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WO2010051489A1 - Alimentation pour animaux comprenant des algues génétiquement modifiées - Google Patents

Alimentation pour animaux comprenant des algues génétiquement modifiées Download PDF

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Publication number
WO2010051489A1
WO2010051489A1 PCT/US2009/062843 US2009062843W WO2010051489A1 WO 2010051489 A1 WO2010051489 A1 WO 2010051489A1 US 2009062843 W US2009062843 W US 2009062843W WO 2010051489 A1 WO2010051489 A1 WO 2010051489A1
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Prior art keywords
genetically modified
feedstock
organism
enzyme
photosynthetic organism
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Inventor
Michael Mendez
Craig Behnke
Linda Davis
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Sapphire Energy Inc
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Sapphire Energy Inc
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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
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G OR C10K; LIQUIFIED PETROLEUM GAS; USE OF ADDITIVES TO FUELS OR FIRES; FIRE-LIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/04—Liquid carbonaceous fuels essentially based on blends of hydrocarbons
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • 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
    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen

Definitions

  • a biomass-degrading enzyme may be, but is not limited to, a galactanase, xylanase, protease, carbohydrase, lipase, reductase, oxidase, transglutaminase, phytase, or any mixture thereof.
  • the biomass-degrading enzyme is a carbohydrase, for example, an ⁇ -amylase, ⁇ -amylase, endo- ⁇ - glucanase, endoxylanase, ⁇ -mannanase, ⁇ -galactosidase, or pullulanase.
  • the genetic modification to the organism impairs photosynthetic capability of the organism.
  • an organism in which that photosynthetic capability has been impaired due to a genetic modification is still considered an NVPO if the organism was photosynthetic prior to the genetic modification.
  • the genetically modified non-vascular photosynthetic organism has a higher lipid, fatty acid, or isoprenoid content relative to an unmodified organism of the same species.
  • Useful organisms may be prokaryotic or eukaryotic.
  • the feedstock comprises one or more of alfalfa, barley, blood meal, beet, bone meal, brewer grain, brewer's yeast, broom grass, carrot, cattle manure, clover, coffee, corn, corn glutten meal, distiller grains, poultry fat, grape, hominy feed, hop leaves, spent hops, molasses, oats, algae, peanuts, potato, poultry litter, poultry manure, rape meal, rye, safflower, sorghum, soybean, soy, sunflower meal, timothy hay, or triticale.
  • the feedstock comprises one or more of alfalfa, barley, blood meal, beet, bone meal, brewer grain, brewer's yeast, broom grass, carrot, cattle manure, clover, coffee, corn, corn glutten meal, distiller grains, poultry fat, grape, hominy feed, hop leaves, spent hops, molasses, oats, algae, peanuts, potato, poultry litter, poultry manure, rape meal, rye, safflower, sorghum, soybean, soy, sunflower meal, timothy hay, or triticale.
  • the increased nutrient availability may be one or more of increased digestible protein, increased digestible carbohydrate, or increased mineral availability.
  • the non-ruminant animal may also be an aquatic species such as a fish or crustacean.
  • aquatic species such as a fish or crustacean.
  • fish include, but are not limited to trout, salmon, tilapia, catfish, perch, bluebill or carp.
  • crustaceans include lobster, crabs, crayfish and shrimp.
  • the non-ruminant animal may also be an aquatic species such as a fish or crustacean.
  • aquatic species such as a fish or crustacean.
  • fish include, but are not limited to trout, salmon, tilapia, catfish, perch, bluebill or carp.
  • crustaceans include lobster, crabs, crayfish and shrimp.
  • any of the improved feedstocks described herein are suitable for consumption by an animal.
  • the improved feedstocks described herein are suitable for consumption by an animal to be used as food by humans or producing a product to be used as food by humans.
  • Figure 4 is a graphic representation of additional nucleic acid constructs.
  • Figure 6 shows gas chromatography - mass spectrometry analysis of C. reinhardtii transformed with FPP synthase and bisabolene synthase.
  • Biomass degrading enzymes can improve the nutrient value of an existing feedstock by breaking down complex components of the feedstock (e.g. indigestible components) into components that can be absorbed and used by the animal.
  • a biomass-degrading enzyme can be expressed and retained in the NVPO or secreted or expelled (i.e. produced ex vivo) from the NVPO.
  • Genetically modified NVPOs that provide the biomass degrading enzymes can also be utilized by the animal for the inherent nutrient value of the NVPO.
  • a composition can comprise a feedstock, a genetically modified non-vascular photosynthetic organism, and a biomass-degrading enzyme that is ex vivo to the genetically modified non-vascular photosynthetic organism.
  • a genetically modified non-vascular photosynthetic organism is modified to increase expression of a naturally occurring biomass-degrading enzyme.
  • NVPO neoid rich lipids, fatty acids and carbohydrates.
  • Genetically modified NVPOs that express such nutrient rich components can be added to an existing feedstock to supplement the nutritional value of the feedstock.
  • such genetically modified NVPOs can comprise as much as 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the improved feedstock.
  • NVPOs can be genetically modified to produce or increase production of one or more fatty acids, lipids or hydrocarbons.
  • a genetically modified NVPO comprises at least one exogenous nucleic acid encoding an enzyme in an isoprenoid biosynthesis pathway.
  • a genetically modified NVPO can have a higher content of fatty acids, lipids or hydrocarbons (e.g. isoprenoids) than an unmodified NVPO of the same species. Therefore in one aspect a composition can comprise a feedstock and a genetically modified NVPO wherein the NVPO has a higher lipid, fatty acid, or isoprenoid content relative to an unmodified NVPO of the same species.
  • the biosynthetic enzymes can also be one found in a mevalonate pathway.
  • the enzyme can be farnesyl pyrophosphate synthase, geranyl geranyl phosphate synthase, squalene synthase, thioesterase, or fatty acyl-CoA desaturase.
  • a genetically modified NVPO can be used for a purpose (e.g. in producing a recombinant product or biofuel) and the remaining portion thereof can be used for an improved feedstock. Therefore an improved feedstock can comprise a portion of a genetically modified NVPO.
  • a composition of an animal feed ingredient can comprise whole and/or defatted algae (e.g. after removal of fatty acids, lipids or hydrocarbons, e.g. after hexane extraction) or a mixture of whole and defatted algae, which provides both the feed enzyme and the inherent nutritive value of the algae.
  • Methods of generating, modifying, supplementing or improving a feedstock composition are also disclosed herein.
  • the methods can comprise combining a genetically modified NVPO or a portion thereof with a feedstock to generate the improved feedstock.
  • the method comprises removing a lipid, fatty acid, isoprenoid, or carbohydrate from a genetically modified NVPO.
  • the remaining genetically modified NVPO, or a portion thereof can be combined with a feedstock to generate the improved feedstock composition.
  • the modified NVPO does not express an exogenous phytase.
  • the genetically modified NVPO or a portion thereof can comprise at least one nucleic acid (e.g. an exogenous nucleic acid).
  • the nucleic acid can be a vector.
  • the nucleic acid encodes a biomass degrading enyzme.
  • the biomass-degrading enzyme can be a galactanase, xylanase, protease, carbohydrase, lipase, reductase, oxidase, transglutaminase, or phytase.
  • the biomass-degrading enzyme can be a carbohydrase, for example, an ⁇ -amylase, ⁇ -amylase, endo- ⁇ -glucanase, endoxylanase, ⁇ -mannanase, ⁇ -galactosidase, or pullulanase.
  • the genetically modified NVPO can be grown under heterotrophic conditions or autotrophic conditions.
  • the genetically modified NVPO can be grown in darkness.
  • the feedstock or improved feedstock to which the NVPO is added can comprise one or more of the following of alfalfa, barley, blood meal, beet, bone meal, brewer grain, brewer's yeast, broom grass, carrot, cattle manure, clover, coffee, corn, corn glutten meal, distiller grains, poultry fat, grape, hominy feed, hop leaves, spent hops, molasses, oats, algae, peanuts, potato, poultry litter, poultry manure, rape meal, rye, safflower, sorghum, soybean, soy, sunflower meal, timothy hay, or triticale.
  • a composition can comprise a feedstock and a genetically modified NVPO.
  • the NVPO e.g. genetically modified NVPO
  • the NVPO can be S. dimorphus, S. obliquus, C. reinhardtii, D. salina or H. pluvalis.
  • the NVPO is a NVPO other than Po ⁇ hyridium.
  • the biosynthetic enzyme can be in an isoprenoid biosynthesis pathway.
  • two enzymes are encoded by one or more exogenous nucleic acids.
  • a genetically modified non-vascular photosynthetic organism comprises at least one nucleic acid encoding a first and a second enzyme, wherein the first enzyme is an enzyme in an isoprenoid biosynthesis pathway, and the second enzyme is a biomass-degrading enzyme.
  • the first and second enzymes are encoded on a vector. In some aspects, the first and second enzymes are encoded on a single vector.
  • the biomass-degrading enzyme can be a galactanase, xylanase, protease, carbohydrase, lipase, reductase, oxidase, transglutaminase, or phytase.
  • the biomass-degrading enzyme is a carbohydrase.
  • the biomass-degrading enzyme is a protease.
  • the biomass-degrading enzyme is a phytase.
  • nucleic acid refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. f0033
  • a regulatory element as the term is used herein, broadly refers to a nucleotide sequence that regulates the transcription or translation of a nucleic acid or the localization of a polypeptide to which it is operatively linked.
  • a regulatory clement may be native or foreign to the nucleotide sequence encoding the polypeptide.
  • Such elements include, but are not limited to, an RBS, a leader, a polyadenylation sequence, a pro-peptide sequence, a promoter, a signal peptide sequence, a transcription terminator, an enhancer, an initiation (start) codon, a splicing signal for intron excision and maintenance of a correct reading frame, a STOP codon, an amber or ochre codon, or an IRES.
  • the typical vector may comprise regulatory elements such as a promoter or enhancer, a transcriptional and translational start signal and a transcriptional and translational stop signal.
  • a vector may comprise various linkers for the purpose of introducing one or more restriction sites that facilitate ligation of regulatory elements and coding regions into the vector.
  • a vector can comprise sequences and regulatory elements indigenous to NVPO such as viral sequences and bacterial sequences which are naturally associated with the NVPO.
  • a vector can be engineered to express one or more proteins wherein the expressed protein can be an individual protein, a pro-protein or a fusion protein.
  • a vector can be engineered to express proteins comprising additional elements that allow the expressed proteins to be targeted to an organelle, targeted to the cell surface, anchored on the cell surface, or secreted to the environment.
  • a vector can be engineered to express proteins comprising elements that allow compartmentalization (i.e., targets a polypeptide to the cytosol, nucleus, chloroplast membrane or cell membrane). Such signals are well known in the art and have been widely reported (see, e.g., U.S. Pat. No. 5,776,689).
  • Vectors can be engineered to express a selectable marker or reporter for efficient selection of a host organism (e.g. green algae) transformed by the vector.
  • a selectable marker or reporter can be operably linked downstream or upstream of one or more regulatory elements.
  • reporter or "selectable marker” refers to an encoded polypeptide that confers a detectable phenotype.
  • a reporter generally encodes a detectable polypeptide, for example, a green fluorescent protein or an enzyme such as luciferase, which, when contacted with an appropriate agent (a particular wavelength of light or luciferin, respectively) generates a signal that can be detected by eye or using appropriate instrumentation (Giacomin, F/ ⁇ «/ Sci.
  • a reporter or selectable marker can be a cell surface marker.
  • a selectable marker generally is a molecule that, when present or expressed in a cell, provides a selective advantage (or disadvantage) to the cell containing the marker, for example, the ability to grow in the presence of an agent that otherwise would kill the cell.
  • a selectable marker can provide a means to isolate a genetically modified host organism that expresses the marker (see, for example, Bock, R. (2001) J.
  • Non limiting examples of selectable markers include those that confer antimetabolite resistance, for example, dihydrofolate reductase, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13: 143-149, 1994); neomycin phosphotransferase, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera- Estrella, EMBOJ.
  • selection markers may include kanamycin, phlcomycin, bleomycin and zeocin.
  • Additional selectable markers include those that confer herbicide resistance, for example, phosphinothricin acetyltransferase gene, which confers resistance to phosphinothricin (White et al., Nucl. Acids Res. 18:1062, 1990; Spencer et al., Theor. Appl. Genet.
  • a composition can comprise a feedstock and a genetically modified NVPO.
  • the genetically modified NVPO can be genetically modified to produce a biomass-degrading enzyme such as a phytase.
  • the phytase is a phytase of bacterial or fungal origin.
  • the biomass-degrading enzyme is an enzyme other than a phytase.
  • enzymes of the present invention may be modified to exhibit characteristics which are useful in feed production and/or supplementation. For example, as some feeds are heated during processing, enzymes may be engineered to withstand high temperatures for extended periods of time. An enzyme of the present invention may be engineered to withstand elevated temperatures of 40-100 0 C for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or more minutes. In some instances, thermostable biomass-degrading enzymes are also contemplated for use herein such as those disclosed in WO/2003/062409.
  • Non limiting examples of enzymes that make up the MEP pathway include l-deoxy-D-xyIulose-5-phosphate synthase (DXS) and 1-deoxy-D- xylulose-5-phosphate reductoisomerase (DXR).
  • DXS l-deoxy-D-xyIulose-5-phosphate synthase
  • DXR 1-deoxy-D- xylulose-5-phosphate reductoisomerase
  • a genetically modified NVPO is defatted (e.g. depleted or partially depleted of lipid, fatty acids or hydrocarbons, including but not limited to isoprenoids and carotenoids) prior to use as an animal feed.
  • the genetically modified NVPO can first be used as a source of producing a biofuel or other product (e.g. a pharmaceutical product) and the remaining defatted NVPO can then be used for animal feed. Therefore a genetically modified NVPO can be lysed (for example by mechanical, chemical, enzymatic or other means) prior to use as an animal feed.
  • a genetically modified NVPO can be dried prior to or after lysis and used as an animal feed.
  • Transforming DNA (SEQ ID NO. 16 ) is shown graphically in FIG. IA.
  • the segment labeled “Transgene” is the endo- ⁇ - glucanase gene (SEQ ID NO. 16) encoding the endo- ⁇ -glucanase protein (SEQ ID NO. 15)
  • the segment labeled "psbA 5' UTR” is the 5' UTR and promoter sequence for the psbA gene from C. reinhardtii
  • the segment labeled "psbA 3' UTR” contains the 3' UTR for the psbA gene from C.
  • transgene in FIG 4 and is regulated by the 5' UTR and promoter sequence for thepsbA gene from C reinhardtii and the 3' UTR for the psbA gene from C reinhardtii, and the segment labeled "Resistance Marker” is the streptomycin resistance encoding gene from bacteria, which is regulated by the 5' UTR and promoter sequence for the atpA gene from C reinhardtii and the 3' UTR sequence for the rbcL gene from C reinhaidtii
  • the FPP synthase transgene cassette is targeted to the psbA loci of C reinhardtii via the segments labeled "Homology A" and "'Homology B,” which are identical to sequences of DNA flanking the psbA loci on the 5' and 3' sides, respectively
  • the bisabolene synthase lransgene cassette is targeted to the 3HB locus of C reinhardtii via the segments labeled "Hom
  • Transgcne 2 is the xylanasc from T. reesei (BDI l SEQ ID NO: 31), the promoter and 5' UTR for the psbD gene from S. dimorphus, and the 3' UTR for the psbA gene from S. dimorphus.
  • the vector was prepared without Transgene 2. The transgene expression cassette and selection marker are targeted to the 5.
  • Transforming DNA was introduced into S. dimorphus via particle bombardment with DNA carried on 550 nm gold particles at 500psi and a shooting distance of 4cm. Transformants were selected by growth on TAP-CAM agar medium under constant light 50-10OuE at room temperature for approximately 2 weeks. Transformants were streaked onto TAP-CAM agar medium to ensure single colony isolation and grown for 4 days under constant light. Transformants were analyzed by PCR screening for homoplasmy.
  • Samples were mixed 1 :4 with loading buffer (XT Sample Buffer with ⁇ - mercaptoethanol, Bio-Rad), heated to 98°C for 5 min, cooled to 23°C, and proteins were separated by SDS-PAGE, followed by transfer to PVDF membrane.
  • the membrane was blocked with Starting Block T20 Blocking Buffer (Thermo Scientific) for 15 min, incubated with horseradish peroxidase-linked anti-FLAG antibody (diluted 1 :2,500 in Starting Block T20 Blocking Buffer) at 23 0 C for 2 hours, washed three times with TBST. Proteins were visualized with chemiluminescent detection.
  • PCR was used to identify transformed strains.
  • 10 6 algae cells (from agar plate or liquid culture) were suspended in 10 mM EDTA and heated to 95 0 C for 10 minutes, then cooled to near 23°C.
  • a PCR cocktail consisting of reaction buffer, MgCl 2 , dNTPs, PCR primer pair(s) DNA polymerase, and water was prepared.
  • PCR was conducted as described in Example 1 with the addition of SEQ ID NO. 32, SEQ ID NO. 33. and SEQ ID NO. 34 as the reverse primers for FD6, FD7 and FDl 1, respectively (Table 3).
  • Algae lysate in EDTA was added to provide template for reaction. Magnesium concentration is varied to compensate for amount and concentration of algae lysate in EDTA added. Annealing temperature gradients were employed to determine optimal annealing temperature for specific primer pairs.
  • reinhardtii is selected for expression of all three proteins using suitable markers and protein expression as confirmed by Western blot analysis.
  • the activity of each enzyme can be confirmed by FRET analysis using diluted cell lysates and suitable FRET substrates.
  • the genetically modified C.reinhardtii is then grown into a biomass at a density of about 500 million cells per ml using a suitable growing system.
  • the biomass is then added to the feedstock composition to generate an improved feedstock.
  • the genetically modified algae are added to the feedstock at a ratio of 1 :20.
  • the improved feedstock composition is then exposed to natural sunlight for a holding period of three days with active mixing and mulching 3-4 times a day.
  • the improved feedstock is suitable for providing nutrients to ruminants.
  • the biomass and wet media used to culture the biomass is then added to the feedstock composition to generate an improved feedstock.
  • the genetically modified wet algae are added to the feedstock at a ratio of 1 :5.
  • the improved feedstock composition is then exposed to natural sunlight for a holding period of three days with active mixing and mulching 3-4 times a day.
  • the improved feedstock is then suitable for providing nutrients to ruminants.
  • the genetically modified C. reinhardtii is selected for expression of all seven proteins using suitable markers and protein expression is confirmed by Western blot analysis.
  • the activity of all four biosynthetic enzymes can be confirmed by adding HMG-CoA as a substrate to a lysate of the genetically modified C. reinhardtii and detecting the presence of Isopentenyl-5-pyrophosphate using mass spectrometry analysis.
  • the activity of each biodegrading enzyme can be confirmed by FRET analysis using diluted cell lysates and suitable FRET substrates.
  • the genetically modified C. reinhardtii is then grown into a biomass using a suitable growing system at a density of about 500 million cells per ml.

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Abstract

La présente invention concerne des procédés de production d’un organisme photosynthétique, ledit organisme photosynthétique produisant une ou plusieurs enzymes qui favorisent la dégradation ou la digestion d’une biomasse, aboutissant à une composition plus nutritive à destination de l’alimentation pour animaux. L’invention concerne également des procédés de production d’un organisme photosynthétique, l’organisme photosynthétique produisant un ou plusieurs acides gras ou lipides et apportant une valeur nutritive supplémentaire. Les acides gras et les lipides produits peuvent également être utilisés pour la production de biocarburant et les organismes photosynthétiques après usage peuvent alors être utilisés comme alimentation pour animaux. L’invention concerne également des compositions d’aliments pour animaux comprenant des organismes photosynthétiques modifiés.
PCT/US2009/062843 2008-10-31 2009-10-30 Alimentation pour animaux comprenant des algues génétiquement modifiées Ceased WO2010051489A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010111707A1 (fr) * 2009-03-27 2010-09-30 Sapphire Energy, Inc. Enzymes produisant des variants d'isoprénoïdes et leurs utilisations
WO2011034863A1 (fr) * 2009-09-15 2011-03-24 Sapphire Energy, Inc. Système de transformation du génome chloroplastique des espèces scenedesmus et dunaliella
CN104068284A (zh) * 2014-07-16 2014-10-01 山东新希望六和集团有限公司 用于治疗小鹅瘟的配合饲料及其制备方法
US9175256B2 (en) 2010-12-23 2015-11-03 Exxonmobil Research And Engineering Company Production of fatty acids and fatty acid derivatives by recombinant microorganisms expressing polypeptides having lipolytic activity
CN105166383A (zh) * 2015-09-17 2015-12-23 怀宁县鑫冶岭家庭农场 一种猪饲料
CN106942513A (zh) * 2017-04-07 2017-07-14 临泽县锐翔科技开发有限责任公司 一种育肥鹅用中草药配合饲料
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WO2010111707A1 (fr) * 2009-03-27 2010-09-30 Sapphire Energy, Inc. Enzymes produisant des variants d'isoprénoïdes et leurs utilisations
US8987433B2 (en) 2009-03-27 2015-03-24 Sapphire Energy, Inc. Variant isoprenoid producing enzymes and uses thereof
WO2011034863A1 (fr) * 2009-09-15 2011-03-24 Sapphire Energy, Inc. Système de transformation du génome chloroplastique des espèces scenedesmus et dunaliella
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US9175256B2 (en) 2010-12-23 2015-11-03 Exxonmobil Research And Engineering Company Production of fatty acids and fatty acid derivatives by recombinant microorganisms expressing polypeptides having lipolytic activity
CN104068284A (zh) * 2014-07-16 2014-10-01 山东新希望六和集团有限公司 用于治疗小鹅瘟的配合饲料及其制备方法
CN105166383A (zh) * 2015-09-17 2015-12-23 怀宁县鑫冶岭家庭农场 一种猪饲料
CN106942513A (zh) * 2017-04-07 2017-07-14 临泽县锐翔科技开发有限责任公司 一种育肥鹅用中草药配合饲料
CN115851779A (zh) * 2022-10-29 2023-03-28 昆明理工大学 一种葡萄糖-6-磷酸脱氢酶基因RkZWF1及其应用
CN115851779B (zh) * 2022-10-29 2024-03-26 昆明理工大学 一种葡萄糖-6-磷酸脱氢酶基因RkZWF1及其应用

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