WO1999045122A1 - Modification du metabolisme des acides gras dans des plantes - Google Patents

Modification du metabolisme des acides gras dans des plantes Download PDF

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WO1999045122A1
WO1999045122A1 PCT/US1999/004999 US9904999W WO9945122A1 WO 1999045122 A1 WO1999045122 A1 WO 1999045122A1 US 9904999 W US9904999 W US 9904999W WO 9945122 A1 WO9945122 A1 WO 9945122A1
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promoter
plant
fatty acid
coa
group
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PCT/US1999/004999
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Oliver P. Peoples
Laura Boynton
Gjalt W. Huisman
Maurice Moloney
Nii Patterson
Kristi Snell
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Metabolix, Inc.
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Priority to EP99912316A priority Critical patent/EP1060250A1/fr
Priority to AU30714/99A priority patent/AU750057B2/en
Priority to CA2322729A priority patent/CA2322729C/fr
Priority to MXPA00008674A priority patent/MXPA00008674A/es
Priority to JP2000534653A priority patent/JP2002505107A/ja
Publication of WO1999045122A1 publication Critical patent/WO1999045122A1/fr

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    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • 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
    • 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
    • C12N15/8247—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 involving modified lipid metabolism, e.g. seed oil composition

Definitions

  • the present invention is generally in the field of transgenic plant systems for the production of polyhydroxyalkanoate materials, modification of triglycerides and fatty acids, and methods for altering seed production in plants.
  • Crops have been genetically modified for improvements in both input and output traits.
  • tolerance to specific agrochemicals has been engineered into crops, and specific natural pesticides, such as the Bacillus thuringenesis toxin, have been expressed directly in the plant.
  • specific natural pesticides such as the Bacillus thuringenesis toxin
  • crops are being modified to increase the value of the product, generally the seed, grain, or fiber of the plant.
  • Critical metabolic targets include the modification of starch, fatty acid, and oil biosynthetic pathways.
  • PHA polyhydroxyalkanoate
  • PHAs are natural, thermoplastic polyesters and can be processed by traditional polymer techniques for use in an enormous variety of applications, including consumer packaging, disposable diaper linings and garbage bags, food and medical products.
  • transgenic plant systems that produce PHA polymers having a well-defined composition.
  • Careful selection of the PHA biosynthetic enzymes on the basis of their substrate specificity allows for the production of PHA polymers of defined composition in transgenic systems (U.S. Patent Nos. 5,229,279; 5,245,023; 5,250,430; 5,480,794; 5,512,669; 5,534,432; 5,661,026; and 5,663,063).
  • each PHA group is produced by a specific pathway.
  • three enzymes are involved: ⁇ - ketothiolase, acetoacetyl-CoA reductase, and PHA synthase.
  • the homopolymer PHB for example, is produced by the condensation of two molecules of acetyl-coenzyme A to give acetoacetyl-coenzyme A. The latter then is reduced to the chiral intermediate R-3-hydroxybutyryl-coenzyme A by the reductase, and subsequently polymerized by the PHA synthase enzyme.
  • the PHA synthase notably has a relatively wide substrate specificity which allows it to polymerize C3-C5 hydroxy acid monomers including both 4-hydroxy and 5-hydroxy acid units. This biosynthetic pathway is found in a number of bacteria such as Alcaligenes eutrophus, A.
  • PHAs are produced for example by many different Pseudomonas bacteria. Their biosynthesis involve the ⁇ -oxidation of fatty acids and fatty acid synthesis as routes to the hydroxyacyl-coenzyme A monomeric units. The latter then are converted by PHA synthases which have substrate specificities favoring the larger C6-C14 monomeric units (Peoples & Sinskey, 1990). In the case of the PHB-co-HX copolymers which usually are produced from cells grown on fatty acids, a combination of these routes can be responsible for the formation of the different monomeric units.
  • the enzymes PHA synthase, acetoacetyl-CoA reductase, and ⁇ - ketothiolase, which produce the short pendant group PHAs in A. eutrophus, are coded by an operon comprising the phbC-phbA-phbB genes; Peoples et al, 1987; Peoples & Sinskey, 1989).
  • the PHA synthases responsible for production of the long pendant group PHAs have been found to be encoded on thepha locus, specifically by the pha A andphaC genes (U.S. Patent Nos. 5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem.
  • PHA biosynthetic genes have been isolated and characterized or identified from genome sequencing projects. Examples ofknown PHA biosynthetic genes are disclosed in the following references: Aeronomas caviae (Fukui & Doi, 1997, J. Bacteriol. 11 A.21-30); Alcaligenes eutrophus (U.S. Patent Nos. 5,245,023; 5,250,430; 5,512,669; and 5,661,026; Peoples & Sinskey, J. Biol. Chem. 264:15298-03 (1989)); Acinetobacter (Schembri et. al., FEMS Microbiol. Lett.
  • Rhizobium etli (Cevallos et. al., J. Bacteriol. 178: 1646-54 (1996)); R. meliloti (Tombolini et. al,
  • PHA synthases suitable for producing PHB-co-HH copolymers comprising from 1-99% HH monomers are encoded by the Rhodococcus ruber, Rhodospirillum rubrum, Thiocapsiae violacea, and Aeromonas caviae PHA synthase genes.
  • PCT WO 95/20614 for producing biological polymers equivalent to the chemically synthesized copolymers described in PCT WO 95/20614, PCT WO 95/20615, and PCT WO 95/20621 have been identified in a number of Pseudomonas and other bacteria (Steinbuchel & Wiese, Appl. Microbiol. Biotechnol. 37:691-97 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 36:507-14 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 40:710-16 (1994); Lee et al., Appl. Microbiol. Biotechnol.
  • a hydratase such as D-specific enoyl-CoA hydratase, for example, the hydratase obtained ⁇ XOX ⁇ Aeromonas caviae
  • a ⁇ -oxidation enzyme system Some plants have a ⁇ -oxidation enzyme system which is sufficient to modify polymer synthesis when the plants are engineered to express the hydratase.
  • Figure 1 is a schematic of fatty acid ⁇ -oxidation routes to produce polyhydroxyalkanoate monomers.
  • Figure 2 is a schematic showing plasmid constructs pSBS2024 and pSBS2025.
  • Figures 3 A and 3B are schematics showing plasmid constructs pCGmfl24 and pCGmfl25.
  • Figures 4A and 4B are schematics showing plasmid constructs pmfl249 and pmfl254.
  • Figures 5 A and 5B are schematics showing plasmid constructs pCGmf224 and pCGmf225.
  • Figures 6A and 6B are schematics showing plasmid constructs pCGmflP2S and pCGmf2PlS.
  • Fatty acid oxidation systems typically comprise several enzyme activities including a ⁇ -ketothiolase enzyme activity which utilizes a broad range of ⁇ -ketoacyl-CoA substrates.
  • the methods described herein include the subsequent incorporation of additional transgenes, in particular encoding additional enzymes involved in fatty acid oxidation or polyhydroxyalkanoate biosynthesis.
  • the methods include the incorporation of transgenes encoding enzymes, such as NADH and/or NADPH acetoacetyl- CoenzymeA reductases, PHB synthases, PHA synthases, acetoacetyl-CoA thiolase, hydroxyacyl-CoA epimerases, delta3-cis-delta2-trans enoyl-CoA isomerases, acyl-CoA dehydrogenase, acyl-CoA oxidase and enoyl-CoA hydratases by subsequent transformation of the transgenic plants produced using the methods and DNA constructs described herein or by traditional plant breeding methods.
  • transgenes encoding enzymes, such as NADH and/or NADPH acetoacetyl- CoenzymeA reducta
  • the fatty acid oxidation transgenes are expressed from a seed specific promoter, and the proteins are expressed in the cytoplasm of the developing oilseed.
  • fatty acid oxidation transgenes are expressed from a seed specific promoter and the expressed proteins are directed to the plastids using plastid targeting signals.
  • the fatty acid oxidation transgenes are expressed directly from the plastid chromosome where they have been integrated by homologous recombination.
  • the fatty acid oxidation transgenes may also be expressed throughout the entire plant tissue from a constitutive promoter.
  • transgenes are also useful to be able to control the expression of these transgenes by using promoters that can be activated following the application of an agrochemical or other active ingredient to the crop in the field. Additional control of the expression of these genes encompassed by the methods described herein include the use of recombinase technologies for targeted insertion of the transgenes into specific chromosomal sites in the plant chromosome or to regulate the expression of the transgenes.
  • the methods described herein involve a plant seed having a genome including (a) a promoter operably linked to a first DNA sequence and a 3'- untranslated region, wherein the first DNA sequence encodes a fatty acid oxidation polypeptide and optionally (b) a promoter operably linked to a second DNA sequence and a 3 '-untranslated region, wherein the second DNA sequence encodes a fatty acid oxidation polypeptide.
  • Expression of the two transgenes provides the plant with a functional fatty acid ⁇ -oxidation system having at least ⁇ -ketothiolase, dehydrogenase and hydratase activities in the cytoplasm or plastids other than peroxisomes or glyoxisomes.
  • the first and/or second DNA sequence may be isolated from bacteria, yeast, fungi, algae, plants, or animals. It is preferable that at least one of the DNA sequences encodes a polypeptide with at least two, and preferably three, enzyme activities.
  • Transformation Vectors DNA constructs useful in the methods described herein include transformation vectors capable of introducing transgenes into plants.
  • Several plant transformation vector options are available, including those described in “Gene Transfer to Plants” (Potrykus, et al., eds.) Springer- Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins” (Owen, et al., eds.) John Wiley & Sons Ltd. England (1996); and “Methods in Plant Molecular Biology: A Laboratory Course Manual” (Maliga, et al. eds.) Cold Spring Laboratory Press, New York (1995).
  • Plant transformation vectors generally include one or more coding sequences of interest under the transcriptional control of 5' and 3' regulatory sequences, including a promoter, a transcription termination and/or polyadenylation signal, and a selectable or screenable marker gene.
  • the usual requirements for 5' regulatory sequences include a promoter, a transcription termination and/or a polyadenylation signal.
  • additional RNA processing signals and ribozyme sequences can be engineered into the construct (U.S. Patent No. 5,519,164). This approach has the advantage of locating multiple transgenes in a single locus, which is advantageous in subsequent plant breeding efforts.
  • Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser & Fraley, Science 244: 1293-99 (1989)).
  • the 5' end of the transgene may be engineered to include sequences encoding plastid or other subcellular organelle targeting peptides linked in-frame with the transgene.
  • Suitable constitutive plant promoters include the cauliflower mosaic virus 35S promoter (CaMV) and enhanced CaMN promoters (Odell et.
  • actin promoter McElroy et al., Plant Cell 2:163-71 (1990)
  • Adhl promoter Fromm et. al, Bio/Technology 8:833-39 (1990); Kyozuka et al, Mol. Gen. Genet. 228:40-48 (1991)
  • ubiquitin promoters the Figwort mosaic virus promoter, mannopine synthase promoter, nopaline synthase promoter and octopine synthase promoter.
  • Useful regulatable promoter systems include spinach nitrate-inducible promoter, heat shock promoters, small subunit of ribulose biphosphate carboxylase promoters and chemically inducible promoters (U.S. Patent No. 5,364,780 to Hershey et al.).
  • the transgenes are expressed only in the developing seeds.
  • Promoters suitable for this purpose include the napin gene promoter (U.S. Patent Nos. 5,420,034 and 5,608,152), the acetyl-CoA carboxylase promoter (U.S. Patent Nos. 5,420,034 and 5,608,152), 2S albumin promoter, seed storage protein promoter, phaseolin promoter (Slightom et. al., Proc. Nail. Acad. Sci. USA 80: 1897-1901 (1983)), oleosin promoter (Plant et. al., Plant Mol. Biol. 25: 193-205 (1994); Rowley et al., Biochim. Biophys.
  • suitable agronomic plant hosts using these vectors can be accomplished with a variety of methods and plant tissues.
  • Representative plants useful in the methods disclosed herein include the Brassica family including napus, rappa, sp. carinata andjuncea; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards including Sinapis alba; and flax.
  • Representative tissues for transformation using these vectors include protoplasts, cells, callus tissue, leaf discs, pollen, and meristems
  • Representative transformation procedures include Agrobacterium-mediated transformation, biolistics, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome-mediated transformation, and silicon fiber-mediated transformation (U.S. Patent No. 5,464,765; "Gene Transfer to Plants” (Potrykus, et al, eds.) Springer-Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins” (Owen, et al., eds.) John Wiley & Sons Ltd.
  • the following procedures can be used to obtain a transformed plant expressing the transgenes subsequent to transformation: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene at such that the level of desired polypeptide is obtained in the desired tissue and cellular location.
  • Brassica napus can be transformed as described, for example, in U.S.
  • Patent Nos. 5,188,958 and 5,463,174 Other Brassica such as rappa, carinata and juncea as well as Sinapis alba can be transformed as described by Moloney et. al., Plant Cell Reports 8:238-42 (1989). Soybean can be transformed by a number of reported procedures (U.S. Patent Nos.
  • Patent Nos. 5,004,863 and 5,159,135) Sunflower can be transformed using a combination of particle bombardment and Agrob ⁇ cterium infection (EP 0 486 233 A2; U.S. Patent No. 5,030,572). Flax can be transformed by either particle bombardment or Agrobacterium-mediated transformation.
  • Recombinase technologies include the cre-lox, FLP/FRT, and Gin systems.
  • Selectable marker genes useful in practicing the methods described herein include the neomycin phosphotransferase gene nptll (U.S. Patent Nos. 5,034,322 and 5,530, 196), hygromycin resistance gene (U.S. Patent No.
  • EP 0 530 129 Al describes a positive selection system
  • Screenable marker genes useful in the methods herein include the ⁇ -glucuronidase gene (Jefferson et. al., EMBO J. 6:3901-07 (1987); U.S. Patent No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et. al., Trends Biochem Sci. 20:448-55 (1995); Pang et. al, Plant Physiol. H2:893-900 (1996)). Some of these markers have the added advantage of introducing a trait, such as herbicide resistance, into the plant of interest, thereby providing an additional agronomic value on the input side.
  • more than one gene product is expressed in the plant.
  • This expression can be achieved via a number of different methods, including (1) introducing the encoding DNAs in a single transformation event where all necessary DNAs are on a single vector; (2) introducing the encoding DNAs in a co- transformation event where all necessary DNAs are on separate vectors but introduced into plant cells simultaneously; (3) introducing the encoding DNAs by independent transformation events successively into the plant cells i.e. transformation of transgenic plant cells expressing one or more of the encoding DNAs with additional DNA constructs; and (4) transformation of each of the required DNA constructs by separate transformation events, obtaining transgenic plants expressing the individual proteins and using traditional plant breeding methods to incorporate the entire pathway into a single plant.
  • Yeast possesses a peroxisomal localized fatty acid degradation pathway that proceeds via intermediate R-3- hydroxyacyl CoA (Hiltunen, et al. J. Biol. Chem. 267: 6646-53 (1992); Filppula, et al. J. Biol. Chem. 270:27453-57 (1995)), such that no epimerase activity is required to produce PHAs.
  • Plants like other higher eukaryotes, possesses a ⁇ -oxidation pathway for fatty acid degradation localized subcellularly in the peroxisomes (Gerhardt, "Catabolism of Fatty Acids [ ⁇ and ⁇ Oxidation]” in Lipid Metabolism in Plants (Moore, Jr., ed.) pp. 527-65 (CRC Press, Boca Raton, Florida 1993)).
  • Fatty acids are synthesized as saturated acyl-ACP thioesters in the plastids of plants (Hartwood, "Plant Lipid Metabolism” in Plant Biochemistry (Dey et al., eds.) pp. 237-72 (Academic Press, San Diego 1997)).
  • acyl-ACP thioesters Prior to export from the plastid into the cytosol, the majority of fatty acids are desaturated via a ⁇ 9 desaturase.
  • the pool of newly synthesized fatty acids in most oilseed crops consists predominantly of oleic acid (cis 9- octadecenoic acid), stearic acid (octadecanoic acid), and palmitic acid (hexadecanoic acid).
  • the PHA synthase substrates are C4-C16 R-3- hydroxyacyl CoAs.
  • conversion to R-3- hydroxyacyl CoA thioesters using fatty acids degradation pathways necessitates the following sequence of reactions: conversion of the acyl CoA thioester to tr ⁇ r ⁇ -2-enoyl-CoA (reaction 1), hydration of tra «s-2-enoyl-CoA to R-3-hyddroxy acyl CoA (reaction 2a, e.g.
  • yeast system operates through this route and the Aeromonas caviae D-specific hydratase yields C4-C7 R-3- hydroxyacyl-CoAs), hydration of tr «s , -2-enoyl-Co A to S-3-hydroxy acyl CoA (reaction 2b), and epimerization of S -3 -hydroxyacyl CoA to R-3- hydroxyacyl CoA (reaction 5, e.g. cucumber tetrafunctional protein, bacterial systems).
  • 3 -hydroxyacyl CoA is not polymerized by PHA synthase forming PHA, it can proceed through the remainder of the ⁇ -oxidation pathway as follows: oxidation of 3 -hydroxyacyl CoA to form ⁇ -keto acyl CoA (reaction 3) followed by thio lysis in the presence of CoA to yield acetyl CoA and a saturated acyl CoA thioester shorter by two carbon units (reaction 4).
  • the acyl CoA thioester produced in reaction 4 is free to re-enter the ⁇ - oxidation pathway at reaction 1 and the acetyl-CoA produced can be converted to R-3 -hydroxyacyl CoA by the action of ⁇ -ketothiolase (reaction 7) and NADH or NADPH acetoacetyl-CoA reductase (reaction 6).
  • This latter route is useful for producing R-3-hydroxybutyryl-CoA, R-3- hydroxyvaleryl-CoA and R-3-hydroxyhexanoyl-CoA.
  • R-3- hydroxyacids of four to sixteen carbon atoms produced by this series of enzymatic reactions can be polymerized by PHA synthases expressed from a transgene, or transgenes in the case of the two subunit synthase enzymes,
  • Acyl CoA thioesters also can be degraded to a ⁇ -keto acyl CoA and converted to R-3 -hydroxyacyl CoA via a NADH or NADPH dependent reductase (reaction 6).
  • Multifunctional enzymes that encode S-specific hydratase, S-specific dehydrogenase, ⁇ -ketothiolase, epimerase and ⁇ 3 -cis-A 2 -frans-enoyl CoA isomerase activities have been found in bacteria such as Escherichia coli (Spratt, et al., J. Bacteriol. 158:535-42 (1984)) and Pseudomonas fragi (Immure, et al., J. Biochem. 107: 184-89 (1990)).
  • the multifunctional enzyme complexes consist of two copies of each of two subunits such that catalytically active protein forms a heterotetramer.
  • the hydratase, dehydrogenase, epimerase, and A 3 -cis-A 2 -trans-enoy ⁇ CoA isomerase activities are located on one subunit, whereas the thiolase is located on another subunit.
  • mutant genes also could be used in some embodiments of the methods described herein.
  • Mammals such as rat, possess a trifunctional ⁇ -oxidation enzyme in their peroxisomes that contains hydratase, dehydrogenase, and A 3 -cis-A 2 - trans-en yl CoA isomerase activities.
  • the trifunctional enzyme from rat liver has been isolated and has been found to be monomeric with a molecular weight of 78 kDa (Palosaari, et al., J. Biol. Chem. 265:2446-49 (1990)).
  • thiolase activity is not part of the multienzyme protein (Schultz, "Oxidation of Fatty Acids” in Biochemistry ofLipids, Lipoproteins and Membranes (Vance et al., eds) p. 95 (Elsevier, Amsterdam (1991)).
  • Epimerization in rat occurs by the combined activities of two distinct hydratases, one which converts R-3 -hydroxyacyl CoA to trans-2- enoyl CoA, and another which converts tr «5-2-enoyl CoA to S-3- hydroxyacyl CoA (Smeland, et al., Biochemical and Biophysical Research Communications 160:988-92 (1989)).
  • Mammals also possess ⁇ -oxidation pathways in their mitochondria that degrade fatty acids to acetyl CoA via intermediate S-3-hydroxyacyl CoA (Schultz, "Oxidation of Fatty Acids” in Biochemistry ofLipids, Lipoproteins and Membranes (Vance et al., eds) p. 96 (Elsevier, Amsterdam (1991)).
  • Yeast possesses a multifunctional enzyme, Fox2, that differs from the ⁇ -oxidation complexes of bacteria and higher eukaryotes in that it proceeds via a R-3 -hydroxyacyl CoA intermediate instead of S -3 -hydroxyacyl CoA (Hiltunen, et al, J. Biol. Chem. 267:6646-53 (1992)).
  • Fox2 possesses R- specific hydratase and R-specific dehydrogenase enzyme activities. This enzyme does not possess the ⁇ -cz ' s- ⁇ trflra-enoyl CoA isomerase activity needed for degradation of ⁇ 9-c s-hydroxyacyl CoAs to form R-3- hydroxyacyl CoAs.
  • the gene encoding/ x2 from yeast has been isolated and sequenced and encodes a 900 amino acid protein. The DNA sequence of
  • Plants have a tetrafunctional protein similar to the yeast Fox2, but also encoding a ⁇ -c ⁇ s-A 2 -trans-enoyl CoA isomerase activity (Muller et , al , J Biol Chem 269 20475-81 (1994))
  • the DNA sequence of the cDNA and ammo acid sequence of the encoded polypeptide is shown in SEQ ID NO 3 and SEQ ID NO 4 IV.
  • Eukaryotic acyl CoA dehydrogenase s are targeted to the mitochondria via leader peptides on the N- terminus of the protein that are usually 20-60 ammo acids long (Horwich, Current Opinion in Cell Biology, 2 625-33 (1990))
  • leader peptides on the N- terminus of the protein that are usually 20-60 ammo acids long (Horwich, Current Opinion in Cell Biology, 2 625-33 (1990))
  • mutagenesis of key residues in the leader sequence have been demonstrated to prevent the import of the mitochrond ⁇ al protein
  • Saccharomyces cerevisiae Fl-ATPase was prevented by mutagenesis of its leader sequence, resulting in the accumulation of the modified precursor
  • peroxisomal proteins do not contain the tripeptide at the C-terminal end of the protein. For these proteins, it has been suggested that targeting occurs via the tripeptide in an internal position within the protein sequence (Gould, et al., J. Cell Biol. 108:1657-64 (1989)) or via an unknown, unrelated sequence (Brickner, et al, J. Plant Physiol. Hi: 1213-21 (1997)).
  • transgenic plants can be grown using standard cultivation techniques.
  • the plant or plant part also can be harvested using standard equipment and methods.
  • the PHAs can be recovered from the plant or plant part using known techniques such as solvent extraction in conjunction with traditional seed processing technologies, as described in PCT WO 97/15681, or can be used directly, for example, as animal feed, where it is unnecessary to extract the PHA from the plant biomass.
  • compositions and methods of preparation and use thereof described herein are further described by the following non-limiting examples.
  • Primer 2 5' caa ccc gaa ggt gcc gcc att 3' (SEQ ID NO:6)
  • a 1.1 kb DNA fragment was purified from the PCR reaction and used as a probe to screen a P. putida genomic library constructed in plasmid pBKCMV using the lambda ZAP expression system (Stratagene). Plasmid pMFXl was selected from the positive clones and the DNA sequence of the insert containing the/ ⁇ oAB genes and flanking sequences determined. This is shown in SEQ ID NO:7. A fragment containing faoAB was subcloned with the native P. putida ribosome binding site intact into the expression
  • Enzymes in the FaoAB multienzyme complex were assayed as follows. Hydratase activity was assayed by monitoring the conversion of NAD to NADH using the coupling enzyme L- ⁇ -hydroxyacyl CoA dehydrogenase as previously described, except that assays were run in the presence of CoA (Filppula, et al., J. Biol. Chem. 270:27453-57 (1995)). Severe product inhibitation of the coupling enzyme was observed in the absence of CoA.
  • the assay contained (1 mL final volume) 60 ⁇ M crotonyl CoA, 50 ⁇ M Tris-CI, pH 9, 50 ⁇ g bovine serum albumin per mL, 50 mM KCl, 1 mM NAD, 7 ⁇ g L-specific ⁇ -hydroxyacyl CoA dehydrogenase from porcine heart per mL, and 0.25 mM CoA.
  • the assay was initiated with the addition of FaoAB to the assay mixture.
  • a control assay was performed without substrate to determine the rate of consumption of NAD in the absence of the hydratase generated product, S-hydroxybutyryl CoA.
  • Hydroxyacyl CoA dehydrogenase was assayed in the reverse direction with acetoacetyl CoA as the substrate by monitoring the conversion of NADH to NAD at 340 nm (Binstock, et al, Methods in Enzymology, 71:403 (1981)).
  • the assay contained (1 mL final volume) 0.1 M KH 2 PO 4 , pH 7, 0.2 mg bovine serum albumin per mL, 0.1 mM NADH, and 33 ⁇ M acetoacetyl CoA.
  • the assay was initiated with the addition of FaoAB to the assay mixture.
  • enzyme samples were diluted in 0.1 M KH 2 PO 4 , pH 7, containing 1 mg bovine serum albumin per mL.
  • a control assay was performed without substrate acetoacetyl CoA to detect the rate of consumption of NADH in the crude due to enzymes other than hydroxyacyl CoA dehydrogenase.
  • One unit of activity is defined as the consumption of
  • HydroxyacylCoA dehydrogenase was assayed in the forward direction with crotonyl CoA as a substrate by monitoring the conversion of NAD to NADH at 340 nm (Binstock, et al., Methods in Enzymology, 71:403 (1981)).
  • the assay mixture contained (1 mL final volume) 0.1 M KH 2 PO 4 , pH 8, 0.3 mg bovine serum albumin per mL, 2 mM ⁇ -mercaptoethanol, 0.25 mM CoA, 30 ⁇ M crotonyl CoA, and an aliquot of FaoAB.
  • the reaction was preincubated for a couple of minutes to allow in situ formation of S- hydroxybutyryl CoA.
  • the assay then was initiated by the addition of NAD (0.45 mM).
  • a control assay was performed without substrate to detect the rate of consumption of NAD due to enzymes other than hydroxyacyl CoA dehydrogenase.
  • Thiolase activity was determined by monitoring the decrease in absorption at 304 nm due to consumption of substrate acetoacetyl CoA as previously described with some modifications (Palmer, et al., J. Biol. Chem. 266: 1-7 (1991)).
  • the assay contained (final volume 1 mL) 62.4 mM Tris-Cl, pH 8.1, 4.8 mM MgCl 2 , 62.5 ⁇ M CoA, and 62.5 ⁇ M acetoacetyl CoA.
  • the assay was initiated with the addition of FaoAB to the assay mixture.
  • a control sample without enzyme was performed for each assay to detect the rate of substrate degradation of pH 8.1 in the absence of enzyme.
  • Epimerase activity was assayed as previously described (Binstock, et al., Methods in Enzymology, 71:403 (1981)) except that R-3 -hydroxyacyl CoA thioesters were utilized instead of D,L-3 -hydroxyacyl CoA mixtures.
  • the assay contained (final volume 1 mL) 30 ⁇ M R-3 -hydroxyacyl CoA, 150 mM KH 2 PO 4 (pH 8), 0.3 mg/mL BSA, 0.5 mM NAD, 0.1 mM CoA, and 7 ⁇ g/mL L-specific ⁇ -hydroxyacyl CoA dehydrogenase from porcine heart.
  • the assay was initiated with the addition of FaoAB.
  • 2xTY medium contains (per L) 16 g tryptone, 10 g
  • a starter culture was grown overnight and used to inoculate (1% inoculum) fresh medium (100 mL in a 250 mL Erienmeyer flask for small scale growths; 1.5L in a 2.8L flask for large scale growths).
  • Cells were induced with 0.4 mM IPTG when the absorbance at 600 nm was in the range of 0.4 to 0.6.
  • Cells were cultured an additional 4 h prior to harvest. Cells were lysed by sonication, and the insoluble matter was removed from the soluble proteins by centrifugation.
  • FaoAB in DH5 ⁇ /pMFX3 contained dehydrogenase and thiolase activity values of 4.3 and 0.99 U/mg, respectively, which is significantly more than the 0.0074 and 0.0033 U/mg observed for dehydrogenase and thiolase, respectively, in control strain DH5 ⁇ /pTRCN.
  • FaoAB was purified from DH5 ⁇ /pMFX3 using a modified procedure previously described for the purification of FaoAB from Pseudomonas fragi (Imamura, et al., J. Biochem. 107:184-89 (1990)). Thiolase activity (assayed in the forward direction) and dehydrogenase activities (assayed in the reverse direction) were monitored throughout the purification. Three liters of DH5 ⁇ /pMFX3 cells (2 X 1.5 L aliquots in 2.8 L Erienmeyer flasks) were grown in 2 x TY medium using the cell growth procedure previously described for preparing cells for enzyme activity analysis.
  • the resulting supernatant was adjusted to 56% saturation with (NH 4 ) 2 SO 4 and the insoluble pellet was isolated by centrifugation and dissolved in 10 mM KH 2 PO 4 , pH 7. The sample was heated at 50°C for 30 min. and the soluble proteins were isolated by
  • Enzyme Substrate Activity (U/mg) hydratase crotonyl CoA 8 8 dehydrogenase (forward) crotonyl CoA 0 46 dehydrogenase (reverse) acetoacetyl CoA 29 thiolase acetoacetyl CoA 9 9 epimerase R-3-hydroxyoctanyl CoA 0 022 epimerase R-3- hydroxyhexanyl CoA 0 0029 epimerase R-3- hydroxybutyryl CoA 0 000022
  • FaoAB protein Following purification of the FaoAB protein as described in Example 1, a sample was separated by SDS-PAGE The protein band corresponding to the FaoA (SEQ ID NO 31) and FaoB (SEQ ID NO 26) was excised and used to immunize New Zealand white rabbits with complete Freunds adjuvant Boosts were performed using incomplete Freunds at three week
  • soybean oleosin promoter fragment (SEQ ID NO:l 1; Rowley et al, 1997) was simplified with primers that flank the DNA sequence.
  • SEQ ID NO: 12 contains sequences that are complementary to the 5' end (underlined).
  • 5' -CA TTTAAA 7GGTT AAGGTGAAGGT AGGGCT-3 ' contains sequences homologous to the 3' end (underlined) of the promoter fragment.
  • the restriction sites Xbal (in italics) and Swal (in italics) were incorporated at the 5' end of JA408 and npl, respectively, to facilitate cloning.
  • the primers were used to amplify a 975 bp promoter fragment, which then was cloned into Small site of pUC 19 (see Figure 2).
  • the resulting plasmid, pCSPI was cut with S ⁇ tl and Swal and ligated to the soybean terminator (SEQ ID NO:28).
  • the soybean oleosin terminator was amplified by PCR using the following primers:
  • JA410 5 '-AAGCTTACGTGATGAGTATTAATGTGTTGTTATG-3 ' (SEQ ID NO:29) and
  • plasmid pSBS2025 5 '-TCTAGACAATTCATCAAATACAAATCACATTGCC-3 ' (SEQ ID NO:30) and the 225 bp fragment cloned into the Sall-Swal site of pCSPI to obtain plasmid pSBS2025 ( Figure 6).
  • Plasmid pBmf2 was constructed in a similar process ( Figures 5A and 5B).
  • a second set of synthetic primers were designed. Primers np4
  • the plasmid was referred to as pBmf2. Both plasmids were individually cut with Hindlll and their inserts cloned in plasmids pSBS2024 and pSBS2025, which had previously been linearized with the same restriction enzyme. As a result, the following
  • 26 plasmids were generated: pmfl24 and pmfl25 ( Figures 3A and 3B) and pmf224 and pmf225 ( Figures 5 A and 5B) containing the Fao genes (mfl and mf2) fused to either the phaseolin or soybean promoters.
  • DNA sequence analysis confirmed the correct promoter-coding sequence-termination sequence fusions for pmfl24, pmfl25, pmf224, and pmf225.
  • promoter-coding sequence fusions were independently cloned into the binary vectors, pCGN1559 (McBride and Summerfelt, 1990) containing the CaMV 35S promoter driving the expression of NPTII gene (conferring resistance to the antibiotic kanamycin) and pSBS2004 containing a parsley ubiquitin promoter driving the PPT gene, which confers resistance to the herbicide phosphinothricine.
  • Binary vectors suitable for this purpose with a variety of selectable markers can be obtained from several sources.
  • phaseolin-mf21 fusion cassette was released from the parent plasmid with Xbal and ligated with pCGN1559, which had been linearized with the same restriction enzyme.
  • the resulting plasmid was designated pCGmfl24 ( Figures 3 A and 3B).
  • Plasmid pCGmfl25 containing the soybean-mfl fusion was constructed in a similar way ( Figures 3 A and 3B), except that both pmfl25 and pCGNl 559 were cut with BamHI before ligation.
  • pmf!249 an pmf!254 The plasmid pSBS2004 was linearized with BamHI fragment containing the soybean-mfl fusion. This plasmid was designated pmfl254 ( Figures 4 A and 4B). Similarly, the Xbal phaseolin-mfl fusion fragment was ligated to pSBS2004 which had been linearized with the same restriction enzyme. The resulting plasmid was designated pmfl249 ( Figures 4A and 4B).
  • phaseolin-mf2 and soybean-mf2 fusions were constructed by
  • Plasmid pCGmf2PlS was assembled in similar manner.
  • the phaseolin-mf2 fusion was released from pmf!224 by cutting with BamHI and cloned at the BamHI site of pCGN1559.
  • the resulting plasmid, pCGmfB224 was linearized with Xbal and ligated to the Xbal fragment of pmfl25 containing the soybean-mfl fusion ( Figures 6 A and 6B).
  • Brassica seeds were surface sterilized in 10% commercial bleach (Javex, Colgate-Palmolive) for 30 min. with gentle shaking. The seeds were washed three times in sterile distilled water. Seeds were placed in germination medium comprising Murashige-Skoog (MS) salts and vitamins, 3% (w/v) sucrose and 0.7% (w/v) phytagar, pH 5.8 at a density of 20 per plate and maintained at 24 °C and a 16 h light / 8 h dark photoperiod at a light intensity of 60-80 //Em ' V 1 for four to five days.
  • MS Murashige-Skoog
  • each of the constructs, pCGmfl24, pCGmfl25, pCGmf224, pCGmflP2S, and pCGmf2PlS were introduced into Agrobacterium tumefacians strain EHAIOI (Hood et al., J. Bacteriol. 168:1291-1301 (1986)) by electroporation. Prior to transformation of cotyledonary petioles, single colonies of strain EHAIOI harboring each construct were grown in 5 ml of minimal medium supplemented with 100 mg kanamycin per liter and 100 mg gentamycin per liter for 48 hr at 28 °C. One milliliter of bacterial
  • cotyledons were excised from 4 day old, or in some cases 5 day old, seedlings, so that they included approximately 2 mm of petiole at the base.
  • Individual cotyledons with the cut surface of their petioles were immersed in diluted bacterial suspension for 1 s and immediately embedded to a depth of approximately 2 mm in co-cultivation medium, MS medium with 3% (w/v) sucrose and 0.7% phytagar and enriched with 20 ⁇ M benzyladenine.
  • the inoculated cotyledons were plated at a density of 10 per plate and incubated under the same growth conditions for 48 h.
  • the cotyledons then were transferred to regeneration medium comprising MS medium supplemented with 3% sucrose, 20 ⁇ M benzyladenine, 0.7% (w/v) phytagar, pH 5.8, 300 mg timentinin per liter, and 20 mg kanamycin sulfate per liter.
  • regeneration medium comprising MS medium supplemented with 3% sucrose, 20 ⁇ M benzyladenine, 0.7% (w/v) phytagar, pH 5.8, 300 mg timentinin per liter, and 20 mg kanamycin sulfate per liter.
  • regenerant shoots obtained were cut and maintained on "shoot elongation" medium (MS medium containing, 3% sucrose, 300 mg timentin per liter, 0.7% (w/v) phytagar, 300 mg timentinin per liter, and 20 mg kanamycin sulfate per liter, pH 5.8) in Magenta jars.
  • NPTII neomycin phosphotransferase
  • Seeds from the FaoA and FaoB transgenic lines can be analyzed for expression of the fatty acid oxidation polypeptides by western blotting using the anti-FaoA and anti-FaoB antibodies.
  • the FaoB polypeptide (SEQ ID NO:26) is not functional in the absence of the FaoA gene product; however, the FaoAB gene product has enzyme activity.
  • Each of the constructs, pCGmfl24, pCGmfl25, pCGmf224, pCGmf225, pCGmflP2S, and pCGmf2PlS were introduced into Agrobacterium tumefaciens strain EHAIOI (Hood et al. 1986) by electroporation. Prior to transformation of cotyledonary petioles, single colonies of strain EHA101 harboring each construct were grown in 5 mL of minimal medium supplemented with 100 mg kanamycin per liter, and 100 mg gentamycin per liter for 48 h at 28 °C. One milliliter of bacterial suspension was pelletized by centrifugation for 1 min in a microfuge.
  • the pellet was resuspended in 1 mL minimal medium.
  • cotyledons were excised from four-day-old, or in some cases five-day-old, seedlings so that they included approximately 2 mm of petiole at the base.
  • Individual cotyledons with the cut surface of their petioles were immersed in diluted bacterial suspension for 1 s and immediately embedded to a depth of approximately 2 mm in co-cultivation medium, MS medium with 3% sucrose and 0.7% phytagar, enriched with 20 ⁇ M benzyladenine.
  • the inoculated cotyledons were plated at a density of 10 per plate and incubated under the same growth conditions for 48 h. After
  • the cotyledons then were transferred to regeneration medium, which comprised MS medium supplemented with 3% sucrose, 20 ⁇ M benzyladenine, 0.7% phytagar, pH 5.8, 300 mg timentinin per liter, and 20 mg kanamycin sulfate per liter.
  • regeneration medium comprised MS medium supplemented with 3% sucrose, 20 ⁇ M benzyladenine, 0.7% phytagar, pH 5.8, 300 mg timentinin per liter, and 20 mg kanamycin sulfate per liter.
  • regenerant shoots were obtained, cut, and maintained on "shoot elongation" medium (MS medium containing 3% sucrose, 300mg timentin per liter, 0.7% phytagar, and 20 mg kanamycin per liter, pH 5.8) in Magenta jars.
  • the elongated shoots then were transferred to "rooting" medium, which comprised MS medium, 3% sucrose, 2 mg indole butyric acid per liter, 0.7% phytagar and 500 mg carbenicillin per liter.
  • rooting medium comprised MS medium, 3% sucrose, 2 mg indole butyric acid per liter, 0.7% phytagar and 500 mg carbenicillin per liter.
  • the plantlets were transferred to potting mix (Redi Earth, W.R. Grace and Co. Canada Ltd.). The plants were maintained in a misting chamber (75% RH) under the same growth conditions.
  • NPT II neomycin phosphotransferase
  • the fate of the transforming DNA was investigated for sixteen randomly selected transgenic lines. Southern DNA hybridization analysis showed that the FaoA and/or FaoB were integrated into the genomes of the
  • Seed fatty acid methyl esters were prepared by placing ten seeds of B. napus in 15 x 45-mm screw capped glass tubes and heating at 80 °C in 0.75 mL of IN methanolic HC1 reagent (Supelco, PA) and 10 ⁇ L of 1 mg 17:0 methyl ester (internal standard) per mL overnight.
  • the FAMES were extracted with 0.3 mL hexane and 0.5 mL 0.9% NaCl by vortexing vigorously. The samples were allowed to stand to separate the phases, and 300 ⁇ L of the organic phase was drawn and analyzed on a Hewlett-Packard gas chromatograph.
  • Fatty acid profile analysis indicated the presence of an additional component or enhanced component in the lipid profile in all of the transgenic plants expressing the FaoA gene SEQ ED NO:24 which was absent from the control plants. This result again proves conclusively that the FaoA gene is being transcribed and translated and that the FaoA polypeptide SEQ ID NO: 27 is catalytically active. This peak also was observed in eleven additional transgenic plants harboring SoyP-FaoA, PhaP-FaoA-SoyP-FaoB, SoyP- FaoA-PhaP-FaoB genes and a sterile (PhaP-FaoA) plant cross-pollinated with SoyP-FaoB.
  • SoyP-FaoA sterile
  • FaoA gene shows functional expression of the FaoA gene and that even the very low levels of expression are sufficient to change the lipid profile of the seed.
  • Adapting the methods described herein one of skill in the art can express these genes at levels intermediate between that obtained with the phaseolin promoter and the soybean oleosin promoter using other promoters such as the Arabidopsis oleosin promoter, napin promoter, or cruciferin promoter, and can use inducible promoter systems or recombinase technologies to control when fatty acid oxidation transgenes are expressed.
  • Example 7 Yeast ⁇ -oxidation Multi-functional Enzyme Complex
  • S. cerevisiae contains a ⁇ -oxidation pathway that proceeds via R-
  • The/ox2 gene from yeast encodes a hydratase that produces R-3 -hydroxyacyl CoA from tr raOenoyl-CoA and a dehydrogenase that utilizes R-3-hydroxyacyl-CoA to produce ⁇ -keto acyl CoAs.
  • the fox2 gene (sequence shown in SEQ ED NO: 1) was isolated from S. cerevisiae genomic DNA by PCR in two pieces. Primers N-fox2b and N- bamfox2b were utilized to PCR a 1 J kb Smal/BamHI fragment encoding the N-terminal region of Fox2, and primers C-fox2 and C-bamfox2 were utilized to PCR a 1.6 kb BamHI/Xbal fragment encoding the C-terminal Fox2 region. The full fox2 gene was reconstructed via subcloning in vector pTRCN.
  • the fox 1 gene does not possess a ⁇ -ketothiolase activity and this activity must be supplied by a second transgene.
  • Representative sources of such a gene include algae, bacteria, yeast, plants, and mammals.
  • the bacterium Alcaligenes eutrophus possesses a broad specificity ⁇ -ketothiolase gene suitable for use in the methods described herein. It can be readily isolated using the acetoacetyl-CoA thiolase gene as a hybridization probe, as described in U.S. Patent 5,661,026 to Peoples et al. This enzyme also has been purified (Hay wood et al., FEMS Micro. Lett. 52:91 (1988)), and the purified enzyme is useful for preparing antibodies or determining protein sequence information as a basis for the isolation of the gene.
  • the DNA sequence of the cDNA encoding ⁇ -oxidation tetrafunctional protein can be isolated as described in Preisig-Muller et al., J. Biol. Chem. 269:20475-81 (1994).
  • the equivalent gene can be isolated from other plant species including Arabidopsis, Brassica, soybean, sunflower, and corn using similar procedures or by screening genomic libraries, many of which are commercially available, for example from Clontech Laboratories Inc., Palo Alto, California, USA.
  • a peroxisomal targeting sequence P-R-M was identified at the carboxy terminus of the protein.
  • Constructs suitable for expressing in the plant cytosol can be prepared by PCR amplification of this gene using primers designed to delete this sequence.

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Abstract

Cette invention se rapporte à des procédés et à des systèmes pour modifier la biosynthèse et l'oxydation des acides gras dans les plantes, afin de produire de nouveaux polymères. Deux enzymes sont essentielles à cet effet: une hydratase, telle que l'hydratase énoyl-CoA D-spécifique, par exemple l'hydratase tirée de l'Aeromonas caviae, et un système d'enzyme de β-oxydation. Certaines plantes ont un système d'enzyme de β-oxydation qui est suffisant pour modifier la synthèse des polymères, lorsque les plantes sont modifiées génétiquement pour exprimer l'hydratase. Certains exemples montrent que la production de polymère peut se faire par l'expression de ces enzymes dans des plantes transgéniques. Des exemples montrent également que des modifications dans la biosynthèse des acides gras peuvent servir à modifier les phénotypes de la plante, ce qui entraîne une diminution ou une élimination de la production des graines et un accroissement de la biomasse verte de la plante, ainsi qu'une production de polyhydroxyalcanoates.
PCT/US1999/004999 1998-03-06 1999-03-05 Modification du metabolisme des acides gras dans des plantes WO1999045122A1 (fr)

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MXPA00008674A MXPA00008674A (es) 1998-03-06 1999-03-05 Modificacion de metabolismo de acidos grasos en plantas.
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WO2000075350A2 (fr) * 1999-06-08 2000-12-14 Calgene Llc Sequences d'acide nucleique codant des proteines impliquees dans la beta-oxydation des acides gras et procedes d'utilisation
WO2001009364A1 (fr) * 1999-08-03 2001-02-08 Oulun Yliopisto Procede de regulation des (3r)-hydroxyacyl-coa esters cellulaires, molecules precurseurs dans la synthese polyhydroxyalcanoate dans des organismes genetiquement modifies
WO2001023596A2 (fr) * 1999-09-29 2001-04-05 Pioneer Hi-Bred International, Inc. Production de polyhydroxyalcanoate dans des plantes
WO2002072837A1 (fr) * 2001-03-14 2002-09-19 Riken Plante synthetisant du copolyester a partir d'un monomere derive d'un acide gras a chaine courte et procede de production d'un polyester
US6806401B2 (en) 2000-12-27 2004-10-19 Pioneer Hi-Bred International, Inc. OAR polynucleotides, polypeptides and their use in PHA production in plants
US6914170B2 (en) 2000-07-06 2005-07-05 Pioneer Hi-Bred International, Inc. Methods for regulating beta-oxidation in plants
US7098298B2 (en) * 2003-11-21 2006-08-29 Kaneka Corporation Method for producing polyhydroxyalkanoate crystal
US7176349B1 (en) 1999-09-29 2007-02-13 Pioneer Hi-Bred International, Inc. Production of polyhydroxyalkanoate in plants

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* Cited by examiner, † Cited by third party
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US6489461B1 (en) 1999-06-08 2002-12-03 Calgene Llc Nucleic acid sequences encoding proteins involved in fatty acid beta-oxidation and methods of use
WO2000075350A2 (fr) * 1999-06-08 2000-12-14 Calgene Llc Sequences d'acide nucleique codant des proteines impliquees dans la beta-oxydation des acides gras et procedes d'utilisation
WO2000075350A3 (fr) * 1999-06-08 2001-11-29 Calgene Llc Sequences d'acide nucleique codant des proteines impliquees dans la beta-oxydation des acides gras et procedes d'utilisation
WO2001009364A1 (fr) * 1999-08-03 2001-02-08 Oulun Yliopisto Procede de regulation des (3r)-hydroxyacyl-coa esters cellulaires, molecules precurseurs dans la synthese polyhydroxyalcanoate dans des organismes genetiquement modifies
WO2001023596A3 (fr) * 1999-09-29 2002-01-31 Pioneer Hi Bred Int Production de polyhydroxyalcanoate dans des plantes
WO2001023596A2 (fr) * 1999-09-29 2001-04-05 Pioneer Hi-Bred International, Inc. Production de polyhydroxyalcanoate dans des plantes
US7176349B1 (en) 1999-09-29 2007-02-13 Pioneer Hi-Bred International, Inc. Production of polyhydroxyalkanoate in plants
US7341856B2 (en) 1999-09-29 2008-03-11 Pioneer Hi-Bred International, Inc. Production of polyhydroxyalkanoate in plants
US6914170B2 (en) 2000-07-06 2005-07-05 Pioneer Hi-Bred International, Inc. Methods for regulating beta-oxidation in plants
US6806401B2 (en) 2000-12-27 2004-10-19 Pioneer Hi-Bred International, Inc. OAR polynucleotides, polypeptides and their use in PHA production in plants
US7129395B2 (en) 2000-12-27 2006-10-31 Pioneer Hi-Bred International, Inc. OAR polynucleotides, polypeptides and their use in PHA production in plants
US7361807B2 (en) 2000-12-27 2008-04-22 Pioneer Hi-Bred International. Inc. OAR polynucleotides, polypeptides and their use in PHA production in plants
WO2002072837A1 (fr) * 2001-03-14 2002-09-19 Riken Plante synthetisant du copolyester a partir d'un monomere derive d'un acide gras a chaine courte et procede de production d'un polyester
US7098298B2 (en) * 2003-11-21 2006-08-29 Kaneka Corporation Method for producing polyhydroxyalkanoate crystal

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AU750057B2 (en) 2002-07-11
JP2009291204A (ja) 2009-12-17
CA2322729A1 (fr) 1999-09-10

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