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EP1157119A1 - Methode de modulation de la biosynthese de metabolite dans des cellules recombinees - Google Patents

Methode de modulation de la biosynthese de metabolite dans des cellules recombinees

Info

Publication number
EP1157119A1
EP1157119A1 EP00904136A EP00904136A EP1157119A1 EP 1157119 A1 EP1157119 A1 EP 1157119A1 EP 00904136 A EP00904136 A EP 00904136A EP 00904136 A EP00904136 A EP 00904136A EP 1157119 A1 EP1157119 A1 EP 1157119A1
Authority
EP
European Patent Office
Prior art keywords
plant
cell
transcription factor
expression
metabolite
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00904136A
Other languages
German (de)
English (en)
Inventor
Johan Memelink
Cornelia Theodora Elisabeth Van Der Fits
Franciscus Leonardus Menke
Jan Willem Kijne
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.)
Universiteit Leiden
Original Assignee
Universiteit Leiden
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universiteit Leiden filed Critical Universiteit Leiden
Publication of EP1157119A1 publication Critical patent/EP1157119A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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

Definitions

  • secondary metabolites usually do not possess a basal function in cell growth and maintenance. They are a group of chemically very diverse products that often have a restricted taxonomic distribution. Secondary metabolites normally exist as members of closely related chemical families, usually of a molecular weight of less than 1,500, although some bacterial toxins are considerably larger. Two examples of fungal cell secondary metabolites are penicillin and ergotamine.
  • AP2 transcription factors suitable for use in the invention include ORCA1, ORCA2, ORCA3, AtERFl, AtEBP, AtCBFl, AtDREBl, AtDREB2A, AtDREB2B, TINY, NtEREBPl,2,3,4, LePti4,5,6, ABI4.
  • AtERFl AtEBP, AtCBFl, AtDREBl, NtEREBP 1,2,3,4, LePti4,5,6, ORCA1, ORCA-2 and ORCA3, of which ORCA-2 and ORCA3 are most preferred.
  • naturally occuring transcription factors and in particular naturally occuring AP2 transcription factors, may be used that are involved in the growth and/or development of a plant, such as the growth and/or development of the flower, of seed, etc..
  • these will be AP2 transcription factors containing two AP2 domains, although the invention in its broadest sense is not limited thereto.
  • mutants, analogs, variants, parts or fragments etc. e.g. as described below
  • of such naturally occuring transcription factors may also be used.
  • such synthetic AP2 transcription factors may be mutants, analogs, variants, parts or fragments of the abovementioned naturally occuring AP2 transcription factors.
  • Such mutants, analogs, variants, parts or fragments may for instance be derived from the amino acid sequence of such a naturally occuring AP2 transcription factor by addition, substitution, insertion and/or deletion of one or more amino acid residues, e.g. in the part(s) of the sequence of the transcription factor corresponding to the AP2 domain(s); in one of the other parts of the sequence of the transcription factor sequence such as the transcription activation domain(s) and/or linking sequences; or in both.
  • the expression in plant (cell) of an AP2 transcription factor in accordance with the invention may be used to regulate one or more metabolic pathways leading to one or more secondary metabolites, including but not limited to those mentioned hereinabove and below.
  • Such regulation of the metabolic pathway(s) may alter or otherwise influence the entire metabolic pathway(s) - e.g. compared to the native situation, e.g. without exposure to stress - but also one or more separate steps (e.g. enzymatic conversions) thereof, which may include enzymatic conversions belonging to the same or to different metabolic pathways.
  • the transcription factor(s) of the invention may also be expressed in the plant cell under the control of a tissue specific promoter, e.g. specific for expression in leaves, stalks, stems, roots, petals, flowers, reproductive organs, fruits, tubers, bulbs and seeds; or under the control of a promoter that is specific for a certain phase of the development of the plant.
  • the transcription factor(s) of the invention may also be expressed in the plant cell under the control of promoters that are activated by environmental signals, including but not limited to stress-related signals, light, exogenously added chemicals, pathogens or pathogenic signals. Examples of such promoters will be clear to the skilled person.
  • the cell to which the AP2 transcription factor is provided may be in the form of- i.e. may be present in - a cell culture (e.g. in vitro); or may be present in a plant in vivo, including but not limited to any tissue, part or organ of such a plant, such as the leaves, stalks, stems, roots, petals, flowers, reproductive organs, as well as fruits, tubers, bulbs and/or seeds.
  • a cell culture e.g. in vitro
  • a plant in vivo including but not limited to any tissue, part or organ of such a plant, such as the leaves, stalks, stems, roots, petals, flowers, reproductive organs, as well as fruits, tubers, bulbs and/or seeds.
  • the invention relates to a method of modulating in a cell the expression of one or more genes involved in the biosynthesis of a metabolite or a precursor herefor, the method comprising inserting into the cell a nucleotide sequence coding for a transcription factor other than a transcription factor having a bHLH-type or a MYB-type DNA-binding domain, the nucleotide sequence is operably linked to at least one expression regulating sequence, and/or modifying the expression of a nucleotide sequence coding for such a transcription factor already present in the cell, the transcription factor is capable of regulating the expression of at least one gene coding for a gene product involved in the biosynthesis of said metabolite or precursor, and subjecting the cell to conditions where the inserted nucleotide sequence coding for a transcription factor is expressed or the expression of the nucleotide sequence already present in the cell is modulated.
  • the nucleotide sequence coding for the transcription factor may be derived from a cell of a species selected from the group consisting of the Gentianales order and the Cornales order and/or of a species selected from an indole alkaloid producing species.
  • Some non-limiting preferred examples thereof include species selected from the group consisting of species belonging to the Apocynaceae, Alangiaceae, Loganiaceae, Icacinaceae, Cornaceae and Rubiaceae families, and the genus Catharanthus, which is particularly preferred. Most preferred is a Catharanthus roseus cell.
  • step (i) probing a cDNA or genomic library with a probe comprising at least a fragment of a nucleic acid as described above; (ii) identifying a DNA clone that hybridises with said at least a fragment of a nucleic acid; and (iii) isolating the DNA clone identified in step (ii) wherein the nucleic acid sequence is coding for all or a part of said transcription factor.
  • the invention relates to a method of isolating a nucleotide sequence coding for a transcription factor not having a bHLH-type or a MYB-type DNA-binding domain, said transcription factor is capable of regulating in a cell the expression of at least one gene coding for a gene product involved in biosynthesis of a metabolite or a precursor therefor, the method comprising the steps of (i) synthesising an oligonucleotide primer set corresponding to at least a fragment of a sequence selected from the group consisting of SEQ ID NO:l, 2 and 3; and (ii) amplifying cDNA or genomic DNA using said primer set in a polymerase chain reaction; wherein the amplified nucleic acid fragment is coding for all or a part of said transcription factor to obtain a nucleotide sequence coding for a transcription factor not having a bHLH- type or a MYB-type DNA-binding domain.
  • the invention relates to a plant, plant cell or plant material that has been transformed with a genetic construct comprising at least one nucleotide sequence encoding at least one transcription factor, operably linked to an expression regulating sequence, in which the plant (cell), genetic construct, transcription factor and expression regulating sequence may be as defined above.
  • the central intermediate in TIA biosynthesis is the glucoalkaloid strictosidine.
  • This compound is formed from the intermediates tryptamine and horroganin. a reaction catalyzed by the enzyme strictosidine synthase (STR; Fig. 17).
  • Tryptophan decarboxylase (TDC) catalyzes the formation of tryptamine. Tryptophan is derived from chorismate via multiple steps, the first of which is catalyzed by anthranilate synthase (ASA). Secologanin is formed via multiple steps from geraniol.
  • anthranilate synthase (Asa) and D-l-deoxyxylulose-5-phosphate synthase (Dxs) were expressed at higher levels in the O3-OX lines (Fig.14B and D). This indicates that ORCA3 not only regulates multiple genes in the TIA pathway, but in addition genes in primary metabolism that are involved in production of TIA precursors.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nutrition Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne une méthode permettant de moduler dans une cellule l'expression d'au moins un gène intervenant dans la biosynthèse d'un métabolite ou d'un précurseur de ce dernier. Cette méthode consiste à insérer dans une cellule une séquence de nucléotides codant pour un facteur de transcription qui comprend un domaine de liaison d'ADN AP2, et/ou en modifiant l'expression d'une séquence de nucléotides codant pour un tel facteur de transcription déjà présent dans la cellule. Ladite méthode prouve son utilité dans l'amélioration de la biosynthèse de métabolites secondaires chez les plantes, tels que les alcaloïdes, dont les alcaloïdes indoliques de terpène. Cette invention concerne également une méthode permettant d'améliorer la tolérance au stress chez les plantes à l'aide de tels facteurs de transcription.
EP00904136A 1999-02-05 2000-02-07 Methode de modulation de la biosynthese de metabolite dans des cellules recombinees Withdrawn EP1157119A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DK15899 1999-02-05
DKPA199900158 1999-02-05
US11938899P 1999-02-10 1999-02-10
US119388P 1999-02-10
PCT/NL2000/000075 WO2000046383A2 (fr) 1999-02-05 2000-02-07 Methode de modulation de la biosynthese de metabolite dans des cellules recombinees

Publications (1)

Publication Number Publication Date
EP1157119A1 true EP1157119A1 (fr) 2001-11-28

Family

ID=26063424

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00904136A Withdrawn EP1157119A1 (fr) 1999-02-05 2000-02-07 Methode de modulation de la biosynthese de metabolite dans des cellules recombinees

Country Status (6)

Country Link
EP (1) EP1157119A1 (fr)
JP (1) JP2002535993A (fr)
AU (1) AU779162B2 (fr)
CA (1) CA2361678A1 (fr)
NZ (1) NZ513356A (fr)
WO (1) WO2000046383A2 (fr)

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NZ513356A (en) 2004-01-30
WO2000046383A2 (fr) 2000-08-10
WO2000046383A8 (fr) 2001-11-01
AU779162B2 (en) 2005-01-06
WO2000046383A3 (fr) 2001-09-07
CA2361678A1 (fr) 2000-08-10
JP2002535993A (ja) 2002-10-29

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