgB-Proteins besitzt.6. Transformed plant cells and transgenic plants regenerated therefrom, containing the DNA sequence or DNA sequences which are compatible with the DNA sequence of the gcpE or yfgB gene from bacteria or parasites or their analogs or their derivatives, by insertion, Deletion or substitution derived from this sequence hybridize and code for a plastid protein that has the biological activity of the gcpE or yfgB protein.
7. Verwendung nach einem der Ansprüche 1 bis 4 zur Veränderung, insbesondere Erhöhung des Isoprenoidgehalts in Viren, eukaryontischen und prokaryontischen Zellen. 7. Use according to one of claims 1 to 4 for changing, in particular increasing the isoprenoid content in viruses, eukaryotic and prokaryotic cells.
8. Verwendung nach einem der Ansprüche 1 bis 4 zur Bestimmung der enzymatischen Aktivität des gcpE- oder des yfgB-Proteins.8. Use according to one of claims 1 to 4 for determining the enzymatic activity of the gcpE or the yfgB protein.
9. Verwendung nach einem der Ansprüche 1 bis 4 zur Identifizierung von Substanzen, die eine inhibierende Wirkung auf die enzymatische Aktivität des gcpE-Proteins haben.9. Use according to one of claims 1 to 4 for the identification of substances which have an inhibitory effect on the enzymatic activity of the gcpE protein.
10. Verfahren zur Bestimmung der enzymatische Aktivität des gcpE-Proteins aus Bakterien oder Parasiten, dadurch gekennzeichnet, daß Phosphorylierung eines Zuckers oder eines Phosphorzuckers oder einer Vorstufe der Isoprenoidbio Synthese, insbesondere die Phosphorylierung von 2-C-Methyl-D-erythritol, 2-C-Methyl-D-erythritol-phosphat, insbesondere 2-C-Methyl-D-erythritol-4-phosphat, 2-C-Methyl-D-erythrose, 2-C-Methyl- D-erythrose-phosphat, insbesondere 2-C-Methyl-D-erythrose-4-phosphat, CH∑ OH)- C(CH3)=C(OH)-CH2-O-PO(OH)2, CH2(OH)-C(CH3)=C(OH)-CH2-OH, CH2(OH)-CH(CH3)-CO-CH2-O-PO(OH)2, CH2(OH)-CH(CH3)-CO-CH2-OH10. A method for determining the enzymatic activity of the gcpE protein from bacteria or parasites, characterized in that phosphorylation of a sugar or a phosphorus sugar or a precursor of isoprenoid bio synthesis, in particular the phosphorylation of 2-C-methyl-D-erythritol, 2- C-methyl-D-erythritol phosphate, in particular 2-C-methyl-D-erythritol-4-phosphate, 2-C-methyl-D-erythrose, 2-C-methyl-D-erythrose phosphate, in particular 2- C-methyl-D-erythrose-4-phosphate, CH∑ OH) - C (CH 3 ) = C (OH) -CH 2 -O-PO (OH) 2, CH 2 (OH) -C (CH 3 ) = C (OH) -CH 2 -OH, CH 2 (OH) -CH (CH 3 ) -CO-CH 2 -O-PO (OH) 2, CH 2 (OH) -CH (CH 3 ) -CO- CH 2 -OH
CH2=C(CH3)-CO-CH2-O-PO(OH)2, CH2=C(CH3)-CO-CH2-OH, CH2=C(CH3)-CH(OH)-CH2-O-PO(OH)2, CH2=C(CH3)-CH(OH)-CH2-OH, CH2(OH)-C(=CH2)-C(OH)-CH2-O-PO(OH)2, CH2(OH)-C(=CH2)-C(OH)-CH2-OH CHO-CH(CH3)-CH(OH)-CH2-O-PO(OH)2, CHO-CH(CH3)-CH(OH)-CH2-OH,CH2 = C (CH 3 ) -CO-CH 2 -O-PO (OH) 2, CH2 = C (CH 3 ) -CO-CH2-OH, CH 2 = C (CH 3 ) -CH (OH) -CH 2 -O-PO (OH) 2 , CH 2 = C (CH 3 ) -CH (OH) -CH 2 -OH, CH 2 (OH) -C (= CH 2 ) -C (OH) -CH 2 - O-PO (OH) 2 , CH 2 (OH) -C (= CH 2 ) -C (OH) -CH 2 -OH CHO-CH (CH 3 ) -CH (OH) -CH 2 -O-PO ( OH) 2 , CHO-CH (CH 3 ) -CH (OH) -CH 2 -OH,
CH2(OH)-C(OH)(CH3)-CH=CH-O-PO(OH)2, CH2(OH)-C(OH)(CH3)-CH=CH-OH CH(OH)=C(CH3)-CH(OH)-CH2-O-PO(OH)2, CH(OH)=C(CH3)-CH(OH)-CH2-OH, CH3-C(CH3)=CH-CH2-O-PO(OH)2, CH3-C(CH3)=CH-CH2-OH,CH 2 (OH) -C (OH) (CH 3 ) -CH = CH-O-PO (OH) 2, CH 2 (OH) -C (OH) (CH 3 ) -CH = CH-OH CH (OH ) = C (CH 3 ) -CH (OH) -CH2-O-PO (OH) 2 , CH (OH) = C (CH 3 ) -CH (OH) -CH 2 -OH, CH 3 -C (CH 3 ) = CH-CH 2 -O-PO (OH) 2 , CH 3 -C (CH 3 ) = CH-CH 2 -OH,
CH2K.(CH3)-CH2-CH2-O-PO(OH)2, CH2=C(CH3)-CH2-CH2-OH detektiert wird.CH 2 K. (CH 3 ) -CH 2 -CH 2 -O-PO (OH) 2 , CH 2 = C (CH 3 ) -CH 2 -CH 2 -OH is detected.
11. Verfahren zum Screening einer Verbindung, wobei das Verfahren umfaßt: a) Bereitstellen einer Wirtszelle, die einen rekombinanten Expressionsvektor enthält, wobei der Vektor zumindest einen Teil der DNA-Sequenz die für ein gcpE- oder ein yfgB-Protein aus Bakterien oder Parasiten oder für ein Analoges oder Derivat des Polypeptids kodiert, worin eine oder mehrere Aminosäuren deletiert, hinzugefügt oder durch andere Aminosäuren substituiert worden sind, ohne die enzymati- sehe Wirkung des Polypeptids wesentlich zu reduzieren, aufweist und außerdem eine Verbindung, von der vermutet wird, daß sie eine antimykotische, antiparasitäre oder antivirale Wirkung bei Mensch und Tier hat, b) In-Kontakt-Bringen der Wirtszelle mit der Verbindung und c) Bestimmung der antimykotischen, antiparasitären oder antiviralen Wirksamkeit der Verbindung.11. A method of screening a compound, the method comprising: a) providing a host cell containing a recombinant expression vector, the vector comprising at least part of the DNA sequence for a gcpE or a yfgB protein from bacteria or parasites or encodes an analog or derivative of the polypeptide in which one or more amino acids have been deleted, added or substituted by other amino acids without significantly reducing the enzymatic action of the polypeptide, and also has a compound which is suspected to be has an antifungal, antiparasitic or antiviral effect in humans and animals, b) contacting the host cell with the compound and c) Determination of the antifungal, antiparasitic or antiviral activity of the compound.
12. Verfahren zum Screening einer Verbindung, wobei das Verfahren umfaßt: a) Bereitstellen einer Wirtszelle, die einen rekombinanten Expressionsvektor enthält, wobei der Vektor zumindest einen Teil der DNA- Sequenz die für ein gcpE- oder ein yfgB-Protein aus Bakterien oder Parasiten oder für ein Analoges oder Derivat des Polypeptids kodiert, worin eine oder mehrere Aminosäuren deletiert, hinzugefügt oder durch andere Aminosäuren substituiert worden sind, ohne die enzymati- sehe Wirkung des Polypeptids wesentlich zu reduzieren, aufweist und außerdem eine Verbindung, von der vermutet wird, daß sie eine antivirale, antiparasitäre, füngizide oder herbizide Wirkung bei Pflanzen hat, b) In-Kontakt-Bringen der Wirtszelle mit der Verbindung und c) Bestimmung der antiviralen, antiparasitären, füngiziden oder herbiziden Wirksam- keit der Verbindung. 12. A method of screening a compound, the method comprising: a) providing a host cell containing a recombinant expression vector, the vector comprising at least part of the DNA sequence for a gcpE or a yfgB protein from bacteria or parasites or encodes an analog or derivative of the polypeptide in which one or more amino acids have been deleted, added or substituted by other amino acids without significantly reducing the enzymatic action of the polypeptide, and also has a compound which is suspected to be has an antiviral, antiparasitic, fungicidal or herbicidal action on plants, b) bringing the host cell into contact with the compound and c) determining the antiviral, antiparasitic, fungicidal or herbicidal activity of the compound.
EP00935082A 1999-05-21 2000-05-20 Use of genes of the deoxy-d-xylulose phosphate biosynthetic pathway for altering the concentration of isoprenoid Withdrawn EP1179187A1 (en)

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DE19923567A DE19923567A1 (en) 1998-09-22 1999-05-21 Genes of the 1-deoxy-D-xylulose biosynthetic pathway
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ATE373715T1 (en) 1999-08-04 2007-10-15 Adelbert Bacher ISOPRENOID BIOSYNTHESIS
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DE10027821A1 (en) 2000-06-05 2001-12-06 Adelbert Bacher New intermediate in isoprenoid biosynthesis, useful in screening for potential herbicides, comprises mutant encoding-enzymes sequences for imparting herbicide resistance
DE10201458A1 (en) 2001-04-11 2002-10-17 Adelbert Bacher New proteins involved in isoprenoid biosynthesis, useful in screening for inhibitors, also new intermediates, potential therapeutic agents, nucleic acids and antibodies
DE10119905A1 (en) * 2001-04-23 2002-10-24 Jomaa Pharmaka Gmbh Enriching intermediates in the mevalonate-independent pathway of isoprenoid synthesis, useful for therapeutic activation of T cells, comprises altering enzymatic activity in the pathway
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