EP0948603A1 - Sterol-glycosyl transferases - Google Patents

Sterol-glycosyl transferases

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Publication number
EP0948603A1
EP0948603A1 EP97945740A EP97945740A EP0948603A1 EP 0948603 A1 EP0948603 A1 EP 0948603A1 EP 97945740 A EP97945740 A EP 97945740A EP 97945740 A EP97945740 A EP 97945740A EP 0948603 A1 EP0948603 A1 EP 0948603A1
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EP
European Patent Office
Prior art keywords
sterol
plants
transformed
parts
amino acid
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EP97945740A
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German (de)
English (en)
Inventor
Martina Baltrusch
Ernst Heinz
Dirk Warnecke
Frank P. Wolter
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Gesellschaft fur Erwerb und Verwertung Von Schutz
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GVS Gesellschaft fuer Verwertungssysteme GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • 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
    • C12P33/00—Preparation of steroids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to DNA sequences which code for sterol glycosyltransferases, and to their use for changing the content and / or the structure of sterylglycosides and / or their synthetic secondary products in transgenic organisms.
  • Sterylglycosides and the biosynthetic secondary products Steryloligoglycosidede and acylated Sterylglycoside are natural substances found in plants as well as in some fungi and bacteria. Many physiological effects are described for these substances and their secondary products, e.g. Inhibition of vascular permeability, anti-tumor activity, anti-inflammatory and hemostatic effects (Okuyama, E. and Yamazaki, M. (1983) Yakugaku Zasshi 103: 43 ff; Normura, T .; Watanabe, M .; Inoue, K. and Ohata, K. (1978) Japan J. Pharmacol. 28, Suppl.
  • a disadvantage of these substances is that they are present in the organisms only in relatively small quantities and must be hiert by complicated processes specially ⁇ and cleaned. Moreover, some organisms that these substances contain pathogenic to humans and can be cultivated only with great effort, what their potential use as drug de- detergents with, emulsifying agents, as a raw material for plastics and for Her ⁇ position of liposomes when large quantities and must be of higher purity, currently appears to be of little use.
  • the enzymatic synthesis of sterylglycosides in the organisms from sugar nucleotides and sterols with a free OH group is carried out by the sugar-nucleotide-dependent sterol-glycosyltransferases (in short: sterol-glycosyl- transferases) catalyzed.
  • These enzymes can be part of the organisms ⁇ men isolated and purified, but are not available in sufficient quantities and Qulticianen for economic applications.
  • sterol-glucosyltransferase from oats could also be purified to homogeneity (Warnecke and Heinz, 1994). However, no gene or other nucleic acid that codes for a sterol glycosyltransferase is known.
  • nucleic acid sequences are known which are similar to the sequences described in this application. In no case was a sterol glycosyltransferase activity of the associated transcription product shown or even discussed. Such nucleic acid sequences may now be used, the content and / or the composition of sterol glycosides and Vietnamesepro ⁇ Dukten to manipulate in certain organisms and to change so economically relevant characteristics of these organisms positive. So crops can be produced with improved tolerance or resistance to Vermin ⁇ approach to environmental influences such as soil salinity, drought, cold and frost. Microorganisms such as baker's and brewing yeast can also be improved with regard to ethanol and temperature tolerance.
  • the enzyme itself can also be economically usable if it is genetically engineered in large quantities.
  • the application for cholesterol quantification may be mentioned here as an example.
  • the sterol can glycosyltransferases - and this kodie ⁇ leaders DNA sequences - len due to the similarity of solanidine with Stero- - as enzymes, or for the provision of such enzymes, USAGE ⁇ det be used for the synthesis of solanine are responsible in Solanaceen. This could then be used to produce genetically modified solaninar plants or plants. By choosing suitable methods a sol ⁇ che reduction to certain plant parts or stages of development be ⁇ can be limited.
  • the object of the present invention is to isolate nucleic acid fragments to provide with which transgenic organisms can be produced, which have improved economically relevant properties or with which sterylglycosides and their secondary products can be produced in vivo or in vitro.
  • These substances can a) be produced in larger quantities than in the starting organisms; or b) are produced by organisms that are easier and safer to cultivate than those in which these substances occur naturally; or c) be of novel structure and favorable to use natural ⁇ exhibit properties.
  • nucleic acid fragments are provided which code for sterol glycosyltransferases in order to generate chimeric genes.
  • These chimeric genes can be used to cell cultures ⁇ to transform plants, animals or microorganisms and change with their Sterylglycosidsynthese as ⁇ .
  • the invention relates to a first object.
  • An isolated DNA fragment DNA construct contained ⁇ tend least part of a sterol or sterol glycosyltransferase glycosyltransferase coding sequence in a narrow sense (1) or recombinant;
  • a protein is the abuitet ⁇ acid sequences from any of the nucleic shown in Figures 1-3 or 11-22.
  • a chimeric gene that is capable of changing the content of sterol glycosyl transferase or sterol glycosyl transferase in the narrow sense, in particular sterol glucosyl transferase or sterol glucosyl transferase in the narrow sense, in a transformed cell;
  • (10) a chimeric gene which contains a DNA fragment defined in (9) and which is able to determine the content of sterol-glycosyltransferase or sterol-glycosyltransferase in the narrower sense, in particular ⁇ sterol-glucosyltransferase or sterol, in a transformed cell -Glucosyltransferase in the narrower sense, to change;
  • organisms in particular microorganisms such as bacteria and yeasts, the gene or genes, encoding sterol glycosyl or sterol-Gly ⁇ cosyltransferasen in the narrower sense, particularly sterol Glucosyltransfera ⁇ sen or sterol glycosyl in the narrow sense, by transformation deleted or interrupted with suitable chimeric genes;
  • Antisera products from antisera, antibodies or parts thereof, which are directed against proteins as defined in (13).
  • the nucleic acid fragments coding for sterol glucosyltransferases could be isolated from Avena sativa and Arabidopsis thalliana.
  • the deduced amino acid sequences of these nucleic acid fragments show surprisingly little resemblance to the familiar Se ⁇ sequences of steroid hormone glucuronosyltransferases. It is therefore surprising that we were able to isolate completely new nucleic acid fragments with our methods. No other nucleic acid fragments that code for sterol glycosyltransferases have been identified.
  • the iso ⁇ profiled eukaryotic nucleic acid fragments are characterized in that they are surprisingly capable provided ⁇ ersequenzen with corresponding STEU, ⁇ mechanisms in both eukaryotes and prokaryotes Orga, and therefore herein dack without the typical eukaryotic processing and Mo ⁇ , the synthesis effect of enzymatically active sterol-glucosyltransfer lawn .
  • the invention also relates to isolated nucleic acid fragments whose amino acid sequences have ended extract ⁇ defined similarities to the deduced amino acid sequences of the sequences shown in Fig. 12 or 13.
  • the invention also relates to all plasmids, viruses and other vectors which contain these isolated nucleic acid fragments or parts thereof.
  • FIGS. 4 and 18 show striking similarities with the derived amino acid sequence of a piece of genomic DNA from S. cerevisiae (see FIG. 9). This is the chromosome XII Cosmid 9470 (Genbank No. gb U17246). The similarity related to the 3 'region of the reverse reading frame of bp 32961-36557 (gene L9470.23). No function is known for this putative gene. Various parts of this gene are provided with suitable control sequences and, after transformation of E. coli with this chimeric gene, were surprisingly able to detect sterol-glucosyltransferase activity in cell homogenates of the transgenic cells.
  • the invention further relates to the use of Nuklein Textresequen ⁇ zen of Figs. 1-3, 11-13 and 17, or the sequences derived therefrom Amino Textrese ⁇ for the isolation of genes or cDNAs encoding syltransferasen for other sterol Glyco ⁇ .
  • the derivative of oligonucleotides and their use in the PCR method, from the nucleotide or Aminokla ⁇ resequenzen also by comparison to other sequences affected.
  • the invention relates to all plasmids, viruses and other vectors which contain the nucleic acid fragments from FIGS. 1-3, 11-13, 17 or parts thereof or the yeast gene L9470.23 or parts thereof or nucleic acid fragments or parts thereof which are according to the in last paragraph methods described were iso profiled ⁇ containing and the lawn suitable in transformed cells for expression of sterol Glycosyltransfe ⁇ are. Protection claim is also applicable to all organisms (microorganisms, animals, plants, parts since ⁇ of, cell cultures) containing these chimeric genes and the products or extracts from it if the material composition of the Orga ⁇ mechanisms has been altered by the action of these chimeric genes .
  • nucleic acids in the figures are from the 5 'end to the 3' end, that of proteins from the amino terminus to the carboxyter minus.
  • the amino acids are named in the one-letter code.
  • the image ⁇ Ungen serve to illustrate the present invention. They show:
  • Fig. 3 wal ⁇ e comparisons of the partial DNA sequences, and 800 bp of the wal9er lan ⁇ gene DNA fragment with the sequence of the oat HaSGT (Fig. 1) (Fig. 2). The comparison was carried out using the CLUSTAL program (Higgins and Sharp, 1988, Gene 73, 237-244).
  • Fig. 4 amino acid sequence HaSGTP in the one-letter code, which from the DNA sequence of the HaSGT nucleic acid fragment was derived and which codes for a sterol glucosyltransferase with a molecular mass of 71 kD.
  • N-TERMINUS N-terminal amino acid sequence of the purified enzyme
  • HaSGTP amino acid sequence derived from the oat clone HaSGT. The comparison was carried out using the CLUSTAL program (Higgins and Sharp, 1988, Gene 73, 237-244). The * mark identical amino acids. - denote non-existent or unknown amino acids.
  • the organic phases were applied to silica gel 60 plates (Merck, Darmstadt), which with the eluent chloroform: methanol 85:15 (A) or chloroform: methanol: ammonia (25%) 65: 35: 5 (B). were developed.
  • the Rf values of the radioactive, lipophilic reaction products were determined using a Berthold TLC analyzer and compared with authentic standards ⁇ s, which were detected with ⁇ -naphthol-sulfuric acid. Only one product was identified that was identified as sterylglucoside.
  • the Rf value of the steryl glucoside at A in this case deviates from the usual value with this eluent because the eluent was not freshly prepared and the composition had changed due to evaporation.
  • A. The E. coli cells were transformed with the plasmid pBS-ATG (Example 5.).
  • B. The E. coli cells were transformed with the plasmid pBS-HRP (Example 5.).
  • Fig. 7 Western blot of recombinant sterol glucosyl transferases. Expressing each 40 ug protein from E. coli cells, the different parts of the oat HaSGT was subjected to SDS-polyacrylamide gel electrophoresis un ⁇ terworfen and then transferred to a hydrophobic membrane. The immunostaining was carried out with an antiserum against the sterol-glucosyltransferase purified from oats. Lanes 1 and 2: protein from E. coli cells which were transformed with the plasmid pBS-HRP. Lane 3: protein from E. coli cells transformed with the plasmid pBS-HATG. Lane 4: standard proteins with molecular weights of 31, 45, 66 and 97 kD. The proteins were stained with Ponceaurot, the standard proteins with marked with a pen and discarded.
  • Fig. 8 Dunn harshchromatographische analysis of radioactive products of an in vitro enzyme assay which was carried out with cell-free homogenate of transformed with the plasmid pGALHAMl S. cerevisiae cells (In ⁇ game 6). The organic phase was applied to a silica gel 60 plate (Merck, Darmstadt), which was developed with the eluent chloroform: methanol 85:15. The Rf value of the radioactive, lipophilic Christspro ⁇ domestic product was determined using a Berthold TLC analyzer and compared with authentic standards that were detected with ⁇ -naphthol-sulfuric acid. Only one product was identified that was identified as steryl glucoside.
  • Fig. 12 DNA sequence of the DNA fragment Kpcr, which was from the Solanum tuberosum with the PCR method (Example 8.).
  • Fig. 13 DNA partial sequence of the DNA fragment Cpcr, which with the PCR method was isolated from Candida albicans (Example 8.).
  • A Amino acid sequence ApcrP in the one-letter code, which was derived from the DNA sequence of the DNA fragment Aper.
  • B Comparison of the amino acid sequence ApcrP with the oat sequence HaSGTP. The comparison WUR ⁇ de using the program CLUSTAL (Higgins and Sharp, 1988, Gene 73, 237- 244) was performed. The * mark identical amino acids.
  • A Amino acid sequence KpcrP in the one-letter code, which was derived from the DNA sequence of the DNA fragment Kpcr.
  • B Comparison of the amino acid sequence KpcrP with the oat sequence HaSGTP. The comparison WUR ⁇ de using the program CLUSTAL (Higgins and Sharp, 1988, Gene 73, 237- 244) was performed. The * mark identical amino acids.
  • AtSGT DNA sequence of the nucleic acid fragment AtSGT, which was isolated from a cDNA expression gene bank from oat seedlings (Example 9). It has a length of 2353 base pairs (bp) and an open entält Le ⁇ seraster from position 1 to 2023. start or stop codon found at positions 113-115 and 2023-2025.
  • Fig. 19 Comparison of the amino acid sequences HaSGTP and AtSGTP.
  • the Ver ⁇ was immediately performed using the program CLUSTAL (Higgins and Sharp, 1988, Gene 73, 237-244).
  • Fig. 20 Dunn harshchromatographische analysis of radioactive products ei ⁇ nes in vitro enzyme assays, the E. transformed with cell-free homogenate of the ⁇ Plas mid pBS-AtSGT was performed coli cells (Example 10). The organic phase was that corresponds on silica gel 60 plates (Merck, intestinal ⁇ city) coated with an eluent Chloroforr methanol 85:15 was wrapped. The Rf value of the radioactive, lipophilic reaction product was determined with a Berthold TLC analyzer and compared with authentic standards, which were detected with ⁇ -naphthol-sulfuric acid. Only one product was identified that was identified as sterylglucoside.
  • Fig. 21 Partial amino acid sequence of the A HaSGTP Letter B ⁇ ben code.
  • Fig. 22 Partial amino acid sequence of the A AtSGTP Letter B ⁇ ben code.
  • Fig. 23 Partial amino acid sequence in the one-letter code, which was derived from the S. cerevisiae gene L9470.23.
  • the protein was then sequenced directly N- terminal or proteolytically cut to internal Bruchstüc ⁇ ke to obtain.
  • the protein was according to Bauw, G .; van den Bulcke, M .; van Damme, J .; Puype, M .; digested with trypsin 194-196 and the proteolytic breakdown ⁇ pieces with a high-performance liquid chromatography system (130A, Applied Biosystems,: Van Montagu, M. and Vandekerckhove, J. (1988) J. Chem Prot. 7. Rothstadt) separated on a "reverse phase" column (Vydac C4, 300 Angstrom pore diameter, 5 ⁇ m particle size).
  • the peptides were eluted with a linear gradient (0-80% B, solution A: water with 0.1% trifluoroacetic acid, solution B: 70 acetonitrile with 0.09% trifluoroacetic acid) at a flow rate of 0.2 ml / min.
  • the elution pattern of the peptides corresponded to the pattern which typically corresponds to a trypsin self-digest. Even after repeating the experiment several times, no peptide could be assigned to the purified protein due to the retention time. Most peptides were then sequenced. However, the sequences all corresponded to the amino acid sequence of the trypsin.
  • a cDNA expression gene bank from oats was created in order to isolate complete clones of the sterol-glucosyltransferase.
  • Oligonucleotide primers were derived:
  • XXS4T 5'-GATCTAGACTCGAGGTCGACTTTTTTTTTTTTTTTTTT-3 '
  • the polymerase chain reaction (PCR) method was carried out as follows:
  • Reaction mix 46 ⁇ l aqua dest .; 5 ul Boehringer (Mannheim) 10 x PCR buffer; 1 ⁇ l each of 10 mM dATP, dGTP, dCTP, dTDP; 1 ⁇ l each 100 ⁇ M DW1 (or DW2), XXS4T; 0.25 ul Boehringer Taq polymerase; 0.5 ⁇ l cDNA from oat seedlings (see 2., concentration not determined).
  • Reaction conditions 94 ° C, 3 min; 30x (94 "C, 40 s; 53 ° C, 1 min; 72 ° C, 3 min); 72 C °, 10 min.
  • oligonucleotide primer was derived from the sequences of the peptide amino acid sequencing (see 1.):
  • the polymerase chain reaction (PCR) method was carried out as follows:
  • Reaction mix 46 ⁇ l aqua dest .; 5 ul Boehringer (Mannheim) 10 x PCR buffer; 1 ⁇ l each of 10 mM dATP, dGTP, dCTP, dTDP; 1 ⁇ l each 100 ⁇ M DWI, whale; 0.25 ul Boehringer Taq polymerase; 0.5 ⁇ l cDNA from oat germ (see 2nd, concentration not determined).
  • Reaction conditions 94 ° C, 3 min; 30x (94 ° C, 40 s; 53 ° C, 1 min; 72 ° C, 3 min); 72 ° C, 10 min.
  • a successful PCR reaction could only be carried out using the specific reverse primer whale: agarose gel electrophoresis with 15 ⁇ l of the reaction mixture gave a DNA band e of approx. 800 bp length.
  • the cloned piece of DNA (see 3) was labeled and used to screen a cDNA library (see 2) used transferase to complete clones of the sterol glucosyl ⁇ isolate.
  • the labeled sample was then used to search the oat cDNA library.
  • the method is described in the Boehringer DIG System User4s Guide for Filter Hybridization (Plaque Hybridization, Colorimetric Detection with NBT and BCIP). There were 250,000 in Stammionsfä ⁇ hige phage particles scanned (hybridization temperature 69 ° C).
  • HaSGT An approximately 2300 bp long clone (hereinafter referred HaSGT) completeness, ⁇ sequenced dig and double-stranded.
  • This sequence is shown in FIG. 2: The partial sequences (wal ⁇ e and wal9er) of the cloned PCR fragment are over 95% identical to the clone HaSGT (FIG. 3).
  • This clone is 2317 bp long and shows an open reading frame from bp 1 to bp 1971.
  • a start ⁇ codon (ATG) for the translation starts at bp 148. If the open reading ⁇ mimic in an amino acid sequence translated (HaSGTP, Fig.
  • the amino acid sequence shows complete identity with the amino acid sequence of the peptide fragment of the purified protein and almost complete Identitä ⁇ th to the N-terminal amino acid sequence of the purified protein (14 amino acids of 15 are identical, Fig. 5). This agreement clearly shows that the cloned cDNA corresponds to the purified protein.
  • the Un ⁇ ter Kunststoff at an amino acid presumably due to allelic differentiation ⁇ zen.
  • the plasmid which contains the 2317 bp long oat clone in the vector pBluescript I SK (inserted between the EcoRI and the Xhol interface) is called pBS-HaSGT in the following.
  • -It were performed two cloning into appropriate vectors for expression by ⁇ : a) This cloning generated a plasmid (pBS-HATG), which encodes a fusion protein ⁇ , the first amino acids from the pBluescript lacZ OPE ron and the polylinker derived (normal printed, see below) and the following amino acids which after initiating methionine, the sequence in an amino acid ⁇ translated nucleotide sequence of the HaSGT corresponding (under ⁇ painted, see below).
  • the plasmid pBS-HaSGT was cut with the restriction enzymes Eael and Eagl and the linearized part, which contains the vector sequences, ligated together with itself.
  • the resulting plasmid codes for a fusion protein, the beginning of which looks like this: MTMITPSSELTLTKGNKSWSSTAVAADADEPTGG ...
  • Plasmid pBS-HaSGT was used as the matrix DNA. The following were
  • DW 15 GATGAGGAAATTCACTAGTTG
  • a PCR fragment of approximately 500 bp in length was nigt gerei ⁇ on an agarose gel, cut with the restriction enzymes BamHI and NdeI and still ⁇ again gel purified, from which a fragment was isolated from approximately 450 bp in length.
  • the plasmid pBS-HaSGT was cut with the restriction enzymes BamHI and Ndel and a fragment of approximately 4300 bp in length was eluted from an agarose gel. This fragment was ligated with the cut PCR fragment and used to transform E. coli. From the transformier ⁇ th cells plasmid DNA was isolated and partially sequenced.
  • the plasmid DNA codes for the following fusion protein: MTMITPSSELTLTKGNKSWSSTAVAALELVDLDVGGEDGY ...
  • 1 ml lysis buffer 50 mM Tris / HCl pH 8.0; 15% glycerol; 5 mM DTT; 1 mg / ml lysozyme (from Hühnerei, Boehringer, Mannheim); 200 ⁇ M Pefabloc (Merck, Darmstadt); 0.
  • the reaction solution of the in vitro enzyme assay had a volume of 60 ⁇ l and was composed as follows (1/17/1996):
  • a vector was produced which is suitable for the expression of the plant cDNA in Saccharomyces cerevisiae.
  • Part of the oat clone HaSGT was extracted from plasmid pBS- with Sall / Kpnl HaSGT cut out and cloned into the pSP72 Vector (Promega, Heidelberg, Sall / Kpnl).
  • the SalI / Kpnl fragment of the resulting plasmid pSPHAMl comprises the corresponding portion of the HaSGT and was cloned into the vector pGAL4 (XhoI / BamHI).
  • the resulting plasmid was used pGALHAMl and to transform the Saccharomyces cerevisiae strain UTL-7A (MATa, ura3-52, trpl, leu2-3 / 112).
  • the sterol-glucosyltransferase activity was an in vitro enzyme assay performed with cell-free homo ⁇ Genat of the yeast cells to detect the expressed plant sequence.
  • the yeast cells were grown on the following medium (aerobically shaken for 72 h at 29 ° C):
  • the cells of a 30 ml culture are sedimented and taken up in 1 ml of lysis buffer:
  • the reaction solution of the in vitro enzyme assay had a volume of 150 ⁇ l and was composed as follows (March 10, 1996):
  • the radioactivity present in the organic phase was subjected to thin-layer chromatography analysis from parallel samples : the organic phases were applied to silica gel 60 plates (Merck, Darmstadt), which were developed with the eluent chloroform: methanol 85:15.
  • the Rf values determined to the radioactive, lipophilic reaction products WUR ⁇ with a Berthold TLC analyzer and compared with authentic standards that were detected with ⁇ -naphthol-sulfuric acid. Only one product could be identified that was cosid was identified (see Fig. 8). It could thus be demonstrated that the transformed yeast cells express a protein which shows sterol-glucosyltransferase activity. Control cells show no sterol-glucosyltransferae activity.
  • a 6359 bp fragment was isolated from a Cosmid 9470 DNA preparation by cutting with the enzymes Ndel and Spei (Cosmid bp 31384-37744).
  • This sequence einhielt the desired reading frame and was constructed by cloning into the vector pBluescript II KS (with EcoRV ge ⁇ cut) are used for further subcloning.
  • This plasmid was called pBS-HSC.
  • Four subclonings were carried out which should lead to the expression of parts of the open reading frame of different lengths. These clonings are tabulated below:
  • the cells were sedimented from 15 ml overnight culture (15 ml LB ampicillin, 37 ° C., 14 h) and taken up in 1.5 ml lysis buffer (50 mM Tris / HCl pH 8.0; 15% glycerol; 5 mM DTT; 1 mg / ml Lysozyme (from egg, Boehringer, Mannheim). 200 uM Pefabloc (Merck, intestinal ⁇ city) After a 5 minute incubation at 20 ° C, the suspension was placed on ice and the cells by 3x 3 seconds of treatment with the Ultra ⁇ sound rod broken.
  • lysis buffer 50 mM Tris / HCl pH 8.0; 15% glycerol; 5 mM DTT; 1 mg / ml Lysozyme (from egg, Boehringer, Mannheim). 200 uM Pefabloc (Merck, intestinal ⁇ city)
  • the reaction solution of the in vitro enzyme assay had a volume of 100 ⁇ l and was composed as follows (March 22, 1996):
  • the reaction was stopped after 45 min (at 30 ° C) by mixing with 0.5 ml of water and 1.6 ml of ethyl acetate. After phase separation by brief centrifugation, the upper organic phase was removed and the radioactivity contained therein was determined using a scintillation counter:
  • E. coli homogenate with clone 7500 dpm E. coli homogenate with clone 2 10700 dpm
  • organi ⁇ rule phases of assay with untransformed control cells also contain some radioactivity; however, this is not a labeled steryl glucoside.
  • the deduced amino acid sequence of the gene 9470.23 is mentioned in Fol ⁇ constricting ScSGTP (see Fig. 9).
  • Oligonucleotide primers which were used for PCR experiments could be derived from similar amino acid sequence regions between HaSGTP (see 4.) and ScSGTP (see 7.):
  • the polymerase chain reaction method was carried out as follows:
  • Reaction mix 40 ⁇ l distilled water; 5 ul Boehringer (Mannheim) 10 x PCR buffer; 1 ⁇ l each of 10 M dATP, dGTP, dCTP, dTDP; 1 ⁇ l each 100 ⁇ M oligonucleotide primer,
  • Lamda ZAP cDNA bank (Stratagene, Heidelberg) of potato with approx. 10
  • the potato sequence KpcrP is 86% identical to the corresponding part of the oat sequence HaSGTP,
  • the Arabidopsis sequence ApcrP is 90% identical to the corresponding part of the HaSGTP and oat sequence
  • the candidate sequence CpcrP is 64% identical to the corresponding part of the S. cerevisiae sequence ScSGTP.
  • the Arabidopsis PCR clone Aper was using a method as described in the fourth ⁇ be issued for the isolation of complete clones from a lambda Zap Arabidopsis cDNA library (obtained from the Stock Center of the Max Planck Institute for breeding research ⁇ , Cologne) used. It was a 2300 bp long clone (the consequences ⁇ AtSGT called) completely sequenced and double-stranded (Fig. 17). This clone is 2353 bp long and shows an open reading frame from bp 1 to bp 2023. A start codon (ATG) for translation begins at bp 113. If the open reading frame is translated into an amino acid sequence (AtSGTP, FIG. 18), it shows the amino acid sequence is very similar to the oat sequence HaSGTP (see Fig. 19).
  • the cloning produced an Plas ⁇ mid (pBS-AtSGT) encoding for a fusion protein whose first amino ⁇ acids from the pBluescript lacZ operon and the polylinker derived (nor ⁇ printed times, see below) and the following amino acids to those of the open Correspond to the reading frame of the clone AtSGT (underlined, see below).
  • the beginning of the fusion protein looks like this: MTMITPSSELTLTKGNKSWSSTAVAAALELVDPPGCRNSEFGTPLILSFTFWD. ".
  • E. coli cells transformed with the plasmid pBS-AtSGT it was checked whether the corresponding fusion protein was expressed by carrying out an in vitro enzyme assay for the detection of sterol-glucosyltransferase activity with cell homogenates.
  • the cells from 1.5 ml overnight culture (1.5 ml LB-ampicillin, 37 ° C., 14 h) were sedimented and taken up in 1 ml lysis buffer (50 mM Tris / HCl pH 8.0; 15% glycerol; 5 mM DTT ; 1 mg / ml lysozyme (from Hühnerei, Boehringer, Mannheim); 200 ⁇ M Pefabloc (Merck, Darmstadt); 0.1% Triton X100. After ⁇ minute incubation at 20 ° C the suspension was placed on ice and the cells were passed through 3x 3 Seconds of treatment with the ultrasound wand.
  • 1 ml lysis buffer 50 mM Tris / HCl pH 8.0; 15% glycerol; 5 mM DTT ; 1 mg / ml lysozyme (from Hühnerei, Boehringer, Mannheim); 200 ⁇ M Pefabloc (Merck, Darmstadt); 0.1% Triton
  • the reaction solution of the in vitro enzyme assay had a volume of 50 ⁇ l and was composed as follows (11.3.1996):
  • E. coli homogenate, untransformed 100 dpm (blank value)
  • -Stereols are substances that have the following structural features: They consist of a 5 ⁇ -cholestan-3-ß-ol or 5 ⁇ -cholestan-3- ⁇ -ol backbone. This basic structure can be modified on the side chain and / or by various double bonds in the ring systems.
  • -STEROLS IN THE NARROW SENSE are cholesterol, ergosterol, ß-sitosterol, stigmasterol.
  • -STERYLGLYCOSIDES are sterols or sterols in the narrower sense, which are linked to a sugar molecule or to the C3 atom via the oxygen atom.
  • These sugars can e.g. Glucose, galactose, mannose, xylose, arabinose, other sugars or sugar derivatives in furanosidic or pyridosidic form and in an ⁇ or ⁇ linkage.
  • Compounds containing glucuronic acid are excluded from this definition.
  • -FOLGEPRODUKTE sterylglycosides are te to a secondary production ⁇ , which can be synthesized cosiden in organisms, or in in vitro systems enzymatically from Sterylgly- (eg Steryldiglycoside, -triglycoside, oligoglycosides or acylated steryl glycosides).
  • Sterylgly- eg Steryldiglycoside, -triglycoside, oligoglycosides or acylated steryl glycosides.
  • punch Sub ⁇ which can be represented by methods of organic chemistry from sterylglycosides.
  • -STEROL-GLYCOSYL TRANSFERASES are enzymes that transfer a sugar molecule, especially from activated sugars or activated sugar derivatives, especially from sugar nucleotides or sugar derivative nucleotides, to the 0H group on the C3 atom of sterols or sterols in their own sense.
  • STEROL glucosyltransferases are enzymes that carry a glucose molecule, particularly of activated glucose, especially from uridine diphosphate to the OH group on the C3 atom of sterols or sterols in their own sense about ⁇ .
  • STEROL glycosyltransferases in the narrower sense are enzymes, the gene is a sugar molecule, especially from activated sugars or activated sugar derivatives, especially from sugar nucleotides or Zuckerderivatnukleoti- to the OH group on the C3 atom of sterols in the narrower sense übertra ⁇ .
  • the transfer of Glucuronic acid is excluded from this definition.
  • STEROL glucosyltransferases in the narrower sense are enzymes that phosphate is a glucose molecule, particularly of activated glucose, especially from Uridindi ⁇ on the OH group on the C3 atom of sterols in the narrower sense transmitted.
  • SUGAR are hexoses or pentoses in furanosidic or pyranosidic form.
  • SUGAR DERIVATIVES are sugars which have been modified in their structure by oxidation or reduction or by the addition or removal of functional groups.
  • N-Ace 'tylglucosamin and Desoxyri ⁇ bose are sugars which have been modified in their structure by oxidation or reduction or by the addition or removal of functional groups.
  • SUGAR NUCLEOTIDES in the sense used here are substances in which one of the organic bases thymine, adenine, guanine, uracil or cytosine is linked to a ribose or deoxyribose and this sugar is in turn linked to another sugar molecule via two phosphoric acid residues.
  • -PLANT PARTS are parts of a plant such as Leaves, roots, seeds or fruits.
  • -VECTORS are nucleic acid fragments that are capable of replication under certain conditions and for the incorporation of foreign nucleic acid fragments for the purpose of multiplying this fragment or the expression of this fragment (for example for the production of a pro- teins) can be used.
  • Typical examples are plasmids and phages.
  • CHIMARY GENE is a nucleic acid fragment that is composed of different parts and does not naturally occur in this form. It comprises a numeral for a sequence encoding a polypeptide sequence and geeig ⁇ control sequences that allow expression.
  • the coding Se acid sequence may in this case the control sequences with respect to "sense" - or be "anti-sense” orientation.
  • Things can thereby substances (eg proteins, Nu ⁇ small acid fragments, mRNA, DNA, cDNA, cDNA clones, genes) lines, cell components (eg membranes), cells (eg, bacterial cells, plant cells, protoplasts), Zel- or organisms and their descendants his.
  • substances eg proteins, Nu ⁇ small acid fragments, mRNA, DNA, cDNA, cDNA clones, genes
  • cell components eg membranes
  • cells eg, bacterial cells, plant cells, protoplasts

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Abstract

L'invention concerne des fragments d'ADN ou des produits de recombinaison d'ADN isolés comportant au moins une partie d'une séquence codant pour la stérol-glycosyl transférase. On utilise comme stérols des substances qui soit sont des stérols au sens strict (cholestérol, ergostérol, bêta-sitostérol, stigmastérol), soit sont constituées d'un squelette 5-alpha-cholestan-3-bêta-ol ou 5-alpha-cholestan-3-alpha-ol (éventuellement modifié par des chaînes latérales ou des liaisons doubles dans les systèmes cycliques). L'invention concerne d'autre part des séquences d'ADN, ainsi que leurs utilisations pour la modification de la teneur et/ou de la structure des stérylglycosides et/ou des produits secondaires synthétiques dans des organismes transgéniques.
EP97945740A 1996-10-21 1997-10-10 Sterol-glycosyl transferases Withdrawn EP0948603A1 (fr)

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DE19643309 1996-10-21
DE19643309 1996-10-21
PCT/DE1997/002335 WO1998017789A1 (fr) 1996-10-21 1997-10-10 Sterol-glycosyl transferases

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CN104357418B (zh) * 2014-10-11 2017-11-10 上海交通大学 一种糖基转移酶及其突变体在合成人参皂苷Rh2中的应用
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