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  • 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
  • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
  • C12N15/8253—Methionine or cysteine
• • C—CHEMISTRY; METALLURGY
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  • 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
  • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
• • 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
  • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
  • C12N15/8254—Tryptophan or lysine
• • 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
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
• • 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
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/12—Methionine; Cysteine; Cystine
• • 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
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
  • C12P13/227—Tryptophan

Definitions

• the rate of synthesis of the aspartate-family amino acids is regulated primarily by a complex process of feedback inhibition of the activity of some key enzymes in the pathway by the relevant amino acid end product.
• the first enzymatic activity in the pathway that is common to all of the aspartate-family amino acids, aspartate kinase (AK) activity is feedback inhibited by both lysine and threonine.
• lysine also inhibits the activity of the enzyme dihydrodipicolinate synthase (DHPS), the first enzyme of the pathway after the branch point that leads to the synthesis of lysine.
• DHPS dihydrodipicolinate synthase
• Signal sites such as targeting signals or protein cleavage signals are well known m the art, and are easily provided for by incorporating the appropriate nucleic acid codons (encoding the appropriate ammo acids m the desired order) n the nucleic acid encoding the sink protein.
• a sink protein as provided by the invention which functions as sink or reservoir for the desired ammo acid, thereby regulating biosynthesis or reducing the amount or concentration of available of free ammo acid in said cell or cells of said organism by binding it through transcription and translation processes to the sink protein encoded by the additional nucleic acid with which it is provided.
• a sink protein as provided or used according to the invention preferably contains about at least 7,5% lysine, or 2.5% methion e, or 2.5% cysteine, or 1.5% tryptophan, or 6% threonine, or 6.5% valine, or 12.5% leucine, or 5.5% isoleucine, or 6.5% phenylalanme, or 5.5% tyrosine.
• the invention furthermore provides a transgenic cell having been provided with a nucleic acid encoding a protemaceous substance, a sink protein, comprising at least a desired ammo acid allowing regulating the concentration of said ammo acid in said cell.
• said cell has further been provided with a nucleic acid allowing enhanced translation of said sink protein.
• said cell accumulates said sink protein up to more than 2%, preferably 4%, or even more than 5% to more than 7% of the total protein content of said cell. Such levels of expression are also reached by accumulation of sink protein m aggregates or more-or-less regular crystal-like structures.
• an organism which is a plant, preferably a crop plant such as potato, sugar beet, carrot, cassave, canola, alfalfa, legumes, or grammeae species like rice, maize, wheat, sorghum, barley or grass.
• a crop plant such as potato, sugar beet, carrot, cassave, canola, alfalfa, legumes, or grammeae species like rice, maize, wheat, sorghum, barley or grass.
• a method is provided to increase a desired amino acid level in micro-organisms or organisms such as plants.
• (Overproduced free (essential) amino acids are trapped by incorporation in a sink protein, rich in said amino acid, that is expressed at the same time in the plant.
• the translation of this exogenous sink protein is specifically enhanced by simultaneous expression of a protein factor which recognises a sequence domain in the messenger RNA that is produced from the sink protein gene, and as such enhances the efficiency of protein synthesis of this specific protein.
• Free ammo acid level may be increased by introducing at least one gene encoding (a feedback insensitive) enzyme involved in biosynthesis of said ammo acid.
• Such a combination of gene constructs in a plant cell as provided by the invention comprises: (a) a DNA sequence coding for an enzyme having AK activity, (b) DNA sequences enabling high expression of the AK enzyme in all plant cells or plant organs at an early stage during organ development and subsequent production of high levels of threonine, (c) a DNA sequence coding for an enzyme having DHPS activity, and (d) DNA sequences enabling expression of this DHPS enzyme in specific plant cells or plant organ and at a lower level and at a later stage during development compared to the AK expression and subsequent production of high levels of lysine, (e) a DNA sequence coding for a storage protein rich in essential amino acids or a sink protein forming (semi) - crystalline structures, (f) an omega-sequence, or likewise, translational enhancer functionally linked to the coding sequence of this storage protein, (g) DNA sequence enabling high expression of the storage protein gene, either in the whole plant or in specific tissues of the plant (h)
• Both chimeric gene constructs can be subcloned into expression vectors, such as the Ti plasmids of Agroba cteri um tumefa ciens, the preferred plasmid being the binary vector pBINPLUS (Van Engelen, F.A. et al . (1995) Transgenic Research 4: 288-290) .
• expression vectors such as the Ti plasmids of Agroba cteri um tumefa ciens, the preferred plasmid being the binary vector pBINPLUS (Van Engelen, F.A. et al . (1995) Transgenic Research 4: 288-290) .
• Murashige and Skoog basal salts and vitamins supplemented with 10 mg/1 2,4-D and 3% sucrose at pH 5.8 ) in a 60 ml plastic specimen container (Thovadex) . This in several replicates. Embryogenic callus is induced on immature inflorescence segments of greenhouse grown plants after sterilization with 5% hypochlorite and rinsing with sterile water. The basal parts of the inflorescences are cut into 2 segments of 1-2 mm long and placed on MSt5 medium (i.e. MS basal salts and vitamins supplemented with 0.4 mg/1 ( extra) thiamin-HCl, 5 mg/1 2,4-D and 3% sucrose) solidified with 0.8% Daichin agar.
• Transgenic perennial ryegrass plants are obtained and collected after 8 weeks. They are maintained in tubes containing half strength MSO (Creemers-Molenaar et al., Plan t Science 57: 165-172 [1988]) and subsequently characterized molecularly by PCR and Southern hybridization analysis and biochemically by amino acid , enzyme activity and protein analysis as described in Example 4 - 9 to confirm presence and expression of the newly introduced genes.
• reaction mixture is cent ⁇ fuged (5 mm 16,000 g, 4°C) and the light absorbency of the supernatant at 490 nm is measured. For each sample, the nonspecific activity obtained in the absence of L-aspartate is substracted from that obtained in its presence.

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• Proteomics, Peptides & Aminoacids (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
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• 1999
  • 1999-07-30 EP EP99202524A patent/EP1074629A1/de not_active Withdrawn
• 2000
  • 2000-07-28 CN CN 00813534 patent/CN1377416A/zh active Pending
  • 2000-07-28 CA CA002381556A patent/CA2381556A1/en not_active Abandoned
  • 2000-07-28 EP EP00952058A patent/EP1198576A1/de not_active Withdrawn
  • 2000-07-28 AU AU64819/00A patent/AU6481900A/en not_active Abandoned
  • 2000-07-28 WO PCT/NL2000/000541 patent/WO2001009359A1/en not_active Application Discontinuation

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