WO2002063016A1 - Tocopherol cyclase - Google Patents

Tocopherol cyclase Download PDF

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WO2002063016A1
WO2002063016A1 PCT/EP2002/000973 EP0200973W WO02063016A1 WO 2002063016 A1 WO2002063016 A1 WO 2002063016A1 EP 0200973 W EP0200973 W EP 0200973W WO 02063016 A1 WO02063016 A1 WO 02063016A1
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protein
nucleic acid
acid sequence
seq
tocopherol
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PCT/EP2002/000973
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Antoinette Chougnet
Arno Martin Friedlein
Wolf-Dietrich Woggon
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Roche Vitamins Ag
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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
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention is directed to the full-length amino acid and nucleic acid sequences of proteins having tocopherol cyclase activities from different organisms and their use for different purposes, including but not limited to the biotechnological production of vitamin E.
  • Vitamin E biosynthesis involves a series of enzyme-catalyzed reactions, starting with p-hydroxyphenylpyruvate and geranylgeranyl pyrophosphate. These enzymes include p-hydroxyphenyl-pyruvate dioxygenase, phytyl transferase, tocopherol cyclase, and ⁇ -tocopherol methyltransferase, leading to ⁇ . R- ⁇ f-tocopherol. Tocopherols are synthesized in photosynthetic organisms, i. e. , in higher plants, algae and cyanobacteria.
  • Protein extracts were obtained from Anabaena variabilis, but are also obtainable from any photosynthetic organism, such as higher plants, algae and cyanobacteria. After a first purification by anion exchange chromatography, nine active fractions were analyzed on SDS- polyacrylamide gel electrophoresis (SDS-PAGE) (Example 2c). Tryptic in- gel digestion and subsequent fragmentation by tandem mass spectrometry revealed 24 peptide sequences (SEQ ID NOs: 1 -24) . The corresponding full-length protein (SEQ ID NOs: 25-31 ) and DNA sequences (SEQ ID NOs: 32-38) were identified by a search in the Kasuka DNA Research Institute's database. The protein extracts obtained as described above were further purified by different techniques, including but not limited to ammonium sulfate precipitation, hydrophobic interaction chromatography, cation exchange chromatography, hydroxyapatide or HiTrap affinity columns
  • Example 3 After each purification step, the eluted fractions were tested for tocopherol cyclase activity and analyzed on SDS-polyacrylamide gels. The active fractions were then applied to a further purification step.
  • the present invention is directed to amino acid sequences which are identical or at least 60% homologous to SEQ ID NOs: 25, 26, 27, 28, 29, 30, or 31. More particularly, the degree of homology is preferably at least 70%, more preferably at least 80%, and most preferred at least 90%.
  • the present invention comprises isolated nucleic acid sequences coding for such proteins, said nucleic acid sequences being identical or homologous to SEQ ID NOs: 32, 33, 34, 35, 36, 37, or 38.
  • the degree of homology is at least 60%, preferably at least 70%, more preferably at least 80%, and most preferred at least 90%.
  • “Homology” as defined in the present invention means that when the amino/nucleic acid sequences of two proteins/nucleic acid sequences are aligned at least the given percentage is identical. The remainder of the amino/nucleic acids may be different.
  • the homology search can be performed using the BLAST program (Altschul et al., Nucl. Acids Res. 25, 3389-3402, 1997) .
  • tocopherol cyclase or "protein having tocopherol cyclase activity” means proteins exhibiting the above mentioned substrate specificity and cleavage properties, i. e. resulting in the above mentioned products.
  • the proteins of the present invention may be also part of a multienzyme complex with one or more components exhibiting the function of a tocopherol cyclase.
  • a multiple alignment is performed using, e.g. , the ClustalW program (Thompson et al., Nucl. Acids Res. 22, 4673-4680, 1994) or the GAP program (Genetics Computer Group, University of Wisconsin) , in order to define the degree of homology between the aligned sequences or to define conserved regions within the aligned sequences. These conserved regions can be used to construct oligonucleotides, e.g. primers or probes. Such an alignment is also used to define the homology of proteins with tocopherol cyclase activity originating from different organisms, which is also covered by the scope of the present invention.
  • one aspect of the present invention is the provision of primers for the specific amplification of genes or parts thereof coding for proteins having tocopherol cyclase activity.
  • the length of the oligonucleotide is at least 20 bases.
  • nucleic acid sequences of the present invention include both desoxyribonucleic acid sequences as well as antisense ribonucleic acid sequences.
  • the full-length as well as fragments of the claimed nucleic acid sequences can be used as probes for the detection of coding and non- coding regions of a gene coding for a protein having tocopherol cyclase activity.
  • the generation of antisense ribonucleic acids derived from the claimed sequences which can be used, e.g., as probes for in situ hybridization, is another aspect of the present invention.
  • the invention embraces probes for the detection of proteins having tocopherol cyclase activity as defined above, said probes having a length of at least 1 1 nucleotides, preferably at least 14 nucleotides, and most preferably at least 17 nucleotides.
  • the person skilled in the art can use the protein or peptides in order to generate antibodies which do specifically react with a protein having tocopherol cyclase activity and which is identical or homologous to the sequences as claimed in the present invention. It is either possible to produce polyclonal antibodies by immunizing laboratory animals like rabbits, sheeps or goats, preferably with an adjuvant, or to produce monoclonal antibodies by well-known techniques in the art.
  • the antibodies should specifically react with a protein as claimed in the present invention in order to avoid an unspecific crossreaction. This means that the antibodies of the present invention should preferably react with an epitope which is present only on a protein of the present invention.
  • a further embodiment of the present invention is an antibody which specifically reacts with the proteins having tocopherol cyclase activities, especially with those proteins of the amino acid sequences ID NOs: 25-31 or proteins encoded by SEQ ID NOs: 32-38.
  • a preferred use of such antibodies is the detection and/or quantification of tocopherol cyclase in different assay systems, including but not limited to Western blots, immunoprecipitation, and immunohistochemistry.
  • the tocopherol cyclases disclosed are used for an enzymatic cyclization step in a biotechnological and/or biotransformation process for the production of tocopherol, tocotrienols, or tocols.
  • the present invention is directed to a process for the production of tocols, tocopherols or tocotrienols involving an enzymatic cyclization step catalyzed by a protein according to the present invention.
  • a process for the production of tocols, tocopherols or tocotrienols involving an enzymatic cyclization step catalyzed by a protein according to the present invention.
  • such process comprises the catalyzation of the cyclization of 2,3-dimethyl-5-phytyl- l ,4-benzoquinol to ⁇ -tocopherol.
  • Another preferred embodiment of the present invention concerns the introduction of a gene coding for a protein having tocopherol cyclase activity into a suitable host cell.
  • the gene can be selected, for example, from algae such as Anabaena variabilis or Synechocystis.
  • the coding regions of such genes are PCR-amplified, with specific primers, from photosynthetic organisms and inserted into a suitable vector.
  • the vector must fit with the host cell into which the gene is introduced.
  • There are specific vectors available for bacteria, yeasts, plant cells, insect cells or mammalian cells including but not limited to vectors pDS-His, pDS ( EP 821 063 ), pFPMT 121 (Mayer et al., Biotechnol.
  • the DNA coding for a protein having tocopherol cyclase activity is combined with genetic structures which provide the required genetic regulation like promoters, enhancers, ribosomal binding sites, etc.
  • the recombinant vector having the gene coding for a protein having tocopherol cyclase activity and the other required genetic structures is introduced into suitable host cells by methods well-known to the person skilled in the art, e.g. , transformation, transfection, electroporation or microprojectile bombardment.
  • the host cell as used within this method includes prokaryotic cells, such as cells of E. coli, as well as eukaryotic cells, such as cells of plants, mammals, yeasts or other fungi, and algae.
  • Preferred mammalian cells as used in this method are human cells.
  • a recombinant vector suitable for the expression in a host cell comprising a DNA coding for the proteins of the present invention, i. e. such proteins having the amino acid sequence ID NOs: 25-31.
  • the cDNA encoding a protein with tocopherol cyclase activity is integrated into the host cell's chromosomal DNA. The cells obtained by such methods can then be further propagated.
  • the host cell is a prokaryotic cell, preferably E. coli.
  • the host cell is of eukaryotic origin, including but not limited to cells of yeasts or other fungi, plants, and algae. Examples for such yeasts are Hansenula polymorpha and Saccharomyces cerevisiae.
  • the host cell is a mammalian cell, into which a gene coding for a protein of the present invention is introduced.
  • a preferred mammalian cell is a human cell.
  • tocopherol cyclase can also be produced in a cell- free translation, as described or referenced in Jermutus et al. (Curr. Opin. Biotechnol. 9, 534- 548, 1998) and Ryabova et al. (Meth. Mol. Biol. 77, 179- 193, 1998) .
  • the present invention also covers a method for the cell- free translation of proteins having tocopherol cyclase activity as well as proteins obtainable by such cell-free translation, said proteins being used for the production of tocols, tocopherols or tocotrienols.
  • the scope of the present invention also encompasses a food or feed composition comprising vitamin E obtainable by a process involving an enzymatic cyclization step catalyzed by proteins as claimed in the present invention.
  • a further aspect of the present invention is related to a transgenic plant for the production of vitamin E, wherein the plant contains isolated nucleic acid sequences coding for proteins according to the present invention, i. e. proteins having tocopherol cyclase activity, and wherein said proteins are overexpressed to obtain an increased level of such protein.
  • Example 1 Tocopherol cyclase activity assay
  • tocopherol cyclase activity is highly dependent on the composition and strength of the incubation buffer. Tocopherol cyclase activity cannot be detected in Tris buffer alone, but can be restored completely after buffer exchange against phosphate buffer or simply by addition of concentrated phosphate buffer to reach a concentration in phosphate of approximately 100 mM. Similarly, enzyme fractions displaying reduced tocopherol cyclase activity in 15 mM phosphate buffer can be activated by increasing the phosphate concentration to 100 mM.
  • 2,3-Dimethyl-5-phytylcyclohexa-2,5-diene- l ,4-dione 10 mg, 24 ⁇ mol was added to 2,6-di-O-methyl- ⁇ -cyclodextrin (600 mg, 0.45 mmol) dissolved in 10 ml of a 100 mM potassium phosphate buffer, pH 7.0. The mixture was stirred for 5 min, and ascorbic acid (440 mg, 2.5 mmol) was added. The yellow mixture was kept at 4°C.
  • HPLC analysis was performed on a Merck-Hitachi system consisting of a gradient pump L-7100, an autosampler L-7200, a column oven L-7300 set at 40°C, a UN detector L-7400 set at 290 nm, and a fluorescence spectrometer (model Merck Hitachi F- 1050) set at 290 nm excitation wavelength and 330 nm emission wavelength.
  • Chromatographic analysis was performed on a Lichrosphere- 100, RP 18 ( 5 ⁇ m) column (Merck, Darmstadt, Germany) with a flow rate of 1.5 ml/min. The column was equilibrated with a 10:90 mixture of water:methanol.
  • methanol concentration in the elution buffer was linearly increased over 3 min, and was then maintained at 100% for another 9 min. Under these conditions, the retention times of 2,3-dimethyl-5- phytylcyclohexa-2,5-diene- l ,4-diol and of ⁇ -tocopherol were 5.6 min and 7.6- min, respectively.
  • Example 2 Enrichment of tocopherol cyclase from Anabaena variabilis
  • tocopherol cyclase is readily degraded and/or inactivated during the purification process.
  • Stocker reported that no tocopherol cyclase activity is observed in Tris buffer, and that chromatography on various hydrophobic interaction columns also destroys tocopherol cyclase activity.
  • the growth medium contained (per liter) : 0.15 g MgSO 4 -7H 2 O, 0.6 g K 2 HPO 4 , 10 mg Ca(NO 3 )2-4H 2 O, 0.5 g KNO 3 , 0.165 g Na 3 C 6 H 5 O 7 -2H 2 O (sodium citrate) , 4 mg Fe 2 (SO 4 ) 3 -5H 2 O, 2.86 mg HBO 3 ) 1.81 mg MnCl 2 -4H 2 O, 0.222 mg ZnSO 4 -7H 2 O, 0.015 mg MoO 3 , 0.079 mg CuSO -5H 2 O.
  • the growth medium was autoclaved for 30 min at 120°C.
  • the cell pellet was resuspended in 30 mM potassium phosphate buffer, pH 7.0, 5 mM NaCl, 500 mM sucrose (ratio of cells to buffer 1 : 10 w/v), and centrifuged (Kontron, Centricon H-401 , 15 min, 10,000 rpm, 4°C) . This procedure was repeated twice.
  • the final pellet was resuspended ( at a ratio of 100 ml buffer per 10 g wet algae) in the same buffer containing 1 mg/ml lysozyme and EDTA (2 mM) . The mixture was rotated in a water bath for 2 hours at 35°C.
  • spheroplasts isolated, oval cells
  • the spheroplasts were collected by centrifugation ( 10,000 rpm, 15 min, 4°C) after addition of 2 mM MgSO 4 -7H 2 O, washed twice with 30 mM potassium phosphate buffer, pH 7.0, 5 mM NaCl, 500 mM sucrose in order to remove the lysozyme.
  • Example 2b 1 g of the acetone powder ( Example 2b) was suspended by means of a glass-teflon homogenizer in 40 ml of a potassium phosphate buffer, pH 7.0, containing 15 mM dodecyl maltoside and 2 mM 1 ,4-dithio-DL-threitol, followed by stirring of the suspension for 2 hours at 4°C and centrifugation (Kontron H-401 , 10,000 rpm, 15 min, 4°C) .
  • the strength of the phosphate buffer was 100 mM in case the samples were used for activity measurements ( Example 1 ), and 15 mM in case the samples were used for further enrichment of the protein (Examples 2c and 3) .
  • Example 3 Improved purification procedure for tocopherol cyclase
  • All the buffers used contained a mixture of protease inhibitors consisting of benzamidine (Sigma, 100 mg), aminocaproic acid ( Fluka, 100 mg) and trypsin inhibitor from soybean (Fluka, 40 mg, 1 1551 U/mg) dissolved in 10 ml of bidistilled water. This solution was added at a concentration of 0.1 % v/v to all the buffers used in the different purification steps.
  • Example 1 after solubilization in 100 mM potassium phosphate, pH 7.0, 2 mM DDM, 2 mM DTT and removal of the ammonium sulfate by buffer exchange on a Sephadex G-25 column (Amersham Pharmacia Biotech) previously equilibrated with the phosphate-DDM-DTT buffer.
  • Tocopherol cyclase activity was eluted with 2 ml of mixed micelles and further elution with buffer B l .
  • Mixed micelles were obtained as follows: 1.16 g of glycocholic acid (Sigma) were dissolved in 8 ml water and 470 ⁇ l 5 M NaOH under magnetic stirring for 30 min. The pH was set to 7.0-7.5 by addition of 5 ⁇ l cone, acetic acid; to this solution 80 mg of soybean azolectin (Sigma) were added under magnetic stirring and the total volume was adjusted to 10 ml.
  • Tocopherol cyclase activity of the different fractions was analyzed as described in Example 1.
  • the active fractions were pooled, and to 30 ml of the pooled fractions, 70 ml of saturated ammonium sulfate, pH 7.0, was added at 4°C.
  • the ammonium sulfate pellet was kept at 4°C and analyzed by SDS-PAGE.
  • the flow rate was 0.5 ml/min.
  • Tocopherol cyclase activity of the individual fractions was measured after addition of 1 M phosphate buffer pH 7.0 to yield a final phosphate concentration of 100 mM. Tocopherol cyclase activity eluted at the end of the 30% B2 isocratic elution. The three active fractions were analyzed by SDS-PAGE and pooled.
  • a HAP column Econo-Pac CHT-II, bed volume 5 ml, Bio-Rad was equilibrated with buffer A3.
  • the pooled active fractions obtained after SP- Sepharose ( Example 3d) were loaded onto the HAP column and the column was washed with buffer A3 for 5 min. Elution of bound proteins was attained with a linear gradient from 0% to 100% buffer B4 (500 mM potassium phosphate buffer pH 7.2, 2 mM DDM, 2 mM DTT) , followed by washing with buffer B4 for another 20 min.
  • the active fractions (23-26) were analyzed by SDS-PAGE.
  • Example 4 Identification of nine bands from an SDS-polyacrylamide gel by microproteinchemical methods
  • the peptides were eluted in one step with 1 ⁇ l of 60% methanol/5% formic acid/water directly into the nanoelectrospray needle.
  • Electrospray mass spectra were acquired on an API 365 triple quadrupole mass spectrometer (Sciex, Toronto, Canada) equipped with a nanoelectrospray ion source developed by Wilm and Mann.
  • Q l scans were performed with a 0.2 Da mass step.
  • Q l was set to transmit a mass window of 2 Da, and spectra were accumulated with 0.2 Da mass steps. Resolution was set so that the fragment masses could be assigned to better than 1 Da.
  • Fragmentation of a peptide by tandem mass spectrometry yields a stretch of sequence together with its location in the peptide (peptide sequence tag) .
  • Bold letters represent amino acids as derived from mass spectrometry which are identical to the respective amino acid in the Kazusa database (http://www.kazusa.or.jp/cyano/anabaena) , but differ from the amino acid in the respective peptide sequence as deposited in other publicly available databases. Italic letters (see below) represent amino acids as derived from mass spectrometry that differ from the respective amino acid in the Kazusa database.
  • VGGVAADLPYGWVDK SEQ ID NO: 13 LVTNNPIFR SEQ ID NO: 14
  • Example 5 Identification of homologues from other organisms through a BLAST search
  • a non-redundant database for proteins was searched for homologues with a BLAST search (Altschul et al., Nucl. Acids Res. 25 , 3389-3402, 1997) . While such a search for homologues can be performed with every single amino acid sequence ( SEQ ID NOs: 25-3 1 ) or every single DNA sequence (SEQ ID NOs: 32-38) for bands 1 -9 as input, the result is described - by way of example - for band 4 only.
  • BLASTP 2.0.12 revealed 11 sequences showing a higher homology to the reference sequence having tocopherol cyclase activity (in our example the amino acid sequence 27) in comparison to the other sequences shown up with the BLAST search. These sequences are likely to belong to the same enzyme class and to catalyze the same reaction.
  • Example 6 Multiple sequence alignment of known tocopherol cyclases
  • the conserved amino acid stretches may be used to design oligonucleotides which either alone or in combination may be used to clone further ndhH homologues and proteins having tocopherol cyclase activity, respectively, from any living organism in which such a homologue exists (see Example 8) . Also parts of the conserved sequences or less conserved sequences from the alignment (see above) may be used for this purpose, although in the latter case with a chance of success that decreases with a decrease in sequence identity and/or with an increase in the required degeneracy of the derived oligonucleotides.
  • Example 7 Overexpression of genes encoding for proteins having tocopherol cyclase activity in E. coli and yeast
  • the coding regions of the ndhH genes from Anabaena variabilis and Synechocystis PCC6803 were amplified from the respective genomic DNAs by PCR using the primers Av-Tocy-5 and Av-Tocy-3 for the A. variabilis gene and Sy-Tocy-5 and Sy-Tocy-3 for the Synechocystis gene.
  • the primers were designed such that the ATG start codons constitute the second half of a Ndel site (cleavage recognition site CATATG), and that Bam ⁇ il sites (GGATCC) are introduced immediately after the stop codons.
  • Both PCR products were cloned into the pCR ® 2.1 -TOPO vector (Invitrogen) , resulting in plasmids TOPO-Av-Tocy and TOPO-Sy-Tocy.
  • the expression vectors pDS-His and pDS were derived from pDSNdeHis, which is described in Example 2 of European Patent Application EP 821 063.
  • the plasmid pDS-His was constructed from pDSNdeHis by deleting a 857 bp Nhel and Xbal fragment carrying a silent chloramphenicol acetyl transferase gene from E. coli.
  • the plasmid pDS was constructed from pDS-His by replacing a small EcoRl-Bam l fragment with the annealed primers S/D- l and S/D-2.
  • Synechocystis PCC6803 were excised from TOPO-Av-Tocy and TOPO-Sy- Tocy with Bam l and Ndel and ligated into the BamHl-Ndel cleaved vectors pDS-His and pDS, resulting in plasmids pDS-His-Av-Tocy, pDS- Av-Tocy, pDS-His-Sy-Tocy and pDS-Sy-Tocy.
  • E. coli strain M 15 (Villarejo, M.R. and Zabin, I. , J. Bacteriol.
  • ndhH gene For the expression of the ndhH gene, the strains were grown overnight at 37°C in Luria Broth (GibcoBRL, Life Technologies) with 25 mg/1 kanamycin and 100 mg/1 ampicillin. The next day, fresh medium was inoculated with 2% (volume) of the overnight cultures and the new cultures were grown at 37°C. After 3 hours, expression of the cloned genes was induced by addition of IPTG to a final concentration of 2 mM and the growth of the cultures was continued. Samples were taken at various time points and analyzed by SDS-PAGE for appearance of NdhH. Expression of the ndhH gene and genes encoding proteins having tocopherol cyclase activity, respectively, was tested by using the standard assay as described in Example 1.
  • ndhH gene of A. variabilis or a homologous gene encoding for a protein having tocopherol cyclase activity from another organism in H. polymorpha an EcoRI site was added to each end of the gene by PCR using two primers which cover the 5'- and the 3'-end of the gene, with both primers having an extended 5'-end containing an EcoRI restriction site.
  • the PCR product was purified using the QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA, USA) , digested with EcoRI, and purified by agarose gel electrophoresis.
  • the ndhH expression vector used to transform H. polymorpha RB 1 1 (Gellissen et al., in: Smith, A., ed. , Gene Expression in Recombinant Microorganisms. Dekker, New York, pp. 395-439, 1994) , was constructed by inserting the EcoRI-digested and purified PCR product into the multiple cloning site of the H. polymorpha expression vector pFPMT 121 , which is based on an ura3 selection marker from S. cerevisiae, a formate dehydrogenase (FMD) promoter element and a methanol oxidase (MOX) terminator element from H. polymorpha.
  • FMD formate dehydrogenase
  • MOX methanol oxidase
  • the 5'-end of the ndhH gene is fused to the FMD promoter, the 3'-end to the MOX terminator ( Gellissen et al., Appl. Microbiol. Biotechnol. 46, 46-54, 1996; European patent EP 299 108).
  • the constructed plasmids were propagated in E. coli. Plasmid DNA was purified using standard state-of-the-art procedures and for control, the construct was sequenced by standard methods.
  • the expression plasmids were transformed into the H. polymorpha strain RB 1 1 deficient in orotidine-5'-phosphate decarboxylase ( ura3 ) using the procedure for preparation of competent cells and for transformation of yeast as described in Gellissen et al. ( 1996) .
  • Each transformation mixture was plated on YNB (0.14% w/v Difco YNB and 0.5% ammonium sulfate) containing 2% glucose and 1.8% agar and incubated at 37°C. After 4 to 5 days, individual transformant colonies were picked and grown in the liquid medium described above for 2 days at 37°C. Subsequently, an aliquot of this culture was used to inoculate fresh vials with YNB-medium containing 2% glucose. After seven further passages in selective medium, the expression vector was integrated into the yeast genome in multimeric form.
  • YNB 0.14% w/v Difco YNB and 0.5% ammonium sulfate
  • mitotically stable transformants were obtained by two additional cultivation steps in 3 ml non-selective liquid medium (YPD, 2% glucose, 10 g/1 yeast extract, and 20 g/1 peptone) .
  • YPD non-selective liquid medium
  • an aliquot from the last stabilization culture was plated on a selective plate. Single colonies were isolated for analysis of ndhH expression in YNB containing 2% glycerol instead of glucose to derepress the fmd promoter. Expression of the ndhH gene and genes encoding proteins having tocopherol cyclase activity, respectively, was tested by using the standard assay as described in Example 1.
  • the so-prepared gene was ligated into the EcoRI site of the expression cassette of the Saccharomyces cerevisiae expression vector pYES2 (Invitrogen, San Diego, CA, USA) or subcloned between the shortened GAPFL (glyceraldehyde-3-phosphate dehydrogenase) promoter and the pho5 terminator as described by Janes et al. (Curr.
  • the preculture was then added to 500 ml YPD medium (Sherman et al., 1986) and grown under the same conditions. Induction of the gal l promoter was done according to the manufacturer's instructions. Expression of the ndhH gene and genes encoding proteins having tocopherol cyclase activity, respectively, was tested by using the standard assay as described in Example 1.
  • Example 8 Homology cloning of ndhH genes from other organisms, e.g., algae and plants
  • a set of oligonucleotides can be designed from the same peptide, in which one (or more) ambiguous nucleotide(s) is (are) eliminated.
  • two oligonucleotides could be made from a peptide, one with a C and the other with a T in the penultimate position, thus reducing the degeneracy to 32- fold for each oligonucleotide.
  • This strategy can also be used to create more defined 3'-ends whereby mispriming can be effectively reduced.
  • Some conserved stretches might contain a non-conserved amino acid, e.g. where the third position is either isoleucine or valine.
  • Some organisms use preferred codons for certain amino acids. If the preferred codon usage for an organism is known, the degeneracy of the oligonucleotide can be reduced. Pairs of degenerated oligonucleotides are then used to amplify the coding region between them. The oligonucleotide closer to the beginning of the gene must be complementary to the coding strand and the oligonucleotide closer to the end of the gene must be complementary to the non-coding strand.
  • Nested PCR can be performed to confirm a PCR fragment or to obtain a specific fragment out of a mixture of primary products. Genomic DNA can be used as template when using an organism containing no introns or only few or short introns. When large introns are expected, cDNA or RNA ( RT-PCR) should be used as template. Alternatively, genomic DNA can be used for small fragments, which are expected to lie on the same exon.
  • Example 9 Production of antibodies against proteins having tocopherol cyclase activity

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Abstract

La présente invention permet d'obtenir la purification ainsi que des séquences nucléiques et amino-acides pleine longueur de tocophérol cyclase, une enzyme critique dans la biosynthèse de vitamine E. L'invention peut être utilisée pour la production biotechnologique de vitamine E.
PCT/EP2002/000973 2001-02-02 2002-01-30 Tocopherol cyclase WO2002063016A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007120423A2 (fr) * 2006-03-20 2007-10-25 Microbia Precision Engineering Production de composés dérivés de quinones dans des champignons oléagineux
WO2011050675A1 (fr) * 2009-10-30 2011-05-05 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Gènes conférant une tolérance à la sécheresse et au sel et leurs utilisations

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007120423A2 (fr) * 2006-03-20 2007-10-25 Microbia Precision Engineering Production de composés dérivés de quinones dans des champignons oléagineux
WO2007120423A3 (fr) * 2006-03-20 2008-05-29 Microbia Prec Engineering Production de composés dérivés de quinones dans des champignons oléagineux
US8633009B2 (en) 2006-03-20 2014-01-21 Dsm Ip Assets B.V. Production of quinone derived compounds in oleaginous yeast and fungi
WO2011050675A1 (fr) * 2009-10-30 2011-05-05 Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences Gènes conférant une tolérance à la sécheresse et au sel et leurs utilisations

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