JP2009291204A - Modification of fatty acid metabolism in plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a system for modifying fatty acid biosynthesis and oxidation in plants in order to make new polymers. <P>SOLUTION: Two enzymes are essential: a hydratase such as D-specific enoyl-CoA hydratase, for example, the hydratase obtained from Aeromonas caviae, and a β-oxidase system. Some plants have such a β-oxidase system enough to modify polymer synthesis when the plants are engineered to express the hydratase. Examples demonstrate production of polymer by expression of these enzymes in transgenic plants. Examples also demonstrate that modifications in fatty acid biosynthesis can be used to alter plant phenotypes, thereby decreasing or eliminating seed production and increasing green plant biomass, as well as producing polyhydroxyalkanoates. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates generally to 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.

  Methods for producing stable transgenic plants for crop agronomic crops have been developed over the last 15 years. The crop was genetically modified to improve both input and output traits. In the former trait, resistance to specific pesticides was manipulated in crops, and specific natural insecticides (eg Bacillus thuringensis toxin) were expressed directly in plants. There are also significant advances in the development of male sterile lines to produce hybrid plants. With respect to yield traits, crops are modified to increase the value of the product, generally plant seeds, kernels or fibers. Important metabolic targets include modification of starch, fatty acid and oil biosynthetic pathways.

  There is considerable commercial interest in the production of microbial polyhydroxyalkanoate (PHA) biopolymers in plant crops. For example, US Pat. Nos. 5,245,023 and 5,250,430 to Peoples and Sinskey; US Pat. No. 5,502,273 to Bright et al .; US Pat. No. 5,534,432 to Peoples and Sinsky. US Pat. No. 5,602,321 to John; US Pat. No. 5,610,041 to Somerville et al .; PCT WO 91/00917; PCT WO 92/19747; PCT WO 93/02187; PCT WO 93/02194; PCT WO 94 / 12012; Poirier et al., Science 256: 520-23 (1992); van der Leij and Withholt, Can. J. et al. Microbiol. 41 (Addendum): 222-38 (1995); Nawrath and Poirier, The International Symposium on Bacterial Polyhydroxyalkanoate (Eggink et al.) Davo Switzerland (August 18-23, 1996) 44 (1996). PHA is a natural, thermoplastic polyester and by traditional polymer technology for use in numerous applications (including consumer packaging, disposable diaper lining and garbage bags, food, and medical products) Can be processed.

  Initial studies in the production of polyhydroxybutyrate in the chloroplasts of the experimental plant system Arabidopsis thaliana resulted in accumulation of up to 14% by dry weight of leaves as PHB (Nawrath et al., 1993). However, Arabidopsis has no crop agronomic value. Furthermore, in order to economically produce PHA in crop agronomics, production of PHA in seeds is desired, so that current infrastructure for seed harvesting and processing can be utilized. The choice of PHA recovery from plant seeds (PCT WO 97/15681) and end-use applications (Williams and Peoples, CHEMTECH 26: 38-44 (1996)) is significantly influenced by the composition of the polymer. It is therefore advantageous to develop a transgenic plant line that produces a PHA polymer with a well-defined composition.

  Careful selection of PHA biosynthetic enzymes based on their substrate specificity allows the production of PHA polymers of defined composition in transgenic systems (US Pat. No. 5,229,279; No. 5,250,430; No. 5,480,794; No. 5,512,669; No. 5,534,432; No. 5,661,026; And No. 5,663,063).

  In bacteria, each PHA group is produced by a specific pathway. In the case of the short pendant group PHA, three enzymes are involved: β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase. For example, the homopolymer PHB is produced by the condensation of two molecules of acetyl coenzyme A to yield acetoacetyl coenzyme A. The latter is then reduced to the chiral intermediate R-3-hydroxybutyryl coenzyme A by reductase and subsequently polymerized by the PHA synthase enzyme. PHA synthase has in particular a relatively broad substrate specificity that allows the polymerization of C3-C5 hydroxy acid monomers containing both 4-hydroxy acid units and 5-hydroxy acid units. This biosynthetic pathway is found in many bacteria (eg, Alcaligenes eutrophus, A. latus, Azotobacter vinlandii and Zoogloea ramigera). The long pendant group PHA is produced, for example, by many different Pseudomonas bacteria. Their biosynthesis involves fatty acid β-oxidation and fatty acid synthesis as a pathway to hydroxyacyl coenzyme A monomer units. The latter is then converted by PHA synthase with a substrate specificity favoring larger C6-C14 monomer units (Peoples and Sinsky, 1990).

  In the case of PHB-co-HX copolymers normally produced from cell growth with fatty acids, the combination of these pathways may be responsible for the formation of different monomer units. In fact, using analysis of the DNA locus encoding the PHA synthase gene in Aeromonas caviae, which produces the copolymer PHB-co-3-hydroxyhexanoate, D-β-hydroxybutyryl -A gene encoding a D-specific enoyl-CoA hydratase responsible for the production of CoA and D-β-hydroxyhexanoyl-CoA units has been identified (Fukui and Doi, J. Bacteriol. 179: 4821-30 (1997); Fukui Et al., J. Bacteriol. 180: 667-73 (1998)). Other sources of such hydratase genes and enzymes include Alcaligenes, Pseudomonas, and Rhodospirillum.

  A. Enzymes that produce the short pendant group PHA in eutrophus, PHA synthase, acetoacetyl-CoA reductase, and β-ketothiolase are encoded by operons containing the phbC-phbA-phbB gene (Peoples et al., 1987; Peoples and Siskey 1989). In Pseudomonas organisms, the PHA synthase responsible for the production of the long pendant group PHA has been found to be encoded on the pha locus, in particular by the phaA and phaC genes (US Pat. No. 5,245,023 and No. 5,250,430; Huisman et al., J. Biol. Chem. 266: 2191-98 (1991)). With these initial studies, a range of PHA biosynthetic genes have been isolated and characterized or identified from genomic sequencing projects. Examples of known PHA biosynthetic genes are disclosed in the following references: Aeromass caviae (Fukui and Doi, 1997, J. Bacteriol. 179: 4821-30); Alcaligenes eutrophus (US Pat. No. 5,245,023) No. 5,250,430; 5,512,669; and 5,661,026; Peoples and Sinsky, J. Biol. Chem. 264: 15298-03 (1989)); Acinetobacter (Schembri et al., FEMS Microbiol. Lett. 118: 145-52 (1994); Chromatium vinosum (Liebergesell and Steinbuchel, Eur. J. Bio). hem. 209: 135-50 (1992))); Methylobacterium extorquens (Valentin and Steinbuchel, Appl. Microbiol. Biotechnol. 39: 309-17 (1993)); Nocardia corallina (1998) Paracoccus denitrificans (Ueda et al., J. Bacteriol. 178: 774-79 (1996); Yabutani et al., FEMS Microbiol. Lett. 133: 85-90 (1995)); acidophila (Umeda et al., 199 8, Applied Biochemistry and Biotechnology, 70-72: 341-52); Pseudomonas sp. 61-3 (Matsusaki et al., 1998, J. Bacteriol. 180: 6459-67); Nocardia corallina; Pseudomonas aeruginosa (Timm and Steinbuchel, Eur. J. Biochem. 209: 15-30 (1992)); oleovarans (US Pat. Nos. 5,245,023 and 5,250,430; Huisman et al., J. Biol. Chem. 266 (4): 2191-98 (1991); Rhizobium etli (Cevalos et al., J. Biol. Bacteriol.178: 1646-54 (1996)); R meliloti (Tombolini et al., Microbiology 141: 2553-59 (1995)); Rhodococcus ruber (Pieper-Furst and Steinbuchel, Fet. )); Rhodospirillum rubrum (Hustede et al., FEMS Microbiol. Lett. 93: 285-90). 1992)); Rhodobacter sphaeroides (Hustede et al., FEMS Microbiol. Rev. 9: 217-30 (1992)); Biotechnol. Lett. 15: 709-14 (1993); Synechocystis sp. (DNA Res. 3: 109-36) (1996)); Thiocapsiae violacea (Appl. Microbiol. Biotechnol. 38: 493-501 (1993)) and Zoogloea ramigera (Peoples et al., J. Biol. Chem. 262: 97p (e)); Molecular Microbiology 3: 349-57 (1989)). The availability of these genes or published DNA sequences should provide a range of options for PHA production.

  Suitable PHA synthases for producing PHB-co-HH copolymers containing 1-99% HH monomer are the PHA synthases encoded by the genes of Rhodococcus rubber, Rhodospirillum rubrum, Thiocapsiae violacea, and Aeromonas caviae. In addition to R-3-hydroxybutyrate, useful for incorporation of 3-hydroxy acids of 6 to 12 carbon atoms (ie, chemically synthesized copolymers (PCT WO95 / 20614, PCT WO95 / 20615 and PHA synthases (to produce biological polymers equivalent to those described in PCT WO 95/20621) have been identified in many Pseudomonas and other bacteria (Steinbuchel and Wiese, Appl. Microbiol. Biotechnol. 37). : 691-97 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 36: 507-14 (1992); Valentin et al., Appl. Microbiol. Biotec. No. 40: 710-16 (1994); Lee et al., Appl. Microbiol.Biotechnol.42: 901-09 (1995); Kato et al., Appl.Microbiol.Biotechnol.45: 363-70 (1996); Int. J. Biol. Macromol. 16: 115-19 (1994); Valentin et al., Appl.Microbiol.Biotechnol.46: 261-67 (1996)). They can then be easily isolated as described in US Pat. Nos. 5,245,023 and 5,250,430. P. The PHA synthase from oleovorans (US Pat. Nos. 5,245,023 and 5,250,430; Huisman et al., J. Biol. Chem. 266 (4): 2191-98 (1991)) is a long pendant. Suitable for the production of the base PHA. A plant gene encoding β-ketothiolase has also been identified (Volack and Bach, Plant Physiol. 111: 1097-107 (1996)).

US Pat. No. 5,245,023 US Pat. No. 5,250,430 US Pat. No. 5,502,273 US Pat. No. 5,534,432 US Pat. No. 5,602,321 US Pat. No. 5,610,041 International Publication No. 91/00917 International Publication No. 92/19747 International Publication No. 93/02187 International Publication No. 93/02194 International Publication No.94 / 12014 International Publication No. 97/15681 US Pat. No. 5,229,279 US Pat. No. 5,245,023 US Pat. No. 5,480,794 US Pat. No. 5,512,669 US Pat. No. 5,661,026 US Pat. No. 5,663,063 US Pat. No. 5,245,023

Poirier et al., Science 256: 520-23 (1992) van der Leij and Withholt, Can. J. et al. Microbiol. 41 (Addendum): 222-38 (1995) Nawrath and Poirier, The International Symposium on Bacterial Polyhydroxyalkanoate (Edited by Eggink et al.) Davos Switzerland (August 18-23, 1996) Williams and Peoples, CHEMTECH 26: 38-44 (1996). Fukui and Doi, J. et al. Bacteriol. 179: 4821-30 (1997) Fukui et al. Bacteriol. 180: 667-73 (1998) Huisman et al. Biol. Chem. 266: 2191-98 (1991) Fukui and Doi, 1997, J. MoI. Bacteriol. 179: 4821-30 Peoples and Sinsky, J. et al. Biol. Chem. 264: 15298-03 (1989) Schembri et al., FEMS Microbiol. Lett. 118: 145-52 (1994) Liebergesell and Steinbuchel, Eur. J. et al. Biochem. 209: 135-50 (1992) Valentin and Steinbuchel, Appl. Microbiol. Biotechnol. 39: 309-17 (1993) Hall et al., 1998, Can. J. et al. Microbiol. 44: 687-69 Ueda et al. Bacteriol. 178: 774-79 (1996) Yabutani et al., FEMS Microbiol. Lett. 133: 85-90 (1995) Umeda et al., 1998, Applied Biochemistry and Biotechnology, 70-72: 341-52). Matusaki et al., 1998, J. Am. Bacteriol. 180: 6459-67 Timm and Steinbuchel, Eur. J. et al. Biochem. 209: 15-30 (1992) Huisman et al. Biol. Chem. 266 (4): 2191-98 (1991) Cevallos et al. Bacteriol. 178: 1646-54 (1996) Tomobilini et al., Microbiology 141: 2553-59 (1995) Pieper-Furst and Steinbuchel, FEMS Microbiol. Lett. 75: 73-79 (1992) Hustede et al., FEMS Microbiol. Lett. 93: 285-90 (1992) Hustede et al., FEMS Microbiol. Rev. 9: 217-30 (1992) Hustede et al., Biotechnol. Lett. 15: 709-14 (1993) Hustede et al., Synechocystis sp. (DNA Res. 3: 109-36 (1996) Appl. Microbiol. Biotechnol. 38: 493-501 (1993) Peoples et al. Biol. Chem. 262: 97-102 (1987) Peoples and Sinskey, Molecular Microbiology 3: 349-57 (1989). Steinbuchel and 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. 42: 901-09 (1995) Kato et al., Appl. Microbiol. Biotechnol. 45: 363-70 (1996) Abe et al., Int. J. et al. Biol. Macromol. 16: 115-19 (1994) Valentin et al., Appl. Microbiol. Biotechnol. 46: 261-67 (1996) Huisman et al. Biol. Chem. 266 (4): 2191-98 (1991) Vollac and Bach, Plant Physiol. 111: 1097-107 (1996)

  Despite the ability to modify monomer composition through synthesis and substrate selection, it is desirable to modify other characteristics of polymer biosynthesis, such as biosynthesis with respect to fatty acid metabolism.

  Accordingly, it is an object of the present invention to provide a method and DNA construct for introducing a fatty acid oxidase system for manipulating plant cell metabolism.

  Another object of the present invention is to provide a method for enhancing the production of PHA in a plant, preferably its oil seed.

(Summary of the Invention)
Methods and systems for modifying fatty acid biosynthesis and oxidation in plants to make novel polymers are described. Two enzymes are essential: a hydratase such as D-specific enoyl-CoA hydratase (eg, a hydratase obtained from Aeromonas caviae) and a β-oxidase system. Some plants have a β-oxidase system that is sufficient to modify polymer synthesis when the plant is engineered to express hydratase.

  The examples demonstrate the production of polymers in transgenic plants by expression of these enzymes. Examples can also be used to alter plant phenotypes using alterations in fatty acid biosynthesis, thereby reducing or eliminating seed production, increasing green plant biomass, and producing PHA. Demonstrate.

FIG. 1 is a schematic diagram of the fatty acid β-oxidation pathway to produce polyhydroxyalkanoate monomers. FIG. 2 is a schematic diagram showing plasmid constructs pSBS2024 and pSBS2025. FIG. 3A is a schematic diagram showing plasmid constructs pCGmf124 and pCGmf125. FIG. 3B is a schematic diagram showing plasmid constructs pCGmf124 and pCGmf125. FIG. 4A is a schematic diagram showing plasmid constructs pmf1249 and pmf1254. FIG. 4B is a schematic diagram showing plasmid constructs pmf1249 and pmf1254. FIG. 5A is a schematic diagram showing plasmid constructs pCGmf224 and pCGmf225. FIG. 5B is a schematic diagram showing plasmid constructs pCGmf224 and pCGmf225. FIG. 6A is a schematic diagram showing plasmid constructs pCGmf1P2S and pCGmf2P1S. FIG. 6B is a schematic diagram showing plasmid constructs pCGmf1P2S and pCGmf2P1S.

(Detailed description of the invention)
Methods and DNA constructs are provided for manipulating plant cellular metabolism by introducing a fatty acid oxidase system into the cytoplasm or plastid of developing oilseed or green tissue. Fatty acid oxidation systems typically include several enzyme activities, including β-ketothiolase enzyme activity that utilizes a wide range of β-ketoacyl-CoA substrates.

  Surprisingly, it has been found that expression of at least one of these transgenes from the bean phaseolin promoter results in male sterility. Interestingly, these plants were not seeded and instead produced higher levels of biomass than normal (eg leaves, stems, stalks). Thus, the methods and constructs described herein can also be used to create male sterile plants, for example for hybrid production, or the biomass of a tuna (eg, alfalfa or tobacco) The production of Plants created using these methods and DNA constructs are useful for producing polyhydroxyalkanoate biopolymers or for producing new oil compositions.

  The methods described herein include the subsequent incorporation of additional transgenes, in particular transgenes encoding additional enzymes involved in fatty acid oxidation or polyhydroxyalkanoate biosynthesis. For polyhydroxyalkanoate biosynthesis, this method involves the enzyme (by subsequent transformation of transgenic plants produced using the methods and DNA constructs described herein or by traditional plant breeding methods. For example, NADH and / or NADPH acetoacetyl-coenzyme A reductase, PHB synthase, PHA synthase, acetoacetyl-CoA thiolase, hydroxyacyl-CoA epimerase, δ3-cis-δ2-transenoyl-CoA isomerase, acyl-CoA dehydrogenase , Acyl-CoA oxidase, and enoyl-CoA hydratase).

(I. Plant expression system)
In a preferred embodiment, the fatty acid oxidation transgene is expressed from a seed-specific promoter and the protein is expressed in the cytoplasm of the developing oil seed. In another preferred embodiment, the fatty acid oxidation transgene is expressed from a seed specific promoter and the expressed protein is directed to the plastid using a plastid targeting signal. In another preferred embodiment, fatty acid oxidation transgenes are expressed directly from the plastid chromosome, where they are taken up by homologous recombination. The fatty acid oxidation transgene can also be expressed throughout the plant tissue from a constitutive promoter. It is also useful to be able to control the expression of these transgenes by using promoters that can be activated after application of pesticides or other active ingredients to crops in the field. Further control of the expression of these genes included by the methods described herein includes the use of recombinant enzyme technology for targeted insertion of transgenes into specific chromosomal sites of plant chromosomes, or this introduction Examples include regulation of gene expression.

  The methods described herein include plant seeds having a genome comprising: (a) a promoter operably linked to a first DNA sequence and a 3 ′ untranslated region (this first The DNA sequence encodes a fatty acid oxidized polypeptide) and, optionally, (b) a promoter operably linked to a second DNA sequence and a 3 ′ untranslated region (the second DNA sequence is Encodes a fatty acid oxidized polypeptide). Expression of the two transgenes provides the plant with a functional fatty acid β-oxidation system that has at least β-ketothiolase activity, dehydrogenase activity, and hydratase activity in the cytoplasm, or plastids other than peroxisomes or glyoxisomes. The first DNA sequence and / or the second DNA sequence can be isolated from bacteria, yeast, fungi, algae, plants, or animals. It is preferred that at least one DNA sequence encodes a polypeptide having at least two and preferably three enzyme activities.

(Transformation vector)
DNA constructs useful in the methods described herein include transformation vectors that can introduce transgenes into plants. Several plant transformation vector options are available, including “Gene Transfer to Plants” (Edited by Potrykus et al.) Springer-Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: A Prodint (Owen et al.) John Wiley & Sons, Ltd. England (1996); and “Methods in Plant Molecular Biology: A Laboratory Course Manual” (edited by Malliga et al.) Cold Spring Laboratory Press, New York (1995). Plant transformation vectors generally comprise one or more coding sequences of interest under the transcriptional control of 5 ′ and 3 ′ regulatory sequences (including promoters, transcription termination signals and / or polyadenylation signals), and selectable marker genes or Contains a marker gene that can be screened. The usual requirements for 5 ′ regulatory sequences include a promoter, a transcription termination signal and / or a polyadenylation signal. Additional RNA processing signals and ribozyme sequences can be engineered into the construct for expression of two or more polypeptides from a single transcript (US Pat. No. 5,519,164). This approach has the advantage of localizing multiple transgenes at a single locus. This is advantageous in subsequent plant breeding attempts. A further approach is to use vectors for the specific transformation of plant plastid chromosomes by homologous recombination (US Pat. No. 5,545,818), in which case the prokaryotic nature of the plastid genome is Multiple transgenes can be utilized as operons.

(promoter)
A number of plant promoters are known and result in either constitutive expression of the gene of interest or expression regulated by the environment or by development. Plant promoters can be selected to control transgene expression in different plant tissues or organelles. All methods for these are known to those skilled in the art (Gasser and Fraley, Science 244: 1293-99 (1989)). The 5 ′ end of the transgene can be engineered to contain a sequence encoding a plastid or other intracellular organelle targeting peptide linked in frame with the transgene. Suitable constitutive plant promoters include cauliflower mosaic virus 35S promoter (CaMV) and enhanced CaMV promoter (Odell et al., Nature, 313: 810 (1985)), actin promoter (McElroy et al., Plant Cell 2: 163-71). (1990)), AdhI promoter (Fromm et al., Bio / Technology 8: 833-39 (1990); Kyozuka et al., Mol. Gen. Genet. 228: 40-48 (1991)), ubiquitin promoter, sesame mosaic mosaic promoter, These include the mannopine synthase promoter, the nopaline synthase promoter, and the octopine synthase promoter. Useful regulatable promoter systems include spinach nitrate-inducible promoter, heat shock promoter, ribulose bisphosphate carboxylase promoter small subunit, and chemically inducible promoter (US Pat. No. 5,364 to Hershey et al. , No. 780).

  In preferred embodiments of the methods described herein, the transgene is expressed only in the developing seed. Suitable promoters for this purpose include napin gene promoters (US Pat. Nos. 5,420,034 and 5,608,152), acetyl-CoA carboxylase promoter (US Pat. No. 5,420,034). And No. 5,608,152), 2S albumin promoter, seed storage protein promoter, phaseolin promoter (Slighttom et al., Proc. Natl. Acad. Sci. USA 80: 1897-1901 (1983)), oleosin. ) Promoter (Plant et al., Plant Mol. Biol. 25: 193-205 (1994); Rowley et al., Biochim. Biophys. Acta. 1345: 1-4 (1997); US Pat. No. 5,650,5 54; and PCT WO 93/20216), zein promoter, glutelin promoter, starch synthase promoter, and starch branching enzyme promoter.

  Transformation of suitable crop-cultivated plant hosts using these vectors can be accomplished using a variety of methods and plant tissues. Representative plants useful in the methods disclosed herein include napus, rappa, sp. corn; soybean; cotton seed; sunflower; palm; coconut; safflower; groundnut; mustard including Sinapis alba; and flax. Crops harvested as biomass (eg, silage corn, alfalfa, or tobacco) are also useful for the methods disclosed herein. Representative tissues for transformation with these vectors include protoplasts, cells, callus tissues, leaf discs, pollen, and meristems. Exemplary transformation procedures include Agrobacterium mediated transformation, biolistic, microinjection, electroporation, polyethylene glycol mediated protoplast transformation, liposome mediated transformation, and silicon fiber mediated transformation (US Pat. 5, 464, 765; “Gene Transfer to Plants” (Edited by Potrykus et al.) Springer-Verlag Berlin New Heidelberg New York (1995); “Transgenic Plants: A Productivity In Pt. So s Ltd.England (1996); and "Methods in Plant Molecular Biology: A Laboratory Course Manual" (Maliga et al., eds.) Cold Spring Laboratory Press, New York (1995)), and the like.

II. Methods for producing and screening for transgenic plants
In order to generate transgenic plants using the constructs described herein, the following procedure can be used to obtain transformed plants that express the transgene after transformation: on a selective medium The transformed plant cell is regenerated to produce a differentiated plant; the transgene at a level such that the desired polypeptide is obtained at the desired tissue and cell location. A transformed plant that expresses is selected.

  For specific crops useful for performing the described methods, transformation procedures have been established, for example, as described below: “Gene Transfer to Plants” (Edited by Potrykus et al.) Springer-Verlag. Berlin Heidelberg New York (1995); “Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins” (Owen et al.) John Wisley & L. England (1996); and "Methods in Plant Molecular Biology: A Laboratory Course Manual" (edited by Malliga et al.) Cold Spring Laboratory Press, New York (1995).

  Brassica napus can be transformed, for example, as described in US Pat. Nos. 5,188,958 and 5,463,174. Other Brassica (eg, rappa, carinata, and juncea) and Sinapis alba can be transformed as described in Moloney et al., Plant Cell Reports 8: 238-42 (1989). Soybean has been reported in a number of reported procedures (US Pat. Nos. 5,015,580; 5,015,944; 5,024,944; 5,322,783; , 416, 011; and 5,169, 770). Several transformation procedures (pollen transformation (US Pat. No. 5,629,183), silicon fiber mediated transformation (US Pat. No. 5,464,765), protoplast electroporation (US Pat. , 231,019; 5,472,869; and 5,384,253), gene guns (US Pat. Nos. 5,538,877 and 5,538,880), and Agrobacterium-mediated transformation (including EP 0 604 662 A1; PCT WO 94/00977) has been reported for the production of transgenic corn plants. Agrobacterium-mediated procedures are particularly preferred. This is because a single integration event of the transgene construct is more easily obtained using this procedure, and this greatly facilitates subsequent plant breeding. Cotton can be transformed by particle bombardment (US Pat. Nos. 5,004,863 and 5,159,135). Sunflower can be transformed with a combination of particle bombardment and Agrobacterium infection (EP 0 486 233 A2; US Pat. No. 5,030,572). Flax can be transformed by either particle bombardment or Agrobacterium-mediated transformation. Recombinant enzyme techniques include cre-lox systems, FLP / FRT systems, and Gin systems. Methods utilizing these techniques are described, for example, in US Pat. No. 5,527,695 to Hodges et al; Dale and Ow, Proc. Natl. Acad. Sci. USA 88: 10558-62 (1991); Medbury et al., Nucleic Acids Res. 23: 485-90 (1995).

(Selectable marker gene)
Selectable marker genes useful in carrying out the methods described herein include the neomycin phosphotransferase gene nptII (US Pat. Nos. 5,034,322 and 5,530,196), hygromycin Resistance genes (US Pat. No. 5,668,298) and bar genes (US Pat. No. 5,276,268) encoding resistance to phosphinotricin. EP 0 530 129 A1 expresses a transgene that encodes an enzyme that activates an inactive compound added to the growth medium, thereby allowing transformed plants to grow and escape from non-transformed lines. A positive selection system is described that enables Screenable marker genes useful in the methods herein include β-glucuronidase genes (Jefferson et al., EMBO J. 6: 3901-07 (1987); US Pat. No. 5,268,463) and native green Fluorescent protein genes or modified green fluorescent protein genes (Cubitt et al., Trends Biochem Sci. 20: 448-55 (1995); Pang et al., Plant Physiol. 112: 893-900 (1996)). Some of these markers have the added advantage of introducing traits (eg, herbicide tolerance) into the target plant, thereby providing additional crop-cultivation value on the input side.

  In preferred embodiments of the methods described herein, more than one gene product is expressed in the plant. This expression can be achieved through a number of different methods, including the following: (1) A method of introducing coding DNA in a single transformation event, where all the necessary DNA is single A method present on a vector; (2) a method for introducing coding DNA in a co-transformation event, wherein all the necessary DNA is present on a separate vector but is simultaneously introduced into the plant cell. (3) A method of continuously introducing coding DNA into plant cells by independent transformation events (ie, transformation of transgenic plant cells expressing one or more coding DNAs using additional DNA constructs). And (4) transgenes that express individual proteins by transformation of each of the required DNA constructs by separate transformation events. Give a click plant, and a method of incorporating all paths using traditional plant breeding techniques in a single plant.

(III. Β-oxidase pathway)
Production of PHA in the cytosol of plants requires cytosol localization of an enzyme that can produce R-3-hydroxyacyl CoA thioester as a substrate for PHA synthase. Both eukaryotes and prokaryotes have a β-oxidation pathway for fatty acid degradation that consists of a series of enzymes that convert fatty acyl CoA thioesters to acetyl CoA. These pathways proceed through the intermediate 3-hydroxyacyl CoA, but the configuration of this intermediate varies between organisms. For example, the bacterial β-oxidation pathway and the higher eukaryotic peroxisomal pathway degrade fatty acids to acetyl CoA via S-3-hydroxyacyl CoA (Schultz, “Oxidation of Fatty Acids”, Biochemistry of Lipids, Lipoproteins). and Membranes (edited by Vance et al.) 101-06 (Elsevier, Amsterdam 1991)). In Escherichia coli, the epimerase activity encoded by the β-oxidized multifunctional enzyme complex can convert S-3-hydroxyacyl CoA to R-3-hydroxyacyl CoA. Yeast possesses a peroxisome-localized fatty acid degradation pathway that proceeds via the intermediate R-3-hydroxyacyl CoA (Hiltunen et al. J. Biol. Chem. 267: 6646-53 (1992); Filppula et al. J. MoI. Biol.Chem.270: 27453-57 (1995)), so that no epimerase activity is required to produce PHA.

  Plants, like other higher eukaryotes, have a β-oxidation pathway for fatty acid degradation located subcellularly in peroxisomes (Gerhardt, “Catabolism of Fatty Acids [α and β Oxidation]”, Lipid Metabolism). in Plants (Moore, Jr. ed.) pp. 527-65 (CRC Press, Boca Raton, Florida 1993)). Therefore, the production of PHA in the cytosol of the plant is due to the cytosolic expression of the β-oxidation pathway, as well as R-3- Cytosolic expression of appropriate PHA synthase is required to polymerize hydroxyacyl CoA into a polymer.

  Fatty acids are synthesized as saturated acyl-ACP thioesters in plant plastids (Hartwood, “Plant Lipid Metabolism”, Plant Biochemistry (Dey et al.) 237-72 (Academic Press, San Diego 1997)). Prior to transport from the plastid to the cytosol, the majority of fatty acids are desaturated via a Δ9 desaturase. The pool of newly synthesized fatty acids in most oilseed crops consists primarily of oleic acid (cis 9-octadecenoic acid), stearic acid (octadecanoic acid), and palmitic acid (hexadecanoic acid). However, some plants (eg, coconut and palm kernels) synthesize shorter chain fatty acids (C8-14). This fatty acid is released from ACP via thioesterase and subsequently converted to acyl CoA thioester via acyl CoA synthetase localized in the plastid membrane (Andrews et al., “Fatty acid and lipid biosynthesis and degradation” Plant Physiology, Biochemistry, and Molecular Biology (Edited by Dennis et al.), Pages 345-46 (Longman Scientific & Technolical, Essex, England Biol et al., 1990); Diego 1997)).

  Cytosolic conversion of a pool of newly synthesized acyl CoA thioesters via the fatty acid degradation pathway, and conversion of intermediates from these series of reactions to R-3-hydroxyacyl-CoA substrates for PHA synthase, It can be achieved via the enzymatic reaction outlined in FIG. The PHA synthase substrate is C4-C16 R-3-hydroxyacyl CoA. For saturated fatty acyl CoAs, conversion to R-3-hydroxyacyl CoA thioesters using the fatty acid degradation pathway requires the following sequence of reactions: Conversion of acyl CoA thioesters to trans-2-enoyl-CoA Conversion (Reaction 1), Hydration of trans-2-enoyl-CoA to R-3-hydroxyacyl CoA (Reaction 2a, eg, the yeast system operates through this pathway and is specific for Aeromonas caviae D Hydratase yields C4-C7 R-3 hydroxyacyl-CoA), hydration of trans-2-enoyl-CoA to S-3-hydroxyacyl CoA (reaction 2b), and to R-3-hydroxyacyl CoA Epimerization of S-3-hydroxyacyl CoA (reaction 5, eg, cucumber Functional protein, a bacterial system). If 3-hydroxyacyl CoA is not polymerized by PHA synthase to form PHA, it can proceed through the remaining β-oxidation pathway as follows: Oxidation of 3-hydroxyacyl CoA to form β-ketoacyl CoA (Reaction 3) followed by thio-cleavage in the presence of CoA to yield acetyl CoA and a saturated acyl CoA thioester short by 2 carbon units (Reaction 4) The acyl CoA thioester produced in reaction 4 is free to re-enter the β-oxidation pathway of reaction 1 and the acetyl CoA produced is β-ketothiolase (reaction 7) and NADH acetoacetyl-CoA reductase or NADPH acetoacetyl. -Can be converted to R-3-hydroxyacyl CoA by the action of CoA reductase (Reaction 6). This latter route is useful for producing R-3-hydroxybutyryl-CoA, R-3-hydroxyvaleryl-CoA, and R-3-hydroxyhexanoyl-CoA. The R-16-hydroxy acid of 4-16 carbon atoms produced by this series of enzymatic reactions is converted into PHA polymer by PHA synthase expressed from the transgene (or multiple transgenes in the case of two subunit synthase enzymes). Can be polymerized.

For the Δ9 unsaturated fatty acyl CoA, the various series of reactions described are required. As detailed in FIG. 1, three cycles of β-oxidation remove 6 carbon units to yield an unsaturated acyl CoA thioester with a cis double bond in the 3 position. The conversion of the cis double bond at the 3 position to the trans double bond at the 2 position catalyzed by Δ ³ -cis-Δ ² -trans-enoyl CoA isomerase is a series of β-oxidation reactions outlined in FIG. Allows to progress. This enzymatic activity is present in the microbial β-oxidation complex and in the plant tetrafunctional protein but not in yeast fox1.

  The acyl CoA thioester can also be cleaved to β-ketoacyl CoA and converted to R-3-hydroxyacyl CoA via NADH-dependent or NACCP-dependent reductase (Reaction 6).

A multifunctional enzyme encoding S-specific hydratase activity, S-specific dehydrogenase activity, β-ketothiolase activity, epimerase activity, and Δ ³ -cis-Δ ² -trans-enoyl CoA isomerase activity is Escherichia coli (Splatt et al., J. Bacteriol. 158: 535-42 (1984)) and Pseudomonas fragi (Immure et al., J. Biochem. 107: 184-89 (1990)). The multifunctional enzyme complex consists of two copies of each two subunits such that a catalytically active protein forms a heterotetramer. Hydratase activity, dehydrogenase activity, epimerase activity, and Δ ³ -cis-Δ ² -trans-enoyl CoA isomerase activity are present on one subunit, while thiolase is present on another subunit. Organisms (eg, E. coli (Splatt et al., J. Bacteriol. 158: 535-42 (1984)); DiRusso, J. Bacteriol. 172: 6459-68 (1990)) and P. a. The gene encoding the enzyme from Fragi (Sato et al., J. Biochem. 111: 8-15 (1992)) has been isolated and sequenced, and to carry out the methods described herein Is appropriate. In addition, E.I. The E. coli enzyme system has been subjected to site-directed mutagenesis analysis to identify amino acid residues important for individual enzyme activities (He and Yang, Biochemistry 35: 9625-30 (1996); Yang et al., Biochemistry. 34: 6641-47 (1995); He and Yang, Biochemistry 36: 11044-49 (1997); He et al., Biochemistry 36: 261-68 (1997); Yang and Elzinga, J. Biol. 92 (1993)). These mutated genes can also be used in some embodiments of the methods described herein.

Mammals such as rats have trifunctional β-oxidase in their peroxisomes, including hydratase, dehydrogenase, and Δ ³ -cis-Δ ² -trans-enoyl CoA isomerase activity. The trifunctional enzyme was isolated from rat liver and found to be a monomer with a molecular weight of 78 kDa (Palosarai et al., J. Biol. Chem. 265: 2446-49 (1990)). ). Unlike bacterial systems, thiolase activity is not part of a multi-enzyme protein (Schultz, “Oxidation of Fatty Acids” Biochemistry of Lipids, Lipoproteins and Embranes (Vance et al., Ed.), Page 95, Elsevier, 91). Epimerization in rats occurs by a combination of the activities of two different hydratases, one of which converts R-3 hydroxyacyl CoA to trans-2-enoyl CoA and another one trans-2- Enoyl CoA is converted to S-3-hydroxyacyl CoA (Smeland et al., Biochemical and Biophysical Research Communications 160: 988-92 (1989)). Mammals also have a β-oxidation pathway in their mitochondria that degrades fatty acids to acetyl CoA via the intermediate S-3-hydroxyacyl CoA (Schultz, “Oxidation of Fatty Acids” Biochemistry of Lipids, Lipoproteins and Membranes (Vance et al., Ed.), Page 96 (Elsevier, Amsterdam (1991)). Genes encoding mitochondrial β-oxidation activity have been isolated from several animals, including rat mitochondrial long chain acyl CoA hydratase / 3-hydroxyacyl CoA dehydrogenase (GENBANK accession number D16478) and rat mitochondrial thiol. Rases (GENBANK registry numbers D13921 and D00511) are included.

Yeast has a multifunctional enzyme Fox2, which is a bacterial and higher eukaryote in that it proceeds via an R-3-hydroxyacyl CoA intermediate instead of S-3-hydroxyacyl CoA. (Hiltunen et al., J. Biol. Chem. 267: 6646-53 (1992)). Fox2 has R-specific hydratase enzyme activity and R-specific dehydrogenase enzyme activity. This enzyme does not have the Δ ³ -cis-Δ ² -trans-enoyl CoA isomerase activity required to degrade Δ ⁹ -cis-hydroxyacyl CoA to form R-3-hydroxyacyl CoA. This gene from yeast encoding fox2 has been isolated and sequenced and encodes a 900 amino acid protein. The DNA sequence of this structural gene and the amino acid sequence of the encoded polypeptide are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

The plant has a tetrafunctional protein that is similar to yeast Fox2 but also encodes Δ ³ -cis-Δ ² -trans-enoyl CoA isomerase activity (Muller et al., J. Biol. Chem. 269: 20475-81 ( 1994)). The DNA sequence of this cDNA and the amino acid sequence of the encoded polypeptide are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

IV. Targeting enzymes to the cytoplasm of oilseed crops
Manipulating PHA production in the plant cytoplasm requires a step that directs the expression of β-oxidation into the plant cytoplasm. There is no targeting signal in bacterial systems such as faoAB. In fungi, yeast, plants, and mammals, β-oxidation occurs in subcellular organelles. Typically, the gene is expressed in the nuclear chromosome, and polypeptides synthesized in the cytoplasm are directed to these organelles by the presence of specific amino acid sequences. Methods described herein using genes isolated from eukaryotic sources (eg, fatty acid oxidases from eukaryotic sources such as yeast, fungi, plants, and mammals) In order to do this, removal or modification of the subcellular targeting signal is necessary to direct this enzyme to the cytosol. It may be useful to add a signal to direct the protein to the endoplasmic reticulum. Peptides useful in this process are well known in the art. A common approach is to modify the transgene by inserting a specialized DNA sequence into the peptide sequence targeted to the endoplasmic reticulum to form a chimeric gene.

  Eukaryotic acyl-CoA dehydrogenases and other mitochondrial proteins are targeted to the mitochondria via the N-terminal leader peptide of the protein, which is usually 20-60 amino acids long (Horwich, Current Opinion in Cell Biology, 2: 625). ~ 33 (1990)). Despite the lack of a clear consensus sequence for the mitochondrial import leader peptide, mutagenesis of key residues in the leader sequence has been demonstrated to interfere with mitochondrial protein import. For example, the transfer of Saccharomyces cerevisiae F1-ATPase was hampered by mutagenesis of its leader sequence, which resulted in the accumulation of modified precursor proteins in the cytoplasm (Bedwell et al., Mol. Cell. Biol. 9: 1014). -25 (1989)).

  Three eukaryotic peroxisome targeting signals have been reported (Gould et al., J. Cell. Biol. 108: 1657-64 (1989); Brickner et al., J. Plant Physiol., 113: 1213-21 (1997). ). The tripeptide targeting signal S / A / CK / H / RL occurs at the C-terminus of many peroxisomal proteins (Gould et al., J. Cell Biol. 108: 1657-64 (1989)). Mutagenesis of this sequence has been shown to interfere with protein import into the peroxisome. Some peroxisomal proteins do not contain this tripeptide at the C-terminus of the protein. For these proteins, either via a tripeptide at an internal position in 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. 113: 1213-21 (1997)), it has been suggested that targeting occurs. The results of in vitro peroxidase targeting experiments using fragments of acyl CoA oxidase from Candida tropicalis appear to support the latter theory, and there are two distinct targeting signals in the internal amino acid sequence of the polypeptide. It is suggested to exist (Small et al., The EMBO Journal 7: 1167-73 (1988)). In the previous study, it was found that the targeting signal was localized in two regions of 118 amino acids in length and that both regions contained the targeting signal S / A / CK / H / RL. It was not issued. A small number of peroxisomal proteins appear to contain an amino-terminal leader sequence for import into the peroxisome (Brickner et al., J. Plant Physiol. 113: 1213-21 (1997)). These targeting signals can be deleted or altered by site-directed mutagenesis.

(V. Transgenic plant cultivation and harvesting)
Transgenic plants can be grown using standard culture techniques. Plants or plant parts can also be recovered using standard equipment and methods. PHA can be recovered from plants or plant parts using known techniques (eg, solvent extraction in combination with traditional sowing process techniques as described in PCT WO 97/15681) or, for example, Can be used directly as animal food. Here, it is not necessary to extract PHA from the amount of plant organisms.

  Some lines that did not produce seeds produced much higher levels of biomass. Thus, this phenotype can be useful as a means of increasing the amount of green biomass produced per acre for pasture, feed, or other biomass crops. End use includes the most cost-effective production of forage crops, either for animal feed or as an energy crop for generating electrical power. Other uses include increasing biomass levels of crops (eg, alfalfa or tobacco) for subsequent recovery of industrial products (eg, PHA by extraction).

  The compositions described herein and methods for their preparation and methods of use are further described by the following non-limiting examples.

Example 1: Isolation and characterization of Pseudomononas putida faoAB gene and Fao enzyme
PCR, DNA sequencing, E.I. E. coli transformation and plasmid purification. All DNA manipulations were performed using standard methods as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York (1989)). PCR (polymerase chain reaction) using primers 1 and 2 having homology to the gene encoding faoAB derived from Pseudomonas putida from Pseudomonas fragi (fatoB derived from Pseudomonas fragi (Sato et al., J. Biochem. 111: 8-15 (1992)) ). Isolated using a probe generated from putida genomic DNA.
Primer 1: 5′-gat ggg ccg ctc caa ggg tgg-3 ′ (SEQ ID NO: 5)
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 constructed using the λZAP expression system (Stratagene) into a PBKCMV plasmid. It was used as a probe to screen a putida genomic library. Plasmid pMFX1 was selected from positive clones and the DNA and flanking sequences of the insert containing the faoAB gene were determined. This sequence is shown in SEQ ID NO: 7. The fragment containing faoAB was converted to native P.O. Intact using the putida ribosome binding site was subcloned into the expression vector pTRCN to form plasmid pMFX3. Plasmid pMFX1 was digested with BsrG1. The resulting protruding end was smoothed with Klenow. Digestion with HindIII produced a 3.39 kb blunt end / HindIII fragment encoding FaoAB. The expression vector pTRCN was digested with SmaI / HindIII and ligated with the faoAB fragment to form the 7.57 kb plasmid pMFX3.

The enzyme of the FaoAB multienzyme complex was assayed as follows. Hydratase activity was determined as described previously (Filppula et al., J. Biol. Chem. 270: 27453-57 (1995)), except that the assay was performed in the presence of CoA. -Assayed by monitoring the conversion of NAD to NADH using hydroxyacyl CoA dehydrogenase. Vigorous product inhibition of the coupling enzyme was observed in the absence of CoA. This assay consists of 60 μM crotonyl CoA, 50 μM Tris-Cl, pH 9, 50 μg / ml bovine serum albumin, 50 mM KCl, 1 mM NAD, 7 μg / ml porcine heart-derived L-specific β- (to 1 mL final volume). Hydroxyacyl CoA dehydrogenase and 0.25 mM CoA were included. The assay was initiated with the addition of FaoAB into the assay mixture. A control assay was performed without substrate to determine the consumption rate of NAD in the absence of the hydratase product produced, S-hydroxybutyryl CoA. One unit of activity is defined as the consumption of 1 μmol of NAD per minute (ε ₃₄₀ = 6220 M ⁻¹ cm ⁻¹ ).

Hydroxyacyl CoA dehydrogenase was assayed in the reverse direction by monitoring the conversion of NADH to NAD at 340 nm using acetoacetyl CoA as a substrate (Binstock et al., Methods in Enzymology, 71: 403 (1981)). This assay included (in a final volume of 1 mL) 0.1 M KH ₂ PO ₄ , PH7, 0.2 mg / mL bovine serum albumin, 0.1 mM NADH, and 33 μM acetoacetyl CoA. The assay was initiated with the addition of FaoAB to the assay mixture. If necessary, enzyme samples were diluted in 0.1 M KH ₂ PO ₄ , pH 7 containing 1 mg / mL bovine serum albumin. A control assay was performed without the substrate acetoacetyl CoA to detect the consumption rate of NADH in the crude sample by enzymes other than hydroxyacyl CoA dehydrogenase. One unit of activity is defined as the consumption of 1 μmol of NADH per minute (ε ₃₄₀ = 6220 M ⁻¹ cm ⁻¹ ).

Hydroxyacyl CoA dehydrogenase was assayed in the forward direction by monitoring the conversion of NAD to NADH at 340 nm (Binstock et al., Methods in Enzymology, 71: 403 (1981)) using crotonyl CoA as a substrate. This assay mixture consists of 0.1M KH ₂ PO ₄ , PH8, 0.3 mg / mL bovine serum albumin, 2 mM β-mercaptoethanol, 0.25 mM CoA, 30 μM crotonyl CoA, and FaoAB (in a final volume of 1 mL). An aliquot was included. The reaction was preincubated for several minutes to allow in situ formation of S-hydroxybutyryl CoA. The assay was then initiated by the addition of NAD (0.45 mM). A control assay was performed without substrate to detect the rate of consumption of NAD by enzymes other than hydroxyacyl CoA dehydrogenase. One unit of activity is defined as the consumption of 1 μmol of NAD per minute (ε ₃₄₀ = 6220 M ⁻¹ cm ⁻¹ ).

The thiolase activity, as described previously (Palmer et al., J. Biol. Chem. 266: 1-7 (1991)), with some modifications, is measured by absorption of the substrate acetoacetyl CoA at 304 nm. Determined by monitoring the decrease. Assay contained (final volume in 1mL) 62.4mM Tris-Cl, pH 8.1,4.8mM MgCl 2, the 62.5MyuM CoA, and 62.5MyuM acetoacetyl CoA. The assay was initiated by the addition of FaoAB to the assay mixture. A control sample without enzyme was run for each assay to detect the substrate degradation rate at pH 8.1 in the absence of enzyme. One unit of activity is defined as the consumption of 1 μmole of substrate acetoacetyl CoA per minute (ε ₃₄₀ = 16900 M ⁻¹ cm ⁻¹ ).

Epimerase activity was as previously described (Binstock et al., Methods in Enzymology, 71: 403, except that R-3-hydroxyacyl CoA thioester was used in place of the D, L-3-hydroxyacyl CoA mixture. (1981)) assayed. This assay consists of 30 μM 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 μg / mL (in a final volume of 1 mL). An L-specific β-hydroxyacyl CoA dehydrogenase from porcine heart was included. The assay was initiated by the addition of FaoAB.

  Cultures were grown at 30 ° C. in 2 × TY medium for expression of FaoAB in DH5α / pMFX3. 2 × TY medium contains (per liter) 16 g tryptone, 10 g yeast, and 5 g NaCl. Starter cultures are grown overnight and inoculated into fresh medium (100 mL in a 250 mL Erlenmeyer flask for small scale growth; 1.5 L in a 2.8 L flask for large scale growth) (1% inoculum) Used to). When the absorbance at 600 nm reached the range of 0.4 to 0.6, the cells were induced with 0.4 mM IPTG. Prior to harvest, cells were cultured for an additional 4 hours. Cells were lysed by sonication and insoluble material was removed from soluble protein by centrifugation. Acyl CoA dehydrogenase activity was monitored in the reverse direction to ensure the activity of the FaoA subunit (SEQ ID NO: 31), and thiolase activity was assayed to determine the activity of the Fao subunit. FaoAB in DH5α / pMFX3 contained dehydrogenase activity and thiolase activity with values of 4.3 U / mg and 0.99 U / mg, respectively. This was significantly higher than the 0.0074 U / mg and 0.0033 U / mg observed for dehydrogenase and thiolase, respectively, in the control strain DH5α / pTRCN.

FaoAB was purified from DH5α / pMFX3 using a modified procedure previously described for purification of FaoAB from Pseudomonas fragi (Imamura et al., J. Biochem. 107: 184-89 (1990)). Thiolase activity (assayed in the forward direction) and dehydrogenase activity (assayed in the reverse direction) were monitored throughout the purification. Three liters of DH5α / pMFX3 cells (2 × 1.5 L aliquots in a 2.8 L Erlenmeyer flask) were prepared using a cell growth procedure previously described for cells prepared for enzyme activity analysis. Grown in TY medium. Cells (15.8 g) were resuspended in 32 mL of 10 mM KH ₂ PO ₄ , pH 7, and lysed by sonication. Soluble protein was removed from insoluble cell debris by centrifugation (18,000 RPM, 30 min, 4 ° C.). Soluble extracts were made in 50% acetone and the precipitated protein was isolated by centrifugation and redissolved in 10 mM KH ₂ PO ₄ , pH 7. The sample was adjusted to 33% saturation with (NH ₄ ) ₂ SO ₄ and soluble and insoluble proteins were separated by centrifugation. 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 minutes and the soluble protein was isolated by centrifugation and dialyzed against 10 mM KH ₂ PO ₄ , pH 7 in a 6000-8000 molecular weight cut-off membrane (2 × 3 L; 20 hours ). This sample was loaded onto a Toyo Jozo DEAE FPLC column (3 cm × 14 cm) previously equilibrated with 10 mM KH ₂ PO ₄ , pH 7. The protein was eluted at a flow rate of 3 mL / min with a linear gradient (100 mL each; 10 KH ₂ PO ₄ , 0-500 mM NaCl in pH 7). FaoAB eluted between 300 and 325 mM NaCl. This sample was loaded in a 50,000 molecular weight cut-off membrane prior to loading onto a macro-prep hydroxyapatite 18/30 (Biorad) FPLC column (2 cm × 15 cm) previously equilibrated with 10 mM KH ₂ PO ₄ , pH 7. Dialyzed against 10 mM KH ₂ PO ₄ , pH 7 (1 × 2 L; 15 hours). The protein was eluted using a linear gradient (250 mL each; 10-500 mM KH ₂ PO ₄ , pH 7) at a flow rate of 3 mL / min. FaoAB eluted between 70 and 130 mM KH ₂ PO ₄ . Fractions containing activity were concentrated to 9 mL using a MILLIPORE ^™ 100,000 molecular weight cut-off concentrator. The buffer was changed 3 times with 10 mM KH ₂ PO ₄ , pH 7 containing 20% sucrose and frozen at −70 ° C. Enzymatic activity of the purified hydroxyapatite fraction was assayed using a range of substrates. The results are shown in Table 1 below.

Example 2: Production of antibodies against FaoAB and FaoAB polypeptides
After purification of the FaoAB protein as described in Example 1, samples were separated by SDS-PAGE. Protein bands corresponding to FaoA (SEQ ID NO: 31) and FaoB (SEQ ID NO: 26) were excised and New Zealand white rabbits were immunized with Freund's complete adjuvant. Booster immunizations were performed at 3 week intervals with Freund's incomplete adjuvant. Antibodies were recovered from serum by affinity chromatography on a Protein A column (Pharmacia) and tested against antigen by Western blotting procedures. A control extract of Brassica seeds was used to test for cross-reactivity to plant proteins. No cross reaction was detected.

(Example 3: Construction of a plasmid for expression of the Pseudomonas putido faoAB gene in transgenic oil seeds)
(Construction of pSBS2024)
Oligonucleotide primer GVR471, homologous to the sequence flanking the 5'- and 3'-ends (underlined) of the bean phaseolin promoter (SEQ ID NO: 10; Slighttom et al., 1983), respectively.

およびＧＶＲ４７２

And GVR472

を、それぞれ、ＧＶＲ４７１およびＧＶＲ４７２の５’−末端にＫｐｎＩ（斜字体、配列番号８のヌクレオチド１〜７）およびＳｗａＩ（斜字体、配列番号９のヌクレオチド１〜９）を付加するように設計した。これらの制限部位を、クローニングを容易にするために組み込んだ。これらのプライマーを使用して、１．４ｋｂファゼオリンプロモーターを増幅した。このプロモーターを、平滑末端連結によってｐＵＣ１９のＳｍａＩ部位にクローニングした。命名されたプラスミド、ｐＣＰＰＩ（図２を参照のこと）を、ＳａｌＩおよびＳｗａＩを用いて切断し、そしてＳａｌＩ／ＳｗａＩファゼオリンターミネーター（配列番号２７）に連結した。ポリアデニル化シグナルを含むマメのファゼオリンターミネーター配列を、以下のＰＣＲプライマーを使用して増幅した：ＧＶＲ３９６：５’−ＧＡＴＴＴＡＡＡＴＧＣＡＡＧＣＴＴＡＡＡＴＡＡＧＴＡＴＧＡＡＣＴＡＡＡＡＴＧＣ−３’（配列番号２２）
およびＧＶＲ３９７５’−ＣＧＧＴＡＣＣＴＴＡＧＴＴＧＧＴＡＧＧＧＴＧＣＴＡ−３’（配列番号２３）
そして１．２ｋｂフラグメント（配列番号２７）をｐＣＣＰ１のＳａｌＩ−ＳａｌＩ部位にクローニングして、ｐＳＢＳ２０２４を得た（図２）。クローニングのための独特のＨｉｎｄＩＩＩ部位を含む、得られるプラスミドを、ｐＳＢＳ２０２４と命名した（図２）。

Were designed to add KpnI (italic, nucleotides 1-7 of SEQ ID NO: 8) and SwaI (italic, nucleotides 1-9 of SEQ ID NO: 9) to the 5'-ends of GVR471 and GVR472, respectively. These restriction sites were incorporated to facilitate cloning. These primers were used to amplify the 1.4 kb phaseolin promoter. This promoter was cloned into the SmaI site of pUC19 by blunt end ligation. The named plasmid, pCPPI (see FIG. 2) was cut with SalI and SwaI and ligated to the SalI / SwaI phaseolin terminator (SEQ ID NO: 27). A bean phaseolin terminator sequence containing a polyadenylation signal was amplified using the following PCR primers: GVR396: 5′-GATTTAAATGCAAGCTTAATAATAGTATGAACTAAAAGC-3 ′ (SEQ ID NO: 22)
And GVR3975'-CGGTACCTTAGTTGGTAGGGTCTA-3 '(SEQ ID NO: 23)
The 1.2 kb fragment (SEQ ID NO: 27) was cloned into the SalI-SalI site of pCCP1 to obtain pSBS2024 (FIG. 2). The resulting plasmid containing the unique HindIII site for cloning was named pSBS2024 (FIG. 2).

(Construction of pSBS2024)
The soybean oleosin promoter fragment (SEQ ID NO: 11; Rowley et al., 1997) was simplified using primers that flank the DNA sequence.

Primer JA408

は、５’末端（下線を付した）に相補的な配列を含む。
プライマーｎｐ１

Contains a sequence complementary to the 5 'end (underlined).
Primer np1

は、プロモーターフラグメントの３’末端（下線を付した）に相同な配列を含む。制限部位ＸｂａＩ（斜字体）およびＳｗａＩ（斜字体）を、クローニングを容易にするために、ＪＡ４０８およびｎｐＩの５’末端にそれぞれ組み込んだ。プライマーを、９７５ｂｐプロモーターフラグメントを増幅するために使用し、次いでｐＵＣ１９のＳｍａＩ部位にクローニングした（図２を参照のこと）。得られるプラスミド、ｐＣＳＰＩを、ＳａｌＩおよびＳｗａＩを用いて切断し、そしてダイズターミネーター（配列番号２８）に連結した。ダイズオレオシンターミネーターを、以下のプライマーを使用して、ＰＣＲによって増幅した：ＪＡ４１０：５’−ＡＡＧＣＴＴＡＣＧＴＧＡＴＧＡＧＴＡＴＴＡＡＴＧＴＧＴＴＧＴＴＡＴＧ−３’（配列番号２９）
およびＪＡ４１１：５’−ＴＣＴＡＧＡＣＡＡＴＴＣＡＴＣＡＡＡＴＡＣＡＡＡＴＣＡＣＡＴＴＧＣＣ−３’（配列番号３０）
そして、２２５ｂｐフラグメントをｐＣＳＰＩのＳａｌＩ−ＳｗａＩ部位にクローニングして、プラスミドｐＳＢＳ２０２５（図６）を得た。命名されたプラスミド、ｐＳＢＳ２０２５は、クローニングのための独特のＨｉｎｄＩＩＩ部位を有する（図２）。

Contains a sequence homologous to the 3 ′ end (underlined) of the promoter fragment. Restriction sites XbaI (italic) and SwaI (italic) were incorporated at the 5 ′ ends of JA408 and npI, respectively, to facilitate cloning. Primers were used to amplify a 975 bp promoter fragment and then cloned into the SmaI site of pUC19 (see FIG. 2). The resulting plasmid, pCSPI, was cut with SalI and SwaI and ligated to a soybean terminator (SEQ ID NO: 28). The soybean oleosin terminator was amplified by PCR using the following primers: JA410: 5′-AAGCTTACGTGATGGAGTATTAATGTGTGTTATG-3 ′ (SEQ ID NO: 29)
And JA411: 5'-TCTAGACAAATTCATAAAATACAAATCACATTGCC-3 '(SEQ ID NO: 30)
Then, the 225 bp fragment was cloned into the SalI-SwaI site of pCSPI to obtain plasmid pSBS2025 (FIG. 6). The named plasmid, pSBS2025, has a unique HindIII site for cloning (FIG. 2).

(Construction of promoter coding sequence fusion)
The following two oligonucleotide primers were synthesized: np2 homologous to nucleotides 553 to 557 of the 5 ′ flanking sequence of mfl (faoA, SEQ ID NO: 24) of plasmid pmfx3

および３’末端に隣接するヌクレオチド２７００〜２６８３に相補的なｎｐ３

And np3 complementary to nucleotides 2700-2683 adjacent to the 3 ′ end

クローニングを容易にするために、ＨｉｎｄＩＩＩ（イタリック体）部位を、プライマーｎｐ２およびｎｐ３の５’末端に導入した。さらに、植物の翻訳開始コドンを包囲するより有利な配列を得るために、３ｂｐのＡＡＡ配列（太字）を組み込んだ。プライマーｎｐ２およびｎｐ３を使用して、フラグメントを増幅し、そしてｐＵＣ１９のＳｍａＩ部位にクローニングした。生じたプラスミドをｐＣｍｆＩ（図３Ａおよび３Ｂ）と称した。プラスミドｐＢｍｆ２を、同様のプロセスで構築した（図５Ａおよび５Ｂ）。クローニングのために、ｍｆ２（ｆａｏＢ）遺伝子（配列番号２５）の５’および３’末端で、ＨｉｎｄＩＩＩ（イタリック体）を生じるために、合成プライマーの第二のセットを設計した。

To facilitate cloning, a HindIII (italic) site was introduced at the 5 ′ end of primers np2 and np3. In addition, a 3 bp AAA sequence (bold) was incorporated to obtain a more favorable sequence surrounding the plant translation initiation codon. The primers np2 and np3 were used to amplify the fragment and clone it into the SmaI site of pUC19. The resulting plasmid was designated pCmfI (FIGS. 3A and 3B). Plasmid pBmf2 was constructed in a similar process (FIGS. 5A and 5B). For cloning, a second set of synthetic primers was designed to generate HindIII (italicized) at the 5 ′ and 3 ′ ends of the mf2 (faoB) gene (SEQ ID NO: 25).

Primer np4 complementary to 5 '(nucleotides 2732 to 2752 bp) of mf2 (faoB, SEQ ID NO: 25) of plasmid pmfx3

および３’（ヌクレオチド３９０７〜３８８６ｂｐ）配列に相同なプライマーｎｐ５

And primer np5 homologous to the 3 '(nucleotides 3907-3886 bp) sequence

をＰＣＲ反応に使用して、１．１７ｋｂのＤＮＡフラグメントを増幅した。生じたＰＣＲ産物をｐＢｌｕｅｓｃｒｉｐｔのＥｃｏＲＶにクローニングした。このプラスミドを、ｐＢｍｆ２と称した。

Was used in a PCR reaction to amplify a 1.17 kb DNA fragment. The resulting PCR product was cloned into pBluescript EcoRV. This plasmid was called pBmf2.

  Both plasmids were individually cut with HindIII and their inserts were cloned into plasmids pSBS2024 and pSBS2025 pre-linearized with the same restriction enzymes. As a result, the following plasmids were generated: pmf124 and pmf125 (FIGS. 3A and 3B) and pmf224 and pmf225 (FIGS. 5A and 5B). These include the Fao gene (mf1 and mf2) fused with either phaseolin or a soybean promoter. DNA sequence analysis confirmed the correct promoter-coding sequence-termination sequence fusion for pmf124, pmf125, pmf224, and pmf225.

(Example 4: Assembly of promoter-coding sequence fusion into plant transformation vector)
After obtaining the plasmids pmf124, pmf125, pmf224, and pmf225, the promoter-coding sequence fusion was expressed in the binary vector pCGN1559 (McBride and Summerfeld, 1990), which expressed the NPTII gene (confers resistance to the antibiotic kanamycin) Was cloned separately into pSBS2004 (which contains the Parsubiquitin promoter driving the PPT gene conferring resistance to the herbicide phosphinothricin). Suitable binary vectors for this purpose with various selectable markers can be obtained from several sources.

  The phaseolin-mf21 fusion cassette was released from the parental plasmid with XbaI and ligated with pCGN1559 linearized with the same restriction enzymes. The resulting plasmid was named pCGmf124 (FIGS. 3A and 3B). Plasmid pCGmf125 containing the soybean-mfl fusion was constructed in a similar manner except that both pmf125 and pCGN1559 were cut with BamHI prior to ligation (FIGS. 3A and 3B).

(Construction of pmf1249 and pmf1254)
Plasmid pSBS2004 was linearized with a BamHI fragment containing the soybean-mfl fusion. This plasmid was named pmf1254 (FIGS. 4A and 4B). Similarly, the XbaI phaseolin-mf1 fusion fragment was ligated with pSBS2004 linearized with the same restriction enzymes. The resulting plasmid was named pmf1249 (FIGS. 4A and 4B).

(Construction of pCGmf224 and pCGmf225)
Phaseolin-mf2 and soybean-mf2 fusions were constructed by excising the fusion from the vector by cutting with either BamHI or XbaI and cloned into pCGN1559 that had been linearized with either restriction enzyme ( Figures 5A and 5B).

(Construction of pCGmf1P2S and pCGmf2P1S)
Two expression cassettes containing promoter-coding sequence fusions were assembled on the same binary vector as follows. : Plasmid pmf124 containing phaseolin-mf1 fusion was cut with BamHI and cloned into the BamHI site of pCGN1559 to create pCGmfB124. This plasmid was then linearized with XbaI and ligated to the XbaI fragment of pmf225 containing the soybean-mf2 fusion. The final plasmid was named pCGmf1P2S (FIGS. 6A and 6B). Plasmid pCGmF2P1S was assembled in a similar manner. The phaseolin-mf2 fusion was released from pmf224 by cutting with BamHI and cloned at the BamHI site of pCGN1559. The resulting plasmid pCGmfB224 was linearized with XbaI and ligated to the XbaI fragment of pmf125 containing the soybean-mf1 fusion (FIGS. 6A and 6B).

(Example 5: Transformation of Brassica)
Brassica seeds were surface sterilized by gentle shaking in 10% commercial bleach (Javex, Colgate-Palmolive) for 30 minutes. The seeds were washed 3 times in sterile distilled water. Seeds were seeded in germination medium (pH 5.8) containing Murashige-Skoog (MS) salt and vitamins, 3% (w / v) sucrose and 0.7% (w / v) phytoagar, 20 per plate. And was maintained at 24 ° C. and a photoperiod of 16 hours light / 8 hours dark with a light intensity of 60-80 μEm ⁻² s ⁻¹ for 4-5 days.

  Each construct, pCGmf124, pCGmf125, pCGmf224, pCGmf1P2S, and pCGmf2P1S was introduced into Agrobacterium tumefaciens EHA101 strain (Hood et al., J. Bacteriol. 168: 1291-1301). Prior to cotyledon petiole transformation, a single colony of EHA101 strain with each construct was grown for 48 hours at 28 ° C. in 5 ml minimal medium supplemented with 100 mg / l kanamycin and 100 mg / l gentamicin. . 1 ml of bacterial suspension was pelleted by centrifugation for 1 minute in a microfuge tube. The pellet was resuspended in 1 ml minimal medium.

  For transformation, cotyledons were excised from 4 day old (in some cases 5 day old) seedlings to contain a petiole of about 2 mm at the base. Individual cotyledons with their petiole cut surfaces were soaked in the diluted bacterial suspension for 1 second and immediately co-cultured media (3% (w / v) sucrose and 0.7% (w / v) phytoagar). And embedded in 20 μM benzyladenine-enriched MS medium) to a depth of about 2 mm. Inoculated cotyledons were placed at a density of 10 per plate and incubated for 48 hours under the same growth conditions. After co-cultivation, the cotyledons were then treated with 3% (w / v) sucrose, 20 μM benzyladenine, 0.7% (w / v) phytoagar (pH 5.8), 300 mg / l timetinin, and 20 mg / l. Was transferred to a regeneration medium containing MS medium supplemented with kanamycin sulfate.

  After 2-3 weeks, the regenerated shoots obtained were cut and “shoot elongation” medium (3% sucrose, 300 mg / l timetin, 0.7% (w / v) phytoagar in a Magenta jar, It was maintained on MS medium (pH 5.8) containing 300 mg / l timentinin and 20 mg / l kanamycin sulfate. Elongated shoots were transferred to “rooting” medium (containing MS medium, 3% sucrose, 2 mg / l indolebutyric acid, 0.7% phytoagar, and 500 mg / l carbenicillin). After the roots appeared, the plantlets were transferred to a potting mix (Redi Earth, WR Grace and Co.). Plants were maintained under the same growth conditions in a spray chamber (75% relative humidity). After 2-3 weeks of growth, leaf samples were taken for neomycin phosphotransferase (NPTII) assay (Moloney et al., Plant Cell Reports 8: 238-42 (1989)).

  Seeds from the FaoA and FaoB transgenic lines can be analyzed for expression of fatty acid oxidized polypeptides by Western blotting using 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 enzymatic activity.

  Transgenic lines expressing FaoA and FaoB complexes were obtained by crossing FaoA and FaoB transgenic lines expressing individual polypeptides and seeds were obtained by Western blotting and enzyme assay as described Analyze by.

(Example 6: Transformation of B. napus cv. Westar and analysis of transgenic lines)
(Transformation)
The protocol used was taken from the procedure described in Moloney et al. (1989). Brassica napus cv. Westar seeds were surface sterilized by gentle shaking in 10% commercial bleach (Javex, Colgate-Palmive Canada Inc.) for 30 minutes. The seeds were washed 3 times in sterile distilled water. Seeds are placed at a density of 20 per plate in germination medium (pH 5.8) containing Murashige-Skoog (MS) salt and vitamins, 3% sucrose and 0.7% phytoagar, and at 24 ° C. and It was maintained for 4-5 days with a photoperiod of 16 hours light period / 8 hours dark period at a light intensity of 60-80 μEm ⁻² s ⁻¹ .

  Each construct, pCGmf124, pCGmf125, pCGmf224, pCGmf225, pCGmf1P2S, and pCGmf2P1S was introduced into the Agrobacterium tumefaciens strain EHA101 (Hood et al., 1986) by electroporation. Prior to cotyledon petiole transformation, a single colony of strain EHA101 with each construct was grown for 48 hours at 28 ° C. in 5 ml minimal medium supplemented with 100 mg / l kanamycin and 100 mg / l gentamicin. . 1 ml of bacterial suspension was pelleted by centrifugation for 1 minute in a microfuge tube. The pellet was resuspended in 1 ml minimal medium.

  For transformation, cotyledons were excised from 4 day old (in some cases 5 day old) seedlings to contain a petiole of about 2 mm at the base. Individual cotyledons with their petiole-cut surfaces are soaked in a diluted bacterial suspension for 1 second and immediately co-cultured media (containing 3% sucrose and 0.7% phytoagar and enriched with 20 μl M benzyladenine). In MS medium) to a depth of about 2 mm. Inoculated cotyledons were placed at a density of 10 per plate and incubated for 48 hours under the same growth conditions. After co-cultivation, the cotyledons were then placed in a regeneration medium containing MS medium supplemented with 3% sucrose, 20 μM benzyladenine, 0.7% phytoagar (pH 5.8), 300 mg / l timentinine, and 20 mg / l kanamycin sulfate. Moved.

  After 2-3 weeks, regenerated shoots are obtained, cut, and contain “shoot elongation” medium (3% sucrose, 300 mg / l timentin, 0.7% phytoagar, and 20 mg / l kanamycin in Magenta jars. MS medium (pH 5.8)). Elongated shoots were then transferred to “rooting” medium (MS medium containing 3% sucrose, 2 mg / l indolebutyric acid, 0.7% phytoagar, and 500 mg / l carbenicillin). After the roots appeared, the plantlets were transferred to a potting mix (Redi Earth, WR Grace and Co. Canada Ltd.). Plants were maintained under the same growth conditions in a spray chamber (75% RH). After 2-3 weeks of growth, leaf samples were taken for neomycin phosphotransferase (NPTII) assay (Moloney et al., 1989). The results are shown in Table 2 below. The data shows the number of plants confirmed to be transformed.

  The developmental fate of the transformed DNA was investigated in 16 randomly selected transgenic lines. Southern DNA hybridization analysis showed that FaoA and / or FaoB were integrated into the genome of the transgenic line tested.

  Approximately 80% of the pmf124 transgenic plants in which the FaoA gene is expressed from the strong bean phaseolin promoter were observed to be male sterile. The apparent high level expression of the FaoA gene from this promoter results in functional expression of the FaoA gene product, which impairs seed and / or pollen development. This result was very unexpected. This is because the plant cells were not expected to be able to perform the first step of the β-oxidation pathway in the cytosol. However, this result provides a further application for the expression of β-oxidized genes in plants due to male sterility for hybrid production or to prevent seed production. It was also noted that in parallel comparisons with normal transgenic lines, the pmf124 strain produced higher levels of biomass, possibly due to the elimination of seed development. Thus, this phenotype can be useful as a means to increase the amount of green biomass produced per acre for silage, feed, or other biomass crops. Here, the use of an inducible promoter system or recombinase technology can be used to produce seed for planting. Seven of these sterile plants were successfully cross-pollinated with pollen from the pmf225 transgenic line and yielded seeds.

  Northern analysis for seed-derived RNA from the pmf224 strain containing the phaseolin promoter-FaoB construct showed a signal indicating the expected 1.2 kb transcript in all samples tested except the control. Northern analysis of seed-derived RNA from the pmf125 strain containing the weak soybean oleolsin promoter-FaoA construct revealed a transcript of the expected size of 2.1 kb. Western blotting for 300-500 μg protein from about 80% seeds of pmf125 plants, in which the FaoA gene is expressed from the relatively weak soybean oleosin promoter, did not reach a conclusion, but the weak signal was 1 Detected in two transgenic lines.

(Fatty acid analysis)
The seeds from the transgenic lines expressing the FaoA gene from the weak soybean oleosin promoter were given the unexpected results showing the strong metabolic effect of expressing the FaoA gene from the strong bean phaseolin promoter in the seed The fatty acid profile was analyzed. Seeds that expressed only the FaoA gene or seeds that also expressed the FaoB gene from the bean phaseolin promoter were tested. Analysis was performed as described in Millar et al., The Plant Cell 11: 1889-902 (1998). Seed fatty acid methyl ester (FAMES) Ten seeds of napus were placed in a 15 × 45-mm threaded glass tube and 0.75 mL of 1N methanolic HCl reagent (Supelco, PA) and 10 μL of 1 mg / mL 17: 0 methyl ester Prepared by heating at 80 ° C. overnight (internal standard). After cooling the sample, FAMES was extracted by vortexing vigorously with 0.3 mL hexane and 0.5 mL 0.9% NaCl. The sample was allowed to settle to separate the phases and 300 μL of the organic phase was extracted and analyzed on a Hewlett-Packard gas chromatography.

  Fatty acid profile analysis showed the presence of additional or enhanced components in the lipid profile in all transgenic plants expressing the FaoA gene (SEQ ID NO: 24), which were not present in control plants. This result also concludes that the FaoA gene was transcribed and translated, and that the FaoA polypeptide (SEQ ID NO: 27) has catalytic activity. This peak is also seen in 11 additional transgenic plants carrying the SoyP-FaoA gene, PhaP-FaoA-SoyP-FaoB gene, SoyP-FaoA-PhaP-FaoB gene, and sterilized cross-pollinated with SoyP-FaoB (PhaP- FaoA) observed in plants. These data clearly demonstrate that the functional expression of the FaoA gene and even very low levels of expression are sufficient to change the lipid profile of the seed. By adapting the methods described herein, one of skill in the art can use phaseoline using other promoters such as the Arabidopsis oleosin promoter, the napin promoter, or the luciferin promoter. These genes can be expressed at levels intermediate between those obtained between the promoter and the soybean oleosin promoter, and the time when the transgene of fatty acid oxidation is expressed using an inducible promoter system or recombinase technology. It can be controlled.

Example 7: Multifunctional enzyme complex of β-oxidation of yeast
S. cerevisiae contains a β-oxidation pathway that proceeds through R-hydroxyacyl CoA rather than S-3-hydroxyacyl CoA observed in bacteria and higher eukaryotes. The fox2 gene from yeast contains hydratase (which produces R-3-hydroxyacyl CoA from trans-2-enoyl-CoA) and dehydrogenase (which utilizes R-3-hydroxyacyl CoA to produce β-ketoacyl. Code).

  The fox2 gene (sequence shown in SEQ ID NO: 1) Two pieces were isolated from cerevisiae genomic DNA by PCR. The primers N-fox2b and N-bamfox2b were used to PCR a 1.1 kb SmaI / BamHI fragment encoding the N-terminal region of Fox2, and the primers C-fox2 and C-bamfox2 were used to C-terminal Fox2 A 1.6 kb BamHI / XbaI fragment encoding the region was PCRed. The full length fox2 gene was reconstructed via subcloning into the vector pTRCN.

  However, the fox1 gene does not retain β-ketothiolase activity and this activity must be supplied by a second transgene. Representative sources of such genes include algae, bacteria, yeast, plants and mammals. The bacterium Alcaligenes eutrophus has a broad specificity β-ketothiolase gene suitable for use in the methods described herein. It can be easily isolated using the acetoacetyl-CoA thiolase gene as a hybridization probe, as described in US Pat. No. 5,661,026 to Peoples et al. This enzyme has also been purified (Haywood et al., FEMS Micro. Lett. 52:91 (1988)) and the purified enzyme determines protein sequence information as the basis for antibody preparation or gene isolation. Useful for.

Example 8: Plant β-oxidation gene
The DNA sequence of the cDNA encoding the β-oxidized tetrafunctional protein (shown in SEQ ID NO: 4) is described in Preisig-Muller et al. Biol. Chem. 269: 20475-81 (1994). Equivalent genes can be obtained by using similar procedures or by screening genomic libraries (many of these are commercially available from, for example, Clontech Laboratories Inc., Palo Alto, California, USA.) Can be isolated from a variety of plant species, including Arabidopsis, Brassica, soybeans, sunflowers, and corn. The peroxisome target sequence PRM was identified at the carboxy terminus of this protein. Constructs suitable for expression in the cytosol of plants can be prepared by PCR amplification of this gene using primers designed to delete this sequence.

Claims (24)

  A method for manipulating plant metabolism comprising expressing a transsol encoding fatty acid β-oxidase activity in the cytosol, peroxisome or glyoxisome of the plant, or mitochondria, in the mitochondrion .   2. The method of claim 1, wherein the fatty acid [beta] -oxidase is expressed from a gene selected from the group consisting of bacteria, yeast, fungi, plants and mammals.   The method according to claim 1, wherein the fatty acid β-oxidase is expressed from a gene derived from a bacterium selected from the group consisting of Escherichia, Pseudomonas, Alcaligenes and Coryneform.   The method according to claim 3, wherein the gene is Pseudomonas putida faoAB.   A gene of bacterial, fungal, yeast, plant or animal origin encoding an enzyme selected from the group consisting of polyhydroxyalkanoate synthase, acetoacetyl-CoA reductase, β-ketoacyl-CoA thiolase, and enoyl-CoA hydratase The method of claim 1, further comprising the step of expressing wherein the enzymes encoded by these genes are directed to the cytosol, plastids other than peroxisomes or glyoxisomes, or mitochondria of the plant. the method of. A DNA construct for use in a method of manipulating plant cell metabolism comprising:
(A) a promoter region functional in plants;
(B) a structural DNA sequence encoding at least one fatty acid β-oxidase activity; and (c) a 3 ′ untranslated region of a gene naturally expressed in a plant, wherein the untranslated region is a polyadenyl of mRNA. A DNA construct comprising in the same phase a 3 'untranslated region encoding a signal sequence for crystallization.   The DNA construct according to claim 6, wherein the promoter is a seed-specific promoter.   The seed-specific promoter is selected from the group consisting of napin promoter, phaseolin promoter, oleosin promoter, 2S albumin promoter, zein promoter, β-conglycinin promoter, acyl carrier protein promoter, and fatty acid desaturase promoter. Item 8. The DNA construct according to Item 7.   The DNA construct according to claim 6, wherein the promoter is a constitutive promoter.   The DNA construct according to claim 6, wherein the promoter is selected from the group consisting of a CaMV 35S promoter, an enhanced CaMV 35S promoter and a ubiquitin promoter.   A method for enhancing the biological production of polyhydroxyalkanoates in a transgenic plant, wherein the plant cytosol, plastid other than peroxisome or glyoxisome, or heterologous fatty acid β-oxidase in the mitochondria Expressing the encoded gene.   12. The method of claim 11, wherein the transgenic plant is selected from the group consisting of Brassica, corn, soybean, cottonseed, sunflower, palm, coconut, safflower, peanut, mustard, flax, tobacco, and alfalfa.   A transgenic plant or part thereof comprising a cytosol of the plant, a plastid other than peroxisome or glyoxisome, or a heterologous gene encoding fatty acid β-oxidase in the mitochondria.   14. The plant or part thereof of claim 13, wherein the fatty acid [beta] -oxidase is expressed from a gene selected from the group consisting of bacteria, yeast, fungi, plants, and mammals.   The plant according to claim 14 or a part thereof, wherein the fatty acid oxidase is expressed from a gene derived from a bacterium selected from the group consisting of Escherichia, Pseudomonas, Alcaligenes, and Coryneform.   The plant according to claim 15 or a part thereof, wherein the gene is Pseudomonas putida faoAB.   14. The plant of claim 13 or one of them, further comprising a gene encoding an enzyme selected from the group consisting of polyhydroxyalkanoate synthase, acetoacetyl-CoA reductase, β-ketoacyl-CoA thiolase, and enoyl-CoA hydratase. Department.   14. The plant of claim 13 or a part thereof, wherein the plant is selected from the group consisting of Brassica, corn, soybean, cottonseed, sunflower, palm, coconut, safflower, groundnut, mustard, flax, tobacco, and alfalfa. . (A) a promoter region functional in plants;
(B) a structural DNA sequence encoding at least one fatty acid β-oxidase activity; and (c) a 3 ′ untranslated region of a gene naturally expressed in a plant, wherein the untranslated region is mRNA 14. A plant according to claim 13 or a part thereof, comprising a DNA construct comprising in the same phase a 3 'untranslated region encoding a signal sequence for polyadenylation.   The plant or a part thereof according to claim 19, wherein the promoter is a seed-specific promoter.   The seed specific promoter is selected from the group consisting of a napin promoter, phaseolin promoter, oleosin promoter, 2S albumin promoter, zein promoter, β-conglycinin promoter, acyl carrier protein promoter, and fatty acid desaturase promoter. Item 21. The plant according to item 20 or a part thereof.   20. A plant or part thereof according to claim 19, wherein the promoter is a constitutive promoter.   21. The plant or part thereof of claim 20, wherein the promoter is selected from the group consisting of a CaMV 35S promoter, an enhanced CaMV 35S promoter and a ubiquitin promoter.   A method for preventing or suppressing seed production in a plant, comprising the step of expressing a heterologous gene encoding a fatty acid β-oxidase in the cytosol, or plastid other than glyoxisome, or mitochondria of the plant Including.

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