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WO1990010073A1 - Gene d'origine vegetale de tryptophane decarboxylase produisant de la tryptamine - Google Patents

Gene d'origine vegetale de tryptophane decarboxylase produisant de la tryptamine Download PDF

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Publication number
WO1990010073A1
WO1990010073A1 PCT/CA1990/000057 CA9000057W WO9010073A1 WO 1990010073 A1 WO1990010073 A1 WO 1990010073A1 CA 9000057 W CA9000057 W CA 9000057W WO 9010073 A1 WO9010073 A1 WO 9010073A1
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WIPO (PCT)
Prior art keywords
tryptophan decarboxylase
tdc
gene
tryptophan
sequence
Prior art date
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Ceased
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PCT/CA1990/000057
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English (en)
Inventor
Vincenzo De Luca
Normand Brisson
Wolfgang G. W. Kurz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Canada Minister of Natural Resources
National Research Council of Canada
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Canada Minister of Natural Resources
National Research Council of Canada
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Application filed by Canada Minister of Natural Resources, National Research Council of Canada filed Critical Canada Minister of Natural Resources
Publication of WO1990010073A1 publication Critical patent/WO1990010073A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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.)

Definitions

  • a trypta ine producing tryptophan decarboxylase gene of plant origin A trypta ine producing tryptophan decarboxylase gene of plant origin.
  • Tryptophan decarboxylase catalyses the conversion of L-tryptophan to tryptamine.
  • This enzyme has been detected in numerous plant systems and it has been suggested that its primary role is to supply possible precursors for auxin biosynthesis (Baxter, C. & Slaytor, M. (1972) Phytochemistry ⁇ , 2763-2766; Gibson, R.A., Barret, G. & Wightman F. (1972) J. Exp. Bot. 2., pages 775-786; Gross, W. & Klapchek, S. (1979) Z. convincedphysiol. £., pages 359-363).
  • TDC catalyses the synthesis of precursors for the protoalkaloids which have considerable physiological activity in higher animals (Smith, T.A. , (1977) Phytochemistry Vol. 1., pages 171- 175). It is also known that tryptophan-derived tryptamines are also precursors of the tricyclic ⁇ - carboline alkaloids formed by condensation with a one- or two- carbon moiety (Slaytor, M. , & McFarlane, I.J., (1968) Phytochemistry 7_, pages 605-610).
  • TDC produces tryptamine for biosynthesis of the commercially important antineoplastic monoterpenoid indole alkaloids, vinblastine and vincristine (De Luca, V., & Kurz, .G. . (1988), Cell Culture and Somatic Cell Genetics of Plants, Constabel, F. and Vasil, I.K., eds. Academic Press 5_, pages 385-401).
  • the TDC from Catharanthus roseus has been purified to homogeneity. It occurs as a dimer consisting of 2 identical subunits of Mr 54,000 and it requires pyridoxal phosphate for activity (Noe, W. , Mollenschott, C. , & Berlin J. (1984) Plant Mol. Biol. 3., pages 281-288) .
  • the enzyme possesses characteristics of plant aromatic decarboxylases which usually exhibit high substrate specificity.
  • TDC will decarboxylate L-tryptophan and 5-hydroxy-L-tryptophan but is inactive towards L-phenylalanine and L-tyrosine
  • the tyrosine decarboxylases from Syrinqa vulgaris (Chappie, C.C.S., (1984) Ph.D. Thesis, University of Guelph, Guelph, Ontario, Canada), Thalictrum ruqosu and Escholtzia californica (Marques, I.A., & Brodelius, P. (1988) Plant Physiol.
  • the role of aromatic L-amino acid decarboxylase is to produce the major neurotrans itters dopamine and serotonin and, in E . elanogaster, the DDC enzyme serves a second, inducible role, in the sclerotization of the insect cuticle (Christenson, J.G., Dairman, . & Undenfriend, S. (1972) Proc. Natl. Acad. Sci. USA 6J9., pages 343-347; Lovenberg, . , Weissbach, ., & Undenfriend S. (1962) J. Biol. Chem. 237, pages 89-93; Yuwiler, A., Geller, E. & Eiduson, S. (1954) Arch. Biochem. Biophys. jJO., pages 162-173; Brunet, P. (1980) Insect Biochem. .10., pages 467-500).
  • tryptophan decarboxylase gene could be inserted into living organisms by transformation to produce tryptamine and related protoalkaloids, it could supplement a neurotransmitter deficiency.
  • the insertion of this gene in plants could be useful to alter the spectrum of tryptophan- based chemicals normally produced by the plant.
  • the insertion of constitutive expression of tryptophan decarboxylase in Brassica species could sequester the cytoplasmic tryptophan pool for the synthesis of tryptamine and related protoalkaloids and therefore repress the normal synthesis and accumulation of indole glucosinolates.
  • a cDNA clone which includes the complete coding region of tryptophan decarboxylase, preferably tryptophan decarboxylase (E.C. 4.1.1.27) from periwinkle (Catharanthus roseus) .
  • the cDNA clone (1747 bp) was isolated by antibody screening of a cDNA expression library produced from poly A* RNA found in developing seedlings of C_j_ roseus. The clone hybridized to a 1.8 kb RNA from developing seedlings and from young leaves of mature plants.
  • Also within the scope of the present invention is a method for inserting TDC gene into living organisms by transformation.
  • the identity of the clone was confirmed when extracts of transformed E_j_ coli expressed a protein containing tryptophan decarboxylase enzyme activity.
  • the tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da.
  • the amino acid sequence shows a high degree of similarity with the aromatic L- a ino acid decarboxylase (dopa-decarboxylase) and the alpha-methyldopa hypersensitive protein of Drosophila melaqonaster.
  • the tryptophan decarboxylase sequence also showed significant similarity to feline glutamic acid decarboxylase and mouse ornithine decarboxylase suggesting a possible evolutionary link between these amino acid decarboxylases. Furthermore, the protein encoded by the cDNA clone of the present invention is active in. vitro. IN THE DRAWINGS
  • Figure 1 (lane 2) represents the TDC enzymatic activity in extracts of pTDC5-transformed E ⁇ coli, compared to those in control Ej_ coli (lane 1) and that in C_j_ roseus itself (lane 3).
  • Figure 2 represents the hybridization of the pTDC-5 clone to a 1.8 kb mRNA species isolated from periwinkle.
  • Figure 3 shows the nucleotide sequence of the pTDC5 cDNA clone and its deduced amino acid sequence. The putative polyadenylation signal is underlined.
  • Figure 4 shows the amino acid sequence alignments of the protein for the I _ melanogaster alpha methyldopa hypersensitive gene (AMD), C_j_ roseus tryptophan decarboxylase (TDC), and Drosphila DOPA decarboxylase i ⁇ oenzy e 1 (DDC1).
  • AMD I _ melanogaster alpha methyldopa hypersensitive gene
  • TDC C_j_ roseus tryptophan decarboxylase
  • DDC1 Drosphila DOPA decarboxylase i ⁇ oenzy e 1
  • Figure 5 shows hydropathy profile of TDC and DDC1. Other advantages of the present invention will be readily illustrated by referring to the following description.
  • Delicata were germinated and grown for 5 days in the dark as described previously (De Luca, V. , Alvarez- Fernandez, F., Campbell, D., & Kurz, W.G.W. (1988) Plant Physiol. 86., 447-450). Seedlings were harvested after 18 hours of light treatment and total RNA was isolated as described by Jones, J.D.G., Duns uir, P. & Bedrook, J. (1985) EMBO J. 4., 2411-2418.
  • RNA was isolated by chromatography on oligo (dT) " cellulose (Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69., 1408-1412) and double-stranded cDNAs were prepared according to the procedure of Gubler and Hoffman (1983, Gene 25., 263-269). Following ligation with Eco RI linker, the cDNA was inserted into the Eco RI site of the expression vector ZAP (Stratagene, San Diego, Short, J.M., Fernandez, J.M., Sorge, J.A. & Huse, W.D. (1988) Nucl. Acids Res. 16., 7583-7600).
  • a library containing 3.1 X 10 5 recombinant phages was obtained and after amplification, 2 X 10 plaques were screened with specific polyclonal antiserum raised against-TDC. Plasmids (pBluescript) containing a TDC cDNA insert were rescued using the R408 fl helper phage (Short, J.M., Fernandez, J.M., Sorge, J.A. & Huse, W.D. (1988) Nucl. Acids Res.
  • Poly(A) + RNA was isolated from 6 day old developing seedlings and from young leaves of mature plants as described above. These tissues were chosen as a likely source of TDC poly(A) + RNA based on the presence of high levels of TDC enzyme activity (De Luca, V., Alvarez-Fernandez, F., Campbell, D., & Kurz, W.G.W. (1988) Plant Physiol. 86., 447-450). RNA was denatured, fractioned by electrophoresis on formaldehyde/agarose gels, and then transferred to nitrocellulose filters (Maniati ⁇ , T., Fritsch, E.F. & Sambrook, J. (1982) In: Molecular Cloning, A Laboratory Manual. Cold Spring Harbor, New York). Blotted RNA was hybridized to [ 32 PJ- labelled pTDC5 DNA and autoradiography was performed using Kodak XAR-5 films. TDC activity in extracts of E ⁇ _ coli.
  • a culture (100 ml) of the E_ ⁇ coli strain ZL1- blue containing pTDC5 or pBluescript was incubated at 37°C for 2 hours before adding the IPTG inducer at a final concentration of 1 mM. Incubation was continued for an additional 2 hours. Cells were harvested, washed in TE buffer, resuspended and lysed in 3 ml of a buffer containing 0.1 M Hepes, pH 7.5, 1 mM DTT. Debris was removed by centrifugation and the supernatant was desalted by passage over a Sephadex 6-25** column.
  • TDC enzymatic activity in bacterial supernatants was determined by monitoring the conversion of L-[roethylene- 14 C]-tryptophan to [ 14 C]- ryptamine (De Luca, V., Alvarez- Fernandez, F. , Campbell, D., & Kurz, W.G.W. (1988) Plant Physiol. 86., 447-450).
  • Supernatants (30 pi ) were incubated in the presence of 0.1 ⁇ Ci of ["C]-tryptophan (sp. act. 59 mCi/mmol. ) for 30 minutes and reactions were stopped with 100 jul NaOH.
  • Radioactive tryptamine was extracted from the reaction mixture with ethyl acetate and was analyzed by silica gel thin layer chromatography and autoradiography. Determination of TDC enzyme activity in leaves was performed as described previously (De Luca, V., Alvarez-Fernandez, F., Campbell, D., & Kurz, W.G.W. (1988) Plant Physiol. 86, 447-450). TDC enzymatic activity in E_;_ coli.
  • a tryptophan decarboxylase cDNA clone of C. roseus was isolated by the use of antibody screening of an expression library.
  • the antigenicity and enzymatic activity ( Figure 1) of the encoded protein established the identity of the TDC cDNA.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
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Abstract

La présente invention se rapporte à l'isolement et au clonage d'une séquence d'ADNc du gène de tryptophane décarboxylase provenant di Catharanthus roseus et à la mise au point de la séquence d'ADNc dans un vecteur de plasmide capable de transformer des lignées cellulaires qui vont produire l'enzyme tryptophane décarboxylase.
PCT/CA1990/000057 1989-02-24 1990-02-21 Gene d'origine vegetale de tryptophane decarboxylase produisant de la tryptamine Ceased WO1990010073A1 (fr)

Applications Claiming Priority (2)

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US31487989A 1989-02-24 1989-02-24
US314,879 1989-02-24

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WO1990010073A1 true WO1990010073A1 (fr) 1990-09-07

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EP (1) EP0425597A1 (fr)
JP (1) JP2919069B2 (fr)
CA (1) CA2010582C (fr)
WO (1) WO1990010073A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5814520A (en) * 1989-02-24 1998-09-29 National Research Council Canada Tryptamine producing tryptophan decarboxylase gene of plant origin
WO1999006566A1 (fr) * 1997-07-31 1999-02-11 Sanford Scientific, Inc. Production de poinsettia transgenique
WO1999006581A1 (fr) * 1997-07-31 1999-02-11 Sanford Scientific, Inc. Plantes transgeniques employant le gene de tdc (tryptophane decarboxylase) en vue d'une amelioration des cultures
CN105274083A (zh) * 2015-11-20 2016-01-27 中国科学院华南植物园 一种谷氨酸脱羧酶及其编码基因和应用
CN113403351A (zh) * 2021-06-28 2021-09-17 新泰市佳禾生物科技有限公司 一种将l-色氨酸转化为色胺和d-色氨酸的方法

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
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BOSSA, F. ET AL., BIOCHEM. BIOPHYS. RES. COMMUN., vol. 78, 1977, pages 177 - 183
CHAPPLE, C.C.S., PH.D. THESIS, 1984
CLARK, W.C. ET AL., HOL. GEN. GENET., vol. 162, 1978, pages 287 - 297
DE LUCA, V. ET AL., PLANT PHYSIOL., vol. 86, 1988, pages 4474 - 50
EMBO Journal, Volume 5, No. 10, 1986, IRL Press Limited, (Oxford, GB), D.D. EVELETH et al.: "Sequence and Structure of the Dopa Decarlboxylase Gene of Drosophila: Evidence for Novel RNA Splicing Variants", see pages 2664-2668 *
EVELETH, D.D. ET AL., EHBO J., vol. 5, 1986, pages 2663 - 2672
EVELETH, D.D.; HARSH, J.L., GENETICS, vol. 114, 1986, pages 469 - 483
HANECKJEE, R.; BAYLIN, S.B., BIOCHEMISTRY, vol. 22, 1983, pages 6058 - 6063
HARQUES, I.A.; BRODELIUS, P., PLANT PHYSIOL., vol. 88, 1988, pages 52 - 55
HOE, W.; HOLLENSCHOTT, C.; BERLINE J., PLANT MOL. BIOL., vol. 3, 1984, pages 281 - 288
HORGAN, B.A.; JOHNSON, W.A.; HIRSH, J., EHBO J., vol. 5, 1986, pages 3335 - 3342
Journal of Biological Chemistry, Volume 262, No. 19, 5 July 1987, The American Society of Biological Chemists, Inc., (US), V.R. ALBERT et al.: "A Single Gene Codes for Aromatic L-Amino Acid Decarboxylase in both Neuronal and Non-Neuronal Tissues", pages 8404-9411 *
KOBAYASHI, Y.; KAUFMAN, D.L.; TOBIN, A.J., J. MEUROSCI., vol. 7, 1987, pages 2768 - 2772
LÜTCKE, H.A. ET AL., EHBO J., vol. 6, 1987, pages 43 - 48
Plant Molecular Biology, No. 3, 1984, Martinus Nijhoff/Dr W. Junk Publishers, (Dordrecht, NL), W. NOE et al.: "Tryptophan Decarboxylase from Catharanthus Roseus Cell Suspension Cultures: Purification, Molecular and Kinetic Data of the Homo genous Protein, pages 281-288 *
Procl. Natl. Acad. Sci. USA, Volume 86, April 1989, Biochemistry, (US), V. De LUCA et al.: "Molecular Cloning and Analysis of cDNA Encoding a Plant Tryptophan Decarboxylase: Comparison with Animal Dopa Decarboxylases", pages 2582-2586 *
Progress in Catecholamine Research, Part A: Basic Aspects and Peripheral Mechanisms, 1988, Alan R. Liss, Inc., TONG H. JOH et al.: "Molecular Biology of Aromatic L-Amino Acid Decarboxylase and Dopamine beta-Hydroxylase", see pages 30-31 *
WATSON, H.E.E., NUCL. ACIDS RES., vol. 12, 1984, pages 5145 - 5164

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5814520A (en) * 1989-02-24 1998-09-29 National Research Council Canada Tryptamine producing tryptophan decarboxylase gene of plant origin
WO1999006566A1 (fr) * 1997-07-31 1999-02-11 Sanford Scientific, Inc. Production de poinsettia transgenique
WO1999006581A1 (fr) * 1997-07-31 1999-02-11 Sanford Scientific, Inc. Plantes transgeniques employant le gene de tdc (tryptophane decarboxylase) en vue d'une amelioration des cultures
US7119262B1 (en) 1997-07-31 2006-10-10 Sanford Scientific, Inc. Production of transgenic poinsettia
CN105274083A (zh) * 2015-11-20 2016-01-27 中国科学院华南植物园 一种谷氨酸脱羧酶及其编码基因和应用
CN105274083B (zh) * 2015-11-20 2018-11-23 中国科学院华南植物园 一种谷氨酸脱羧酶及其编码基因和应用
CN113403351A (zh) * 2021-06-28 2021-09-17 新泰市佳禾生物科技有限公司 一种将l-色氨酸转化为色胺和d-色氨酸的方法

Also Published As

Publication number Publication date
JPH03505161A (ja) 1991-11-14
CA2010582A1 (fr) 1990-08-24
JP2919069B2 (ja) 1999-07-12
EP0425597A1 (fr) 1991-05-08
CA2010582C (fr) 1998-07-28

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