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WO2000042200A1 - Strictosidine glucosidase isolee a partir de catharanthus roseus et son utilisation pour la production d'alcaloides - Google Patents

Strictosidine glucosidase isolee a partir de catharanthus roseus et son utilisation pour la production d'alcaloides Download PDF

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Publication number
WO2000042200A1
WO2000042200A1 PCT/NL1999/000733 NL9900733W WO0042200A1 WO 2000042200 A1 WO2000042200 A1 WO 2000042200A1 NL 9900733 W NL9900733 W NL 9900733W WO 0042200 A1 WO0042200 A1 WO 0042200A1
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strictosidine
plant
culture system
terpenoid
alkaloids
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Robert Verpoorte
Rob Van Der Heijden
Johan Memelink
Arjen Geerlings
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Universiteit Leiden
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Universiteit Leiden
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)

Definitions

  • the present invention relates to terpenoid- indolee alkaloids, methods and means for their production.
  • alkaloids are pharmaceutically important compounds, they include for instance vincristine and vinblastine, which are used in the therapy of cancer, as well as for instance ajmalicine and serpentine, used in a treatment of cardiovascular diseases .
  • Other pharmaceutically important alkaloids derived from the same pathway include for instance quinine, strychnine, reserpine, yohimbine, quinidine, vincamine, rescinnamine, ajmaline, and campthotecine .
  • Terpenoid-indole alkaloids are produced by a number of plants, including Cathara.nt.hus roseus, Rauvolfia serpentina, and Cinchona officinalis ' Ledgeriana ' .
  • Strictosidine can be formed by the condensation of NYCoganin, an iridoid-glycoside, and tryptamine, derived from the amino acid tryptophan.
  • the enzyme catalyzing this reaction is strictosidin synthase (STR, E.C. 4.3.3.2).
  • Strictosidine is then deglycosylated by strictosidine ⁇ -glucosidase (SGD, E.C. 3.2.1.105) to yield the main product cathenamine.
  • SGD strictosidine ⁇ -glucosidase
  • This compound is the basis for the formation of many known indolee alkaloids.
  • ajmalicine is produced by the enzyme cathenamine reductase using NADPH as a cofactor.
  • This enzyme has been partly purified from a cell suspension culture of C. roseus (Stockigt et al . ,
  • Ajmalicine has also been synthesized from cathenamine by reducing agents (Brown et al . , 1977) . However, most of the cathenamine was reduced to the stereoisomer of ajmalicine, tetrahydroalstonine. Felix et al . (1981) used simultaneously permeabilized and immobilized C. roseus cells to convert cathenamine into ajmalicine and isomers. However, a suitable system for producing these terpenoid indolee alkaloids in a cost efficient and reproducible way has been lacking.
  • SGD strictosidine ⁇ - D-glucosidase
  • roseus produces the indolee alkaloids vinblastine, vincristine and ajmalicine, while Cinchona species accumulate the quinoline alkaloids such as quinine and quinidine. Therefore, SGD may play an important role in channeling the alkaloid biosynthesis in the specific directions. Terpenoid indolee alkaloids are valuable pharmaceuticals. However, they occur only in trace amounts in plants such as Catharanthus roseus .
  • the present invention provides both a key enzyme in the biosynthetic pathway of terpenoid indolee alkaloids, as well as a suitable cell system which can easily be provided with key enzymes from said pathway and which can easily be provided with substrates on which these key enzymes can act .
  • the invention provides a method for producing terpenoid indolee alkaloids using an in vi tro culture system, for instance a culture system comprises plant materials as a sugar and nitrogen source and as a source for precursors for said terpenoid indolee alkaloids.
  • the plant materials should be derived from a plant which produces terpenoid indolee alkaloid precursors in significant amounts would typically be an alkaloid producing plant.
  • Also usefull would be if the material from the plant is suitable as a source for sugars and nitrogen for the culture system to grow on.
  • good sources for both carbon and nitrogen for culture systems will be based on the fruit of a plant, particularly again the fruit of an alkaloid or iridoid producing plant.
  • a suitable genus of plants which include suitable plants having suitable fruits for producing terpenoid indolee alkaloids using an in vi tro culture system is the genus Symphoricarpus .
  • a particularly suitable species from said genus is Symphoricarpus albus .
  • the fruit of a plant producing the precursors for terpenoid indolee alkaloids in this case one would use, as has been done in the examples, the berries from
  • the in vi tro culture system to be used may be any cell culture system, which is capable to propagate and/or grow on a medium which comprises plant materials as a carbon and nitrogen source and as a source for precursors for said terpenoid indolee alkaloids.
  • Such systems typically include eukaryotic cells, in particular plant cells and preferably yeast cells.
  • Yeast cells are preferred, because there is a lot of experience in culturing yeast strains or yeast cells, because they are easy to manipulate and thus easy to provide with additional enzymatic activity and because yeast cell systems are very capable of growing on different sources of carbon and nitrogen as has been well proven.
  • yeast strain to be used in a method according to the invention be provided with additional enzymatic activity, which of course preferably is derived from a biosynthesis pathway for terpenoid indolee alkaloids, and which is preferably provided through genetic engineering, thus leading to transgenic yeast cells.
  • yeast strains such as Pichia, Kluyveromyces, Hansenula and of course Saccharomyces .
  • enzyme activity that can be added to such yeast culture systems should be at least one kind of activity from a biosynthetic pathway for these alkaloids and should preferably be more than one enzyme from said pathways .
  • Two preferred enzymes to provide to the cell culture system are strictosidine glucosidase and/or strictosidine synthase.
  • the invention also provides a culture system for producing terpenoid indolee alkaloids or precursors thereof in vi tro, comprising all necessary elements for carrying out a method according to the invention.
  • the necessary and/or important elements of such a culture system have been disclosed herein before or will be disclosed herein below.
  • such a culture system will of course be used to produce terpenoid indolee alkaloids, in particular pharmaceutically or economically relevant alkaloids as mentioned herein before. It is very useful to use a culture system or to produce a culture system which can produce strictosidine, because strictosidine is a central precursor in the biosynthesis of any terpenoid indolee alkaloid. However, it is also useful to provide the culture system with enzymatic activity which takes the synthesis beyond strictosidine. Whereas it is thus an embodiment of the invention to provide a culture system with enzymatic activity which enables the system to convert strictosidine precursors such as NYCoganin and tryptamine to strictosidine.
  • the culture system with enzymatic activity downstream from strictosidine in the pathway, in particular with strictosidine glucosidase or a functional fragment and/or derivative thereof.
  • the invention also provides an isolated and/or recombinant nucleic acid encoding a strictosidine glucosidase or a functional fragment and/or derivative thereof.
  • Strictosidine glucosidase in Ca -haran us roseus has a coding sequence as depicted in figurel.
  • a functional fragment and/or derivative of strictosidine glucosidase is defined herein as a molecule which is a part of strictosidine glucosidase which still has the enzymatic activity of strictosidine glucosidase. This activity of course may be different in amount, but should remain more or less the same in kind.
  • Such molecules will include the sequence encoding the active site or sites of strictosidine glucosidase, usually having a size of at least 1500 nucleotides base pairs up to full length. For derivatives, as a rule of thumb, one would say that chances are that a molecule having less than 60% homology will not have strictosidine glucosidase activity.
  • a derivative of course has more than 90% homology with strictosidine glucosidase as present in Catharanthus roseus, from which it is known that it is very well capable of converting strictosidine into its active aglucone.
  • strictosidine glucosidase as present in Catharanthus roseus, from which it is known that it is very well capable of converting strictosidine into its active aglucone.
  • a plant cell or any eukaryotic cell for that manner is capable of producing an aglucone which has activity against for instance microbial infections.
  • a nucleic acid according to the invention, encoding strictosidine glucosidase activity is particularly useful when introduced into a host cell, in particularly 1 into a cell which can be used in a culture system according to the invention.
  • a nucleic acid in order to be able to introduce such a nucleic acid in a functional manner into said cell so that it may be transcribed and translated into enzymatic activity is by providing a nucleic acid on a vector.
  • a vector is defined as any vehicle with which a cell can be transformed or transduced or transfected, whereby the genetic material to be introduced is introduced in a functional manner, be it that the genetic material remains episomal or becomes integrated into the genome of the host cell.
  • a culture system according to the invention is typically made up of cells which have been provided with a nucleic acid encoding additional enzymatic activity, in particular from the biosynthesis pathway of terpenoid indolee alkaloids, and particularly preferred are those cells which have been provided with a nucleic acid encoding strictosidine glucosidase activity according to the invention. This can be done for instance with a vector according to the invention.
  • Strictosidine is a central intermediate in the biosynthesis of terpenoid indolee alkaloids. It is formed by the coupling of NYCoganin and tryptamine in a reaction catalyzed by the enzyme strictosidine synthase (STR, E.C. 4.3.3.2.). Tryptamine is formed from the amino acid tryptophan by the action of the enzyme tryptophan decarboxylase (TDC, E.C. 4.1.1.28), and perennialoganin is a product of the terpenoid pathway. The pathway towards strictosidine is the same in all indolee-alkaloid- producing plants.
  • SGD strictosidine ⁇ -glucosidase
  • C. roseus produces the indolee alkaloids vinblastine, vincristine and ajmalicine
  • Cinchona species accumulate the quinoline alkaloids such as quinine and quinidine. Therefore, SGD might play an important role in channeling the alkaloid biosynthesis into a specific direction (Luijendijk, 1998) .
  • Strictosidine from which all indolee alkaloids are derived, is stored in the vacuole of the cell.
  • the next enzyme in the biosynthesis, SGD was found to be located in the endoplasmic reticulum (Geerlings et al , to be published) .
  • an unstable dialdehyde is formed. This dialdehyde undergoes a spontaneous ring closure to yield cathenamine (Stevens, 1994) .
  • Cathenamine can be substrate for cathenamine reductase resulting in ajmalicine.
  • cathenamine or other intermediates in the SGD-catalyzed reaction, is the intermediate in the biosynthesis of other alkaloids, like catharanthine, is not known. All the products formed after hydrolysis of strictosidine are practically insoluble in water and can therefore be observed as a precipitate being formed in incubation mixtures (Luijendijk et al . , 1996 a).
  • SGD The function of SGD for the plant itself is not only the conversion of strictosidine in the biosynthesis of indolee alkaloids, but this enzyme also plays an important role in the defense mechanism of the plant. It was found that the combination of strictosidine together with SGD has an anti-microbial activity. It was suggested that a constitutive "trigger mechanism" in the plant, in which a microbial infection, by damage of the plant cell, results in strictosidine deglucosylation, thus allowing the active aglucone (s) to act against the intruder (Luijendijk, 1996 b) . This mechanism resembles cyanogenesis present in for instance sweet almonds.
  • glucosidases Apart from SGD, which is very selective towards its substrate, other glucosidases have been reported to be present in C. roseus cell cultures (Hemscheidt, 1980; Stevens, 1994). These glucosidases however, were unable to convert strictosidine.
  • a Catharanthus roseus (L.) G Don cell suspension culture that was harvested one week after subculturing was used to isolate SGD. This culture was grown in hormone-free MS medium containing 30 g/1 sucrose. Purification of SGD was based on the purification scheme according to Luijendijk et al . (1998) with some modifications. After protein extraction, a concentration step was performed on a Filtron concentrator containing a 3OK filter followed by a size exclusion column (Sephacryl S-300) . Instead of an anion exchange column (AEC) , a batch procedure with AEC column material (Q-Sepharose) was used.
  • AEC anion exchange column
  • the protein solution in 50 mM Bis-Tris (pH 6.3) buffer, was incubated with the AEC column material for 1 h at 4'C. After this, the AEC material was washed with 0.1 M NaCL in 50 mM Bis-Tris (pH 6.3) buffer on a glass filter. The bound protein was eluted from the AEC column material with 0.4 M NaCl in the same buffer.
  • SGD was visualized in a native gel by incubation in 2 mM strictosidine solution in 0.1 M sodium phosphate buffer, pH 6.3 (Luijendijk et al . , 1996 a). Glycosylation of SGD was determined with purified enzyme using the GlycotrackTM carbohydrate detection kit from Oxford GlycoSystems .
  • Quantitative SGD activity measurements were performed by HPLC, according to Stevens et al . (1992).
  • Qualitative SGD activity was determined by adding 1 ⁇ l of protein solution directly on nitrocellulose. The protein bound to the nitrocellulose was then directly incubated in 0.1 M sodium phosphate buffer (pH 6.3) containing 2 mM strictosidine, until a yellow spot became visible. This staining procedure is based on the same visualization method as described above. Strictosidine necessary for these measurements was prepared enzymatically using immobilized STR (Pfizner and Zenk, 1982) . A specific ⁇ - glucosidase activity was measured by incubating the protein samples at 30 °C with the substrate p-nitrophenyl- ⁇ -D-glucopyranoside (2mM in sodiumphosphate buffer, pH
  • Escherichia coli strain XL-1 (Stratagene) was used as a plasmid host. Saccharomyces cerevisiae strain YPH500
  • PCR reaction with primers T3 (AATTAACCCTCACTAAAGGG) and Sgsecl (TTCGATCACTCAAGAAGCC) was done under the same conditions as described above, but with an annealing temperature of 50"C instead of 35°C.
  • phages were screened on Nylon filters (Hybond-N, Amersham) . Filters were (pre) -hybridized in 50% deionized formamide, 5x SSPE, 5% SDS at 42 °C, washed with 0. lx SSPE, 0.5% SDS at 65 "C and exposed to Fuji-RX films mounted on Kyokko intensifying screens at -80 "C. Positive phages were taken and the procedure was repeated until homogeneity. A total of 7 positive phages were picked up, and were converted to plasmids . Sequence analysis showed that all 7 clones were identical, and the longest clone was selected to continue with.
  • This clone was sequenced completely on both strands by making subclones (see below) . Since this clone was not a full length clone, a second PCR reaction was performed using PCR primers T3 and Sgsecl (see above) . This PCR product was restricted with the restriction enzyme SacII and inserted in pBluescript SK+ .
  • This 313 bp insert was sequenced on both strands and was found to be 100% identical to the 5' end of the already found cDNA clone, except an extra 72 bp part was found on the 5' end. This extra part contained the start codon. Using the restriction enzyme SacII this extra part was cloned in front of the cDNA clone making a full length clone .
  • the predicted amino acid sequence of SGD was analyzed using several computer programs available on the Internet . Homology with other amino acid sequences from different databases was checked with the Blast program of NCBI (http://www.ncbi.nlm.nih.gov/blast/).
  • the Nakai server (PSORT, http://psort.nibb.ac.jp/) was used to analyze the amino acid sequence for protein localization. Secondary structure prediction of the amino acid sequence was analyzed by the SAPS program (Brendel et al . , 1992) .
  • DNA sequencing of the cDNA clones inserted in the EcoRI- Xhol site of pBluescript SK- was done using the T7 DNA sequencingTM kit from Pharmacia using sequence primers T3 and T7.
  • the complete cDNA coding for SGD was sequenced on both strands by making subclones in pBluescript SK+ of the cDNA clone with different restriction enzymes.
  • Secologanin was obtained using the purification method of Stevens, L.H. (1994) .
  • Crudeloidin was a kind gift from Prof. Dr. Cid A.M. Santos (UFPr - Brasil) .
  • Tryptamine was used from Aldrich. Strictosidine necessary for enzyme assays and as a reference compound was prepared enzymatically, using immobilised STR on a CNBr actived Sepharose column (Pfi tzner and Zenk, 1982) . Loganin was obtained from Extrasynthese .
  • Sterile SD minimal medium that was used to grow yeast under selective conditions, contained 6.7 g/L Difco yeast nitrogen base without amino acids, 20 mg/L adenine, 20 mg/L L-histidine, 30 mg/L L-lysine, 20 mg/L L-leucine, 20 mg/L uracil, 20 mg/L tryptophan, 20 g/L glucose, 50mM Hepes (pH 5.8) .
  • Yeast was also grown on a juice of Symphoricarpus albus berries. This juice was prepared by crushing fresh berries of Symphoricarpus albus using a mortar and pestle. The juice was centrifuged for 5 min at 13000 rpm to remove debris, and filter sterilized.
  • Yeast cultures were grown at 30"C by adding some cells from solid medium to a suitable selective minimal liquid SD medium in a sterile erlenmeyer, during a growth period of 2 days. After this growth period, different combinations of the substrates (end-concentration of 2mM)lloganin, tryptamine and loganin were added and left to incubate for another 2 days (substrates were sterilized using a 0.20 ⁇ m filter). Once the incubation period had finished, the medium and cells were taken. Medium was analyzed directly by HPLC, and cells were extracted as described below.
  • the cDNA coding for SGD was inserted in the BamHI/Kpnl restriction sites of the shuttle vector pYPGE15 (Brunelli and Pall, 1993) .
  • the shuttle vector pYADE4 was used as a vector for the cDNA of STR, which was inserted in the BAMHI/Clal restriction site. Saccharomyces cerevisiae
  • YPH500 was used as a host of the recombinant plasmids which contains deficiencies in Tryptophan, Leucine, Lysine, Adenine, and Uracil metabolism (Sikorski and Hieter, 1989) .
  • Yeast transformations were performed by the Lithium Acetate procedure of Ito et al . , (1983) .
  • the cells were collected by centrifugation at 2500 rpm for 5 min. The supernatant was decanted and the cells were resuspended in 1 ml sterile water to wash them, and centrifuged again at 4000 rpm for 2 min. This wash step was repeated twice. Yeast cell walls were then broken by freezing in liquid N 2 and the pellet was resuspended in 170 ⁇ l of 2.35% trichloroacetic acid. The samples were then gently vortexed and centrifuged at 13000 rpm. A volume of 200 ⁇ l supernatant was used to measure the alkaloid concentration by the HPLC system described above. Peaks were identified by their UV spectrum and by collecting them using preparative HPLC (see above) followed by MS-MS.
  • Concentrations were determined using a calibration curve and are expressed in grams per litre medium or in grams per gram cells (FW) .
  • Cells were collected from the medium by centrifugation at 2500 rpm for 5 min. After being washed twice with water, they were frozen in liquid nitrogen, resuspended in 1 ml 0. IM NaPi buffer at pH 6.3 and ground with mortar and pestle. This solution was collected in an eppendorf tube and centrifuged at 13 krpm for 3 min. The supernatant was centrifuged again and 25 ⁇ l was used to measure the enzyme activities. Enzyme activities in the medium were measured by using the medium without further treatment in the enzyme assay.
  • STR was assayed during the protocol by Pennings et al . (1989) .
  • SGD was assayed using the protocol described by Stevens et al . (1992).
  • the enzyme activities were calculated as nkatal per litre of culture medium or per gram fresh weight (FW) .
  • This 1803 bp cDNA clone that was picked up from the cDNA library was not a full length clone, since no start codon was present at the 5 1 end.
  • Another PCR reaction was performed on the cDNA library with primers T3 and Sgsecl. The product (700 bp) found after the PCR reaction was slightly larger than the PCR product found using the 1803 bp clone. Sequence analysis showed 100% homology with the clone already found, however this PCR product contained an extra 72 bp at the 5' end. On this extra part the start codon is located.
  • this PCR product is the complete 5" part of the open reading frame of this clone.
  • the PCR product was fused to the cDNA clone using the restriction site SacII, making a full length clone (1875 bp) .
  • SacII restriction site
  • the nucleotide as well as the predicted amino acid sequence of the full length clone is presented.
  • the cDNA clone was expressed in S . cerevisiae. This yeast culture was found to have a very high SGD activity (2.52 ⁇ 0.05 nkatal/ ⁇ gram cells) , whereas a control culture did not have any SGD activity.
  • indolee alkaloids two genes of the indolee alkaloid biosynthesis were functionally expressed in Saccharomyces cerevisiae. These genes were strictosidine synthase (STR) and strictosidine ⁇ -glucosidase (SGD) from Catharanthus roseus . Expression was accomplished by introducing two shuttle vectors harbouring the cDNA's of these genes into S. cerevisiae . Enzyme activities of SGD and STR were high. SGD activity was found to be continuously expressed during the whole growth phase (2.5 mKat/g cells) .
  • STR strictosidine synthase
  • SGD strictosidine ⁇ -glucosidase
  • STR activity was found inside the cells (13.2 nKat/g cells) , STR activity was found mainly in the medium, peaking at day 3 (25 nkaltal/liter) .
  • C. roseus STR is located in the vacuole. A signal peptide present on the C-terminus of STR is most likely important for correct targetting (Pasquali et al . 1992). S. cerevisiae may not recognize this signal sequence and therefore excrete most of the protein to the medium.
  • SGD is localized in the endoplasmic reticulum
  • Strictosidine could only be detected when both tryptamine and NYCoganin were fed to yeast cells expressing STR. No strictosidine was formed in the medium nor inside the cells after feeding with loganin and tryptamine, showing that yeast is unable to convert loganin into NYCoganin. Stritosidine in the medium could be converted completely by breaking the yeast cells by liquid nitrogen. Like this SGD was able to convert the strictosidine into cathenamine.
  • Figure 1 Nucleotide (1874 base pairs) and predicted amino acid sequence (554 amino acids) sequence of a cDNA coding for strictosidine ⁇ -glucosidase from Catharanthus roseus .

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Abstract

Les alcaloïdes contenant des terpénoïdes-indoles sont des substances pharmaceutiques intéressantes, mais qui ne se trouvent qu'à l'état de traces dans des plantes telles que Catharanthus roseus. L'invention concerne des moyens et des méthodes permettant de produire lesdits alcaloïdes dans un système de culture in vitro. Ledit système de culture comprend du matériel végétal servant de source de précurseurs et partiellement de source de glucose pour lesdits alcaloïdes.
PCT/NL1999/000733 1998-12-04 1999-12-02 Strictosidine glucosidase isolee a partir de catharanthus roseus et son utilisation pour la production d'alcaloides Ceased WO2000042200A1 (fr)

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AU16960/00A AU1696000A (en) 1998-12-04 1999-12-02 Strictosidine glucosidase from catharanthus roseus and its use in alkaloid production

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EP98204116.2 1998-12-04
EP98204116 1998-12-04

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WO2020229516A1 (fr) 2019-05-13 2020-11-19 Danmarks Tekniske Universitet Procédés de production d'un aglycone de strictosidine et d'alcaloïdes d'indole monoterpénoïdes
CN112034058A (zh) * 2020-08-21 2020-12-04 开封康诺药业有限公司 一种长春胺中异构体杂质的检测方法

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US11072613B2 (en) 2016-03-02 2021-07-27 Willow Biosciences, Inc. Compositions and methods for making terpenoid indole alkaloids
WO2020229516A1 (fr) 2019-05-13 2020-11-19 Danmarks Tekniske Universitet Procédés de production d'un aglycone de strictosidine et d'alcaloïdes d'indole monoterpénoïdes
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