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WO2000040730A1 - Proteines associees a la biosynthese de polyhydroxyalcanoate et regions de codage du $i(bacilius megaterium) - Google Patents

Proteines associees a la biosynthese de polyhydroxyalcanoate et regions de codage du $i(bacilius megaterium) Download PDF

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WO2000040730A1
WO2000040730A1 PCT/US2000/000364 US0000364W WO0040730A1 WO 2000040730 A1 WO2000040730 A1 WO 2000040730A1 US 0000364 W US0000364 W US 0000364W WO 0040730 A1 WO0040730 A1 WO 0040730A1
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nucleic acid
acid sequence
seq
protein
sequence encoding
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PCT/US2000/000364
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Maura C. Cannon
Francis C. Cannon
Gabriel J. Mccool
Henry E. Valentin
Kenneth J. Gruys
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University Of Massachusetts
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Priority to MXPA01006958A priority Critical patent/MXPA01006958A/es
Priority to JP2000592426A priority patent/JP2003523170A/ja
Priority to CA002363803A priority patent/CA2363803A1/fr
Priority to AU24949/00A priority patent/AU771433B2/en
Priority to EP00903161A priority patent/EP1141317A1/fr
Publication of WO2000040730A1 publication Critical patent/WO2000040730A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to nucleic acid and amino acid sequences involved in polyhydroxyalkanoate biosynthesis, and more specifically, to polyhydroxyalkanoate biosynthesis sequences isolated from Bacillus megaterium.
  • nucleic acid sequences phaP, phaQ. phaR, phaB, phaC. and their encoded amino acid sequences are disclosed.
  • PHA Polyhydroxyalkanoic acids
  • acetyl-Coenzyme A In this organism two molecules of acetyl-Coenzyme A (CoA) are condensed by ⁇ -ketothiolase (PhaA). followed by a stereo- specific reduction catalyzed by an NADPH dependent acetoacetyl-CoA reductase (PhaB) to produce the monomer D-(-)- ⁇ -hydroxybutyryl-CoA, which is polymerized by PHA synthase (PhaC). These 3 pha genes are coded on the phaCAB operon. which is speculated to be constitutively expressed, but PHA is not constitutively synthesized.
  • PHA inclusion-bodies are 0.2 to 0.5 ⁇ m in diameter, but their structural details are largely unknown. They were described originally for some species of Bacillus (6, 8, 15, 30, 47) and later for many more bacteria including Pseudomonas, Alcaligenes and Rhodococcus (5, 11, 12, 25, 42). Those from Bacillus megaterium were shown to contain 97.7% PHA, 1.87% protein and 0.46% lipid with protein and lipid forming an outer layer (15). More recent reports show the presence of a 14 kDa protein (GA14) on PHA inclusion-bodies of R. ruber (36, 37), and a 24 kDa protein (GA24) with similarities to GA14 on the inclusion-bodies of A. eutrophus (48).
  • GA14 14 kDa protein
  • R. ruber 36, 37
  • GA24 24 kDa protein
  • GA14 and GA24 have been named "phasins" due to some similarities with oleosins, which are proteins on the surface of oil bodies in plant seeds (21). Granule associated proteins are wide-spread in PHA accumulating bacteria (49).
  • This invention is the result of a study of PHA inclusion-body associated proteins from Bacillus megaterium and the cloning and analysis of their coding region. The transcription starts were identified, the functional expression of several of the sequences was confirmed in Escherichia coli and in PHA negative mutants of Bacillus megaterium and Pseudomonas putida, and PhaP and PhaC were localized to PHA inclusion-bodies throughout growth.
  • a nucleic acid fragment encoding proteins involved in polyhydroxyalkanoate biosynthesis was isolated from Bacillus megaterium. Nine nucleic acid sequences and their encoded amino acid sequences are disclosed. Sequences encoding PhaB and PhaC display not insignificant percent identity and similarity to known acetoacetyl-CoA reductase and polyhydroxyalkanoate synthase proteins, while sequences encoding PhaP, PhaQ, and PhaR do not display significant similarity to known sequences.
  • YkoY is similar to known toxic anion resistance proteins; YkoZ is similar to known RNA polymerase sigma factors; YkrM is similar to known Na + -transporting ATP synthase proteins; and SspD matches the known B. megaterium spore specific DNA binding protein.
  • FIG. 1 PHA inclusion-body associated proteins. SDS-polyacrylamide gel electrophoresis of proteins released from purified PHA inclusion-bodies. Lane 1, molecular weight markers in kDa, 14, 18, 29, 43, 68 and 97. Lane 2, proteins from inclusion-bodies of cells harvested at late exponential growth phase. Lane 3, same as lane 2 except this part of the gel was stained following 45 minutes transfer of proteins (seen in lane 2) to PVDF membrane. The bands were visualized by staining with Coomassie Blue.
  • pGMl, pGM6, pGM9 and pGM7 indicate the cloned DNA fragments in these plasmids (Table 1).
  • Probes used to identify and clone the pha cluster are indicated by thick short lines under pGMl ; n2 and n5 are degenerate probes; bmp and bmc are homologous probes to the ends of the pGMl fragment.
  • Ruler of sequence in base pairs is for Bacillus megaterium and B. subtilis. Map of yko, sspD and ykr region in the B. subtilis genome; genes with homology to those of Bacillus megaterium in this region are indicted by thick black arrows; non-homologous genes are indicated by thick gray arrows. Gene annotations are horizontal over each gene symbol. Relevant restriction enzyme sites are vertical.
  • Figure 3 Pairwise alignment of PhaC from Bacillus megaterium (this study) and P. oleovorans (SWISS-PROT accession no. P26494); amino acid identities are shown in black. s The Clustal method with PAM250 residue weight table was used.
  • Figure 4. phar.gfp fusion plasmids and precursors. Only relevant restriction sites are shown. Annotations are as Figure 2. In all fusions the c-terminus excluding the stop codon, of either phaC or phaP, is fused to the gfp gene by the pGFPuv polylinker. For more details, see Table 1. 0 Figure 5 (A): Time-course analysis of Bacillus megaterium (pGM16.2) by phase contrast, green fluorescence, light image, and PHA fluorescence. Time (hours) are hours post-inoculation as indicated.
  • pGM16.2 Time-course analysis of Bacillus megaterium (pGM16.2) by phase contrast, green fluorescence, light image, and PHA fluorescence. Time (hours) are hours post-inoculation as indicated.
  • Figure 6 Hydrophilicity plot of PhaP protein.
  • Figure 7 Hydrophilicity plot of PhaQ protein.
  • Figure 8 Hydrophilicity plot of PhaR protein.
  • Figure 9 Pairwise alignment of PhaC from Bacillus megaterium (this study) and T. violacea (SWISS-PROT accession no. P45366); amino acid identities are indicated by a star (*), and amino acid similarities are indicated by a period (.) below the sequences.
  • the ClustalW method with PAM350 residue weight table was used.
  • Figure 10 Proposed biosynthetic pathway for the preparation of C8 copolymers.
  • C-terminal region refers to the region of a peptide, polypeptide, or protein chain from the middle thereof to the end that carries the amino acid having a free a carboxyl group (the C- terminus).
  • CoA refers to coenzyme A.
  • coding sequence refers to the region of continuous sequential nucleic acid triplets encoding a protein, polypeptide, or peptide sequence.
  • encoding DNA refers to chromosomal nucleic acid, plasmid nucleic acid, cDNA, or synthetic nucleic acid which codes on expression for any of the proteins or fusion proteins discussed herein.
  • the term "genome” as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding nucleic acids of the present invention introduced into bacterial host cells can therefore be either chromosomally-integrated or plasmid-localized.
  • the term "genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components of the cell. Nucleic acids of the present invention introduced into plant cells can therefore be either chromosomally- integrated or organelle-localized.
  • Identity refers to the degree of similarity between two nucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is
  • the number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acids) or 50 (for proteins); and multiplied by 100 to obtain a percent identity.
  • microbe or “microorganism” refer to algae, bacteria, fungi, and protozoa.
  • N-terminal region refers to the region of a peptide, polypeptide, or protein chain from the amino acid having a free amino group to the middle of the chain.
  • Nucleic acid refers to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
  • a “nucleic acid segment” is a nucleic acid molecule that has been isolated free of total genomic DNA of a particular species, or that has been synthesized. Included with the term “nucleic acid segment” are DNA segments, recombinant vectors, plasmids, cosmids, phagemids, phage, viruses, etcetera.
  • “Overexpression” refers to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein said polypeptide or protein is either not normally present in the host cell, or wherein said polypeptide or protein is present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide or protein.
  • the term "plastid” refers to the class of plant cell organelles that includes amyloplasts, chloroplasts, chromoplasts, elaioplasts, eoplasts, etioplasts, leucoplasts, and proplastids.
  • chloroplast genome a circular DNA molecule that ranges in size from about 120 to about 217 kb, depending upon the plant species, and which usually contains an inverted repeat region (Fosket, Plant growth and Development, Academic Press, Inc., San Diego, CA, p. 132, 1994).
  • Polyadenylation signal or “polyA signal” refers to a nucleic acid sequence located 3 ' to a coding region that directs the addition of adenylate nucleotides to the 3 ' end of the mRNA transcribed from the coding region.
  • polyhydroxyalkanoate (or PHA) synthase refers to enzymes that convert hydroxyacyl-CoAs to polyhydroxyalkanoates and free CoA.
  • promoter refers to a nucleic acid sequence, usually found upstream (5') to a coding sequence, that controls expression of the coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site for RNA polymerase and/or other factors necessary for start of transcription at the correct site.
  • mRNA messenger RNA
  • a promoter or promoter region includes variations of promoters derived by means of ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences.
  • the promoter region disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its ability to produce mRNA.
  • Regeneration refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant).
  • Transformation refers to a process of introducing an exogenous nucleic acid sequence
  • a vector, recombinant nucleic acid molecule into a cell or protoplast in which that exogenous nucleic acid is inco ⁇ orated into a chromosome or is capable of autonomous replication.
  • a “transformed cell” is a cell whose nucleic acid has been altered by the introduction of an exogenous nucleic acid molecule into that cell.
  • a “transformed plant” or “transgenic plant” is a plant whose nucleic acid has been altered by the introduction of an exogenous nucleic acid molecule into that plant, or by the introduction of an exogenous nucleic acid molecule into a plant cell from which the plant was regenerated or derived.
  • This invention was developed in the pursuit of proteins which are associated with polyhydroxyalkanoate inclusion bodies, and in the pursuit of novel nucleic acid and amino acid sequences from the bacteria Bacillus megaterium.
  • a 7,916 base pair nucleic acid fragment was isolated and sequenced (SEQ ID NO:l). This fragment was found to contain nine open reading frames, five of which encode proteins suspected of being involved in polyhydroxyalkanoate biosynthesis.
  • An embodiment of the invention is a nucleic acid segment at least about 80% identical to SEQ ID NO:l. More preferably, the nucleic acid segment is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:l.
  • the nucleic acid segment may be a nucleic acid segment that hybridizes under stringent conditions to SEQ ID NO:l, or to the complement thereof.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the invention is further directed to nucleic acid segments, proteins, recombinant vectors, recombinant host cells, genetically transformed plant cells, genetically transformed plants, methods of preparing host cells, methods of preparing plants, fusion proteins, and nucleic acid segments encoding fusion proteins.
  • yhaP and PhaP nucleic acid segments, proteins, recombinant vectors, recombinant host cells, genetically transformed plant cells, genetically transformed plants, methods of preparing host cells, methods of preparing plants, fusion proteins, and nucleic acid segments encoding fusion proteins.
  • a nucleic acid segment may comprise a nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:2; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:3; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%), 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3.
  • An isolated polyhydroxyalkanoate inclusion body associated protein may comprise an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%) identical to SEQ ID NO:3; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO: 3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3.
  • the protein is preferably at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3
  • a recombinant vector may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; b) a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:2; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:3; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3; and c) a 3' transcription terminator.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3.
  • the promoter may generally be any promoter, and more preferably is a tissue selective or tissue specific promoter.
  • the promoter may be constitutive or inducible.
  • the promoter may be a viral promoter.
  • the promoter may be a CMV35S, enhanced CMV35S, an FMV35S, a Lesquerella hydroxylase, or a 7S conglycinin promoter.
  • a recombinant host cell may comprise a nucleic acid segment encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the nucleic acid segment is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:2; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:3; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%), 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a genetically transformed plant cell may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; b) a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:2; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:3; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3; c) a 3' transcription terminator; and d) a 3' polyadenylation signal sequence that directs the addition
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:2.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3.
  • the plant may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • a method of preparing host cells useful to produce a polyhydroxyalkanoate inclusion body associated protein may comprise a) selecting a host cell; b) transforming the selected host cell with a recombinant vector having a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:2; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:3; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3; and c) obtaining transformed host cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%), 99.5%), or 100% identical to SEQ ID NO:2.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a method of preparing plants useful to produce a polyhydroxyalkanoate inclusion body associated protein may comprise a) selecting a host plant cell; b) transforming the selected host plant cell with a recombinant vector having a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:2; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:3; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3; c) obtaining transformed host plant cells; and d) regenerating the transformed host plant cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%o, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3.
  • the plant (and plant cell) may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • the invention also relates to fusion proteins.
  • a fusion protein may comprise a green fluorescent protein subunit; and a polyhydroxyalkanoate inclusion body associated protein subunit; wherein the polyhydroxyalkanoate inclusion body associated protein subunit comprises an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%) identical to SEQ ID NO:3; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO:3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3.
  • the polyhydroxyalkanoate inclusion body associated protein subunit is preferably at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3
  • a nucleic acid segment encoding a fusion protein may comprise a nucleic acid sequence encoding a green fluorescent protein subunit; and a nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein subunit; wherein the nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein subunit is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:2; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:2 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:3; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 3 as an antigen, the antibody being immunoreactive with SEQ ID NO:3.
  • the nucleic acid sequence is at least about 82%>, 84%>, 86%>, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:2.
  • the nucleic acid sequence may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein subunit at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:3.
  • a nucleic acid segment may comprise a nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:4; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:4 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO: 5; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO:5.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:4.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%>, 84%>, 86%>, 88%, 90%o, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5.
  • An isolated polyhydroxyalkanoate inclusion body associated protein may comprise an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%) identical to SEQ ID NO:5; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO:5.
  • the protein is preferably at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5
  • a recombinant vector may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; b) a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:4; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:4 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:5; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO:5; and c) a 3' transcription terminator.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:4.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5.
  • the promoter may generally be any promoter, and more preferably is a tissue selective or tissue specific promoter.
  • the promoter may be constitutive or inducible.
  • the promoter may be a viral promoter.
  • the promoter may be a CMV35S, enhanced CMV35S, an FMV35S, a Lesquerella hydroxylase, or a 7S conglycinin promoter.
  • a recombinant host cell may comprise a nucleic acid segment encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the nucleic acid segment is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:4; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:4 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:5; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO: 5.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:4.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%), 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a genetically transformed plant cell may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; b) a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:4; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:4 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:5; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO: 5; c) a 3' transcription terminator; and d) a 3' polyadenylation signal sequence that directs the addition
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:4.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5.
  • the plant may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • a method of preparing host cells useful to produce a polyhydroxyalkanoate inclusion body associated protein may comprise a) selecting a host cell; b) transforming the selected host cell with a recombinant vector having a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:4; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:4 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:5; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO:5; and c) obtaining transformed host cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%o, 99.5%), or 100% identical to SEQ ID NO:4.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a method of preparing plants useful to produce a polyhydroxyalkanoate inclusion body associated protein may comprise a) selecting a host plant cell; b) transforming the selected host plant cell with a recombinant vector having a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:4; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:4 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:5; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO:5; c) obtaining transformed host plant cells; and d) regenerating the transformed host plant cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:4.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%>, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5.
  • the plant (and plant cell) may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • a fusion protein may comprise a green fluorescent protein subunit; and a polyhydroxyalkanoate inclusion body associated protein subunit; wherein the polyhydroxyalkanoate inclusion body associated protein subunit comprises an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%) identical to SEQ ID NO:5; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO:5.
  • the polyhydroxyalkanoate inclusion body associated protein subunit is preferably at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5
  • a nucleic acid segment encoding a fusion protein may comprise a nucleic acid sequence encoding a green fluorescent protein subunit; and a nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein subunit; wherein the nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein subunit is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:4; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:4 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:5; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 5 as an antigen, the antibody being immunoreactive with SEQ ID NO:5.
  • the nucleic acid sequence is at least about 82%, 84%, 86%>, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:4.
  • the nucleic acid sequence may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein subunit at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:5.
  • PhaR and PhaR PhaR
  • a nucleic acid segment may comprise a nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%) identical to SEQ ID NO:6; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 6 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:7; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:6.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%o, 86%>, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7.
  • An isolated polyhydroxyalkanoate inclusion body associated protein may comprise an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%» identical to SEQ ID NO:7; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7.
  • the protein is preferably at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7
  • a recombinant vector may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; b) a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:6; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 6 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:7; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7; and c) a 3' transcription terminator.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:6.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7.
  • the promoter may generally be any promoter, and more preferably is a tissue selective or tissue specific promoter.
  • the promoter may be constitutive or inducible.
  • the promoter may be a viral promoter.
  • the promoter may be a CMV35S, enhanced CMV35S, an FMV35S, a Lesquerella hydroxylase, or a 7S conglycinin promoter.
  • a recombinant host cell may comprise a nucleic acid segment encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the nucleic acid segment is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:6; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:6 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:7; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:6.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%>, 88%), 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a genetically transformed plant cell may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; b) a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:6; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 6 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:7; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7; c) a 3' transcription terminator; and d) a 3' polyadenylation signal sequence that directs the addition
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:6.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%>, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7.
  • the plant may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • a method of preparing host cells useful to produce a polyhydroxyalkanoate inclusion body associated protein may comprise a) selecting a host cell; b) transforming the selected host cell with a recombinant vector having a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%) identical to SEQ ID NO:6; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 6 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:7; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7; and c) obtaining transformed host cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%o, or 100% identical to SEQ ID NO:6.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a method of preparing plants useful to produce a polyhydroxyalkanoate inclusion body associated protein may comprise a) selecting a host plant cell; b) transforming the selected host plant cell with a recombinant vector having a structural nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%) identical to SEQ ID NO:6; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 6 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:7; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7; c) obtaining transformed host plant cells; and d) regenerating the transformed host plant cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:6.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7.
  • the plant (and plant cell) may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • a fusion protein may comprise a green fluorescent protein subunit; and a polyhydroxyalkanoate inclusion body associated protein subunit; wherein the polyhydroxyalkanoate inclusion body associated protein subunit comprises an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%) identical to SEQ ID NO:7; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7.
  • the polyhydroxyalkanoate inclusion body associated protein subunit is preferably at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7
  • a nucleic acid segment encoding a fusion protein may comprise a nucleic acid sequence encoding a green fluorescent protein subunit; and a nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein subunit; wherein the nucleic acid sequence encoding a polyhydroxyalkanoate inclusion body associated protein subunit is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:6; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 6 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:7; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 7 as an antigen, the antibody being immunoreactive with SEQ ID NO:7.
  • the nucleic acid sequence is at least about 82%>, 84%, 86%), 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:6.
  • the nucleic acid sequence may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein subunit at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:7. phaB and PhaB
  • a nucleic acid segment may comprise a nucleic acid sequence encoding a 3-keto-acyl- CoA reductase protein, wherein the nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:8.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9.
  • An isolated 3-keto-acyl-CoA reductase protein may comprise an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%> identical to SEQ ID NO:9; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO:9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9.
  • the protein is preferably at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9
  • a recombinant vector may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; b) a structural nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO: 8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO:8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9; and c) a 3' transcription terminator.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:8.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%o, 99.5%, or 100%) identical to SEQ ID NO:9.
  • the promoter may generally be any promoter, and more preferably is a tissue selective or tissue specific promoter.
  • the promoter may be constitutive or inducible.
  • the promoter may be a viral promoter.
  • the promoter may be a CMV35S, enhanced CMV35S, an FMV35S, a Lesquerella hydroxylase, or a 7S conglycinin promoter.
  • a recombinant host cell may comprise a nucleic acid segment encoding a 3-keto-acyl- CoA reductase protein, wherein the nucleic acid segment is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO:8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%), or 100% identical to SEQ ID NO:8.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:9.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a genetically transformed plant cell may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; b) a structural nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9; c) a 3' transcription terminator; and d) a 3' polya
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:8.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:9.
  • the plant may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • a method of preparing host cells useful to produce a 3-keto-acyl-CoA reductase protein may comprise a) selecting a host cell; b) transforming the selected host cell with a recombinant vector having a structural nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%o identical to SEQ ID NO: 8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9; and c) obtaining transformed host cells.
  • the nucleic acid sequence is at least about 82%, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:8.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli. Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a method of preparing plants useful to produce a 3-keto-acyl-CoA reductase protein may comprise a) selecting a host plant cell; b) transforming the selected host plant cell with a recombinant vector having a structural nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9; c) obtaining transformed host plant cells; and d) regenerating the transformed host plant cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:8.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:9.
  • the plant (and plant cell) may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • the invention also relates to fusion proteins.
  • a fusion protein may comprise a green fluorescent protein subunit; and a 3-keto-acyl-CoA reductase protein subunit; wherein the 3- keto-acyl-CoA reductase protein subunit comprises an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%> identical to SEQ ID NO:9; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO:9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9.
  • the 3-keto-acyl-CoA reductase protein subunit is preferably at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9
  • a nucleic acid segment encoding a fusion protein may comprise a nucleic acid sequence encoding a green fluorescent protein subunit; and a nucleic acid sequence encoding a 3-keto- acyl-CoA reductase protein subunit; wherein the nucleic acid sequence encoding a 3-keto-acyl- CoA reductase protein subunit is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:9 as an antigen, the antibody being immunoreactive with SEQ ID NO:9.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100%) identical to SEQ ID NO:8.
  • the nucleic acid sequence may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein subunit at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9. phaC and PhaC
  • a nucleic acid segment may comprise a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, wherein the nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:l l ; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 11 as an antigen, the antibody being immunoreactive with SEQ ID NO:l l .
  • the nucleic acid sequence is at least about 82%>, 84%>, 86%), 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 11.
  • An isolated polyhydroxyalkanoate synthase protein may comprise an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%> identical to SEQ ID NO:l l; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO: 11 as an antigen, the antibody being immunoreactive with SEQ ID NO: 11.
  • a recombinant vector may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; b) a structural nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%) identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:l l; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using
  • the nucleic acid sequence is at least about 82%>, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%>, 99%o, 99.5%, or 100% identical to SEQ ID NO:l 1.
  • the promoter may generally be any promoter, and more preferably is a tissue selective or tissue specific promoter.
  • the promoter may be constitutive or inducible.
  • the promoter may be a viral promoter.
  • the promoter may be a CMV35S, enhanced CMV35S, an FMV35S, a Lesquerella hydroxylase, or a 7S conglycinin promoter.
  • a recombinant host cell may comprise a nucleic acid segment encoding a polyhydroxyalkanoate synthase protein, wherein the nucleic acid segment is selected from the group consisting of: a nucleic acid sequence at least about 80%o identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:l 1 ; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:l 1 as an antigen, the antibody being immunoreactive with SEQ ID NO:l l.
  • the nucleic acid sequence is at least about 82%>, 84%>, 86%o, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%. 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:l l .
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a genetically transformed plant cell may comprise in the 5' to 3' direction: a) a promoter that directs transcription of a structural nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; b) a structural nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:l 1; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 11 as an antigen, the antibody being immunoreactive with SEQ ID NO:l l; c) a 3' transcription terminator; and d) a 3' polyadenylation signal
  • the nucleic acid sequence is at least about 82%>, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%o, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:l 1.
  • the plant may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • a method of preparing host cells useful to produce a polyhydroxyalkanoate synthase protein may comprise a) selecting a host cell; b) transforming the selected host cell with a recombinant vector having a structural nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80%) identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:l 1 ; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 11 as an antigen, the antibody being immunoreactive with SEQ ID NO:l 1; and c) obtaining transformed host cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:l 1.
  • the host cell may generally be any host cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • a method of preparing plants useful to produce a polyhydroxyalkanoate synthase protein may comprise a) selecting a host plant cell; b) transforming the selected host plant cell with a recombinant vector having a structural nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, wherein the structural nucleic acid sequence is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO: 11 ; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 11 as an antigen, the antibody being immunoreactive with SEQ ID NO:l 1; c) obtaining transformed host plant cells; and d) regenerating the transformed host plant cells.
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%>, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid segment may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: l l .
  • the plant may generally be any plant, and more preferably a monocot, dicot, or conifer.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • a fusion protein may comprise a green fluorescent protein subunit; and a polyhydroxyalkanoate synthase protein subunit; wherein the polyhydroxyalkanoate synthase protein subunit comprises an amino acid sequence selected from the group consisting of: an amino acid sequence at least about 80%> identical to SEQ ID NO: l l ; and an amino acid sequence that is immunoreactive with an antibody prepared using SEQ ID NO: 1 1 as an antigen, the antibody being immunoreactive with SEQ ID NO: l l .
  • the polyhydroxyalkanoate synthase protein subunit is preferably at least about 82%>, 84%>, 86%>, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: l 1
  • a nucleic acid segment encoding a fusion protein may comprise a nucleic acid sequence encoding a green fluorescent protein subunit; and a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit; wherein the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit is selected from the group consisting of: a nucleic acid sequence at least about 80% identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO: l 1 ; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO: 1 1 as an antigen, the antibody being immunoreactive with SEQ ID NO: l l .
  • the nucleic acid sequence is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%o, 98%>, 99%, 99.5%), or 100% identical to SEQ ID NO: 10.
  • the nucleic acid sequence may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence preferably encodes a protein subunit at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: l 1.
  • a method for the preparation of polyhydroxyalkanoate may comprise: a) obtaining a cell comprising: a nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; and a nucleic acid sequence encoding a PHA synthase protein; wherein: the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein is not naturally found in the cell; the nucleic acid sequence encoding a PHA synthase protein is not naturally found in the cell; the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein is selected from the group consisting of: a nucleic acid sequence at least about 80%) identical to SEQ ID NO: 8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein more preferably is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:8.
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence encoding a 3- keto-acyl-CoA reductase protein preferably encodes a protein at least about 82%>, 84%>, 86%>, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9.
  • the nucleic acid sequence encoding a PHA synthase protein more preferably is at least about 82%>, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid sequence encoding a PHA synthase protein may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence encoding a PHA synthase protein preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:l l .
  • the cell may generally be any cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • the polyhydroxyalkanoate may be a homopolymer or copolymer.
  • the polyhydroxyalkanoate may be a polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, polyhydroxyoctanoate, polyhydroxydecanoate, or copolymers thereof.
  • a method for the preparation of polyhydroxyalkanoate may comprise: a) obtaining a plant comprising: a nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; and a nucleic acid sequence encoding a PHA synthase protein; wherein: the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein is not naturally found in the plant; the nucleic acid sequence encoding a PHA synthase protein is not naturally found in the plant; the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:9; and a nucle
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein more preferably is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 8.
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence encoding a 3- keto-acyl-CoA reductase protein preferably encodes a protein at least about 82%>, 84%>, 86%>, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9.
  • the nucleic acid sequence encoding a PHA synthase protein more preferably is at least about 82%>, 84%>, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid sequence encoding a PHA synthase protein may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence encoding a PHA synthase protein preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:l 1.
  • the plant is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • the polyhydroxyalkanoate may be a homopolymer or copolymer.
  • the polyhydroxyalkanoate may be a polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, polyhydroxyoctanoate, polyhydroxydecanoate, or copolymers thereof.
  • a method for the preparation of polyhydroxyalkanoate may comprise: a) obtaining a cell comprising: a nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; and a nucleic acid sequence encoding a PHA synthase protein; wherein: the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein is not naturally found in the cell; the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein more preferably is at least about 82%>, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:8.
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%), or 100% identical to SEQ ID NO:9.
  • the cell may generally be any cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • the polyhydroxyalkanoate may be a homopolymer or copolymer.
  • the polyhydroxyalkanoate may be a polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, polyhydroxyoctanoate, polyhydroxydecanoate, or copolymers thereof.
  • a method for the preparation of polyhydroxyalkanoate may comprise: a) obtaining a plant comprising: a nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; and a nucleic acid sequence encoding a PHA synthase protein; wherein: the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein is not naturally found in the plant; the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein is selected from the group consisting of: a nucleic acid sequence at least about 80%> identical to SEQ ID NO:8; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 8 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80% identical to SEQ ID NO:9; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:9 as
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein more preferably is at least about 82%>, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:8.
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:9.
  • the plant may generally be any plant, and preferably is a tobacco, wheat, potato, Arabidopsis, high oil seed plants such as corn, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • the polyhydroxyalkanoate may be a homopolymer or copolymer.
  • the polyhydroxyalkanoate may be a polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, polyhydroxyoctanoate, polyhydroxydecanoate, or copolymers thereof.
  • PHA biosynthesis methods phaC
  • a method for the preparation of polyhydroxyalkanoate may comprise: a) obtaining a cell comprising: a nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; and a nucleic acid sequence encoding a PHA synthase protein; wherein: the nucleic acid sequence encoding a PHA synthase protein is not naturally found in the cell; the nucleic acid sequence encoding a PHA synthase protein is selected from the group consisting of: a nucleic acid sequence at least about 80%) identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%> identical to SEQ ID NO:l l; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:l 1 as an antigen, the antibody being immunoreactive with S
  • the nucleic acid sequence encoding a PHA synthase protein more preferably is at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid sequence encoding a PHA synthase protein may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence encoding a PHA synthase protein preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 11.
  • the cell may generally be any cell, and preferably is a bacterial, fungal, mammalian, or plant cell.
  • the bacterial cell is preferably an Escherichia coli, Bacillus, Pseudomonas, or Ralstonia eutropha cell.
  • the fungal cell is preferably a Saccharomyces cerevisiae or Schizosaccharomyces pombe cell.
  • the plant cell is preferably a tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as com, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.
  • the polyhydroxyalkanoate may be a homopolymer or copolymer.
  • the polyhydroxyalkanoate may be a polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, polyhydroxyoctanoate, polyhydroxydecanoate, or copolymers thereof.
  • a method for the preparation of polyhydroxyalkanoate may comprise: a) obtaining a plant comprising: a nucleic acid sequence encoding a 3-keto-acyl-CoA reductase protein; and a nucleic acid sequence encoding a PHA synthase protein; wherein: the nucleic acid sequence encoding a PHA synthase protein is not naturally found in the plant; the nucleic acid sequence encoding a PHA synthase protein is selected from the group consisting of: a nucleic acid sequence at least about 80%) identical to SEQ ID NO: 10; a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NO: 10 or the complement thereof; a nucleic acid sequence encoding a protein at least about 80%) identical to SEQ ID NO:l 1; and a nucleic acid sequence encoding a protein that is immunoreactive with an antibody prepared using SEQ ID NO:l 1 as an antigen, the antibody being immunoreactive with SEQ
  • the nucleic acid sequence encoding a PHA synthase protein more preferably is at least about 82%, 84%>, 86%>, 88%, 90%o, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 10.
  • the nucleic acid sequence encoding a PHA synthase protein may be obtained from a natural source, may be mutagenized, may be genetically engineered by mutagenesis or other methods, or may be synthetic.
  • the nucleic acid sequence encoding a PHA synthase protein preferably encodes a protein at least about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO:l 1.
  • the plant may generally be any plant, and preferably is a tobacco, wheat, potato, Arabidopsis, high oil seed plants such as com, soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.
  • the polyhydroxyalkanoate may be a homopolymer or copolymer.
  • the polyhydroxyalkanoate may be a polyhydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, polyhydroxyoctanoate, polyhydroxydecanoate, or copolymers thereof.
  • Polyhydroxyalkanoate may be prepared by a method comprising: a) obtaining a recombinant host cell comprising: a nucleic acid sequence encoding a ⁇ -ketothiolase protein; a nucleic acid sequence encoding a 3-ketoacyl-CoA reductase protein; a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a nucleic acid sequence encoding a ⁇ - hydroxyacyl-CoA dehydrase; and a nucleic acid sequence encoding an acyl-CoA dehydrogenase protein or an enoyl-CoA reductase protein; and b) culturing the recombinant host cell under conditions suitable for the preparation of polyhydroxyalkanoate; wherein: the polyhydroxyalkanoate comprises C6, C8, or CIO monomer subunits; the nucleic acid sequence encoding a 3-keto-acyl-CoA reducta
  • sequences disclosed in the sequence listing may also be used to prepare primers, probes, and monoclonal or polyclonal antibodies.
  • SEQ ID NOS.l, 2, 4, 6, 8, 10, 22, 24, 26, and 28, and the their complementary strands may be used to design oligonucleotide primers and probes.
  • Primers and probes are typically at least 15 nucleotides in length, and more preferably are at least 20, 22, 24, 26, 28, 30, 40, or 50 nucleotides in length. Contiguous nucleotide sequences from a given sequence are chosen based upon favorable hybridization conditions, including minimization of hairpin or other detrimental sequences. The identification of suitable primer or probe sequences is well known to those of skill in the art, and is facilitated by commercially available software such as Mac Vector (Oxford Molecular Group) and Xprimer (http://alces.med.umn.edu/rawprimer.html). Primers and probes may be used for the screening of libraries, for PCR amplification, and other routine molecular biological applications. Primers and probes may also be used for antisense applications.
  • SEQ ID NOS:3, 5, 7, 9, 11, 23, 25, 27, and 29 may be used for the generation of monoclonal or polyclonal antibodies.
  • the entire sequences may be used, or antigenic fragments thereof.
  • portions of the full length sequences may be synthesized and covalently attached to antigenic proteins such as keyhole limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • Portions of the full length sequences may be used for the preparation of multi-antigenic peptides (52).
  • KLH keyhole limpet hemocyanin
  • the generation of monoclonal and polyclonal antibodies is well known to those of skill in the art. The following Examples are included to demonstrate preferred embodiments of the invention.
  • Example 1 Bacterial strains and plasmids
  • PhaP GFP in-frame fusion plasmid.
  • the appropriate antibiotics were included in the media: ampicillin (200 ⁇ g/mL [AMP 200 ]), chloramphenicol (25 ⁇ g/mL [CM 25 ]), erythromycin (200 ⁇ g/mL [EM 200 ]), or tetracycline (12.5 ⁇ g/mL [TC 12 5 ]) for plasmid selection in Escherichia coli; chloramphenicol (12 ⁇ g/mL [CM 12 ]), or erythromycin (1 ⁇ g/mL [EM 1 ]) plus lincomycin (25 ⁇ g/mL [LM 25 ]) for plasmid selection in Bacillus megaterium; chloramphenicol (160 ⁇ g/mL [CM 160 ]), or tetracycline (30 ⁇ g/mL [TC 30 ]) for selection in Pseudomonas.
  • ampicillin 200 ⁇ g/mL [AMP 200 ]
  • chloramphenicol 25 ⁇ g/mL [CM 25
  • Escherichia coli and Pseudomonas putida were transformed by electroporation of competent cells using an electroporator (Eppendorf) and following the manufacturers instructions. Bacillus megaterium was transformed using a biolistic transformation procedure (39).
  • phase contrast microscopy wet mounts of cultures were visualized at x 1,000 magnification in a light microscope with phase contrast attachments (Labophot-2 Microscope,
  • Nile Blue A (Sigma) for 15 minutes at 55°C, destained for 30 seconds in 8% (v/v) acetic acid, water washed, air dried, and viewed at xlOOO magnification under fluorescence using filters; excitation, 446/10 nm; barrier filter, 590 nm; dichroic mirror, 580 nm.
  • To view GFP. wet mounts of cultures with or without 1% (w/v) agarose were viewed at xlOOO magnification under fluorescence using filters; excitation, 390-450 nm; barrier filter, 480-520 nm; dichroic mirror,
  • Bacillus megaterium uses three codons as start codons in protein coding sequences. ATG, TTG, and GTG all encode methionine when present at the start of a coding region. TTG and GTG encode leucine and valine when present within a coding region, respectively. Bacillus megaterium uses TGA, TAA, and TAG as stop codons.
  • Bacillus megaterium sequences starting with TTG or GTG may require mutagenesis to ATG if the sequences are to be expressed in organisms that use ATG exclusively as a start codon.
  • Example 6 Separation of polypeptides associated with PHA inclusion-bodies.
  • Inclusion-bodies were purified (32) followed by suspension in TE buffer (10 mM Tris- HCl pH 8, 1 mM EDTA) with 2% (w/v) SDS.
  • An equal volume of 2x sample buffer (100 mM Tris-HCI (pH 6.8), 4% SDS, 4 mM EDTA, 20% glycerol, 2% 2-mercaptoethanol, 0.1% bromophenol blue) was added prior to boiling for 5 minutes and samples were centrifuged for 3 minutes to pellet PHA; the supernatant was loaded on a 12% SDS-polyacrylamide gel and run at 8 mA overnight at 4°C to separate proteins. The gel was stained with Coomassie Blue for 5 minutes prior to transfer of proteins to a polyvinylidene difluoride membrane using a semi-dry electroblotter at 400 mA for 45 minutes.
  • proteins there were at least thirteen such proteins present in various quantities. Some or all of these proteins could be intrinsic structural components of PHA inclusion-bodies, enzymes involved with PHA metabolism or possibly scaffolding components involved in inclusion-body assembly. Alternatively, they could have been acquired by the inclusion-bodies during the purification procedure. The three most abundant proteins had molecular weights of approximately 14, 20 and 41 kDa.
  • the N-terminal amino acid sequence for the three most prevalent proteins were determined. Membrane carrying the proteins of interest was cut for use in N-terminal amino acid sequence determination by Edman Degradation using a minimum quantity of 200 pmols of each protein.
  • the N-terminal amino acid sequence of the 14 kDa protein was KVFGRXELAAAMKRXGL (SEQ ID NO: 19)
  • the 20 kDa protein was NTVKYXTVIXAMXXQ (SEQ ID NO:20)
  • the 41 kDa proteins was AIPYVQEXEKL (SEQ ID NO:21).
  • AAYACRGTNAAATAYN NACRGTNATYN NGCDATGATG (n2, SEQ ID NO: 12) and GCDATYCCDTAYGTNCARGAAGGHTTYAAA (n5, SEQ ID NO: 13) for the 20 kDa and 41 kDa proteins, respectively ( Figure 1).
  • Both probes used in separate 38°C Southern blotting hybridization experiments, identified a 6.4 kb Hindlll, a 5.2 kb EcoRI, and a 3.7 kb Hindlll to EcoRI DNA fragment of DNA, indicating that the 5' ends of the coding regions for both of these proteins were located less than 3.7 kb apart in the genome.
  • the three fragments were purified from agarose following electrophoresis, and cloned into plasmid pBluescriptllSK.
  • DNA fragments of pGMl, pGM6 and pGM9 were subcloned into pBluescriptllSK, and sequenced, from both ends using universal primers and internally by primer walking on both strands, using dye terminator chemistry, cycle sequencing and an ABI Prism 377 sequencer (Applied Biosystems). Sequence assembly and analysis was performed using Lasergene (DNAStar, Inc.), and Gapped BLAST and PSI BLAST (1).
  • the 3.7 kb fragment contained 5 ORFs ( Figure 2), whose predicted amino acid sequences encode PhaP (20 kDa protein), PhaQ, PhaR, PhaB and PhaC (41 kDa protein).
  • the 20 and 41 - kDa proteins were identified by their N-terminal amino acid sequences. Since the C-terminus for each of these two proteins extended beyond the boundaries of pGMl, the remaining sequence were obtained from plasmids pGM6 and pGM9.
  • Example 8 The pha locus.
  • the 7,916 bp region (SEQ ID NO:l) containing pha genes from Bacillus megaterium was cloned, sequenced and characterized. It was shown to carry 8 complete and 1 incomplete open reading frame ( Figure 2, Tables 3 and 4). Coding sequences in this region were assigned on the basis of homology to known sequences, N-terminal amino acid sequences, putative ribosome binding sites and operon location. The complement and arrangement of genes flanking the pha genes in Bacillus megaterium are very similar to a region of Bacillus subtilis 168 ( Figure 2). This strain is negative for PHA and no known pha genes or sequences occur in its genome, for which the complete sequence is available (24). In place of pha genes in this region of B. subtilis are ykrl, ykrK and ykrL, which, respectively, code for putative proteins similar to two unknown proteins, and a probable heat shock protein.
  • Example 9 The ph ⁇ nucleic acid and encoded protein sequences
  • PhaP The deduced amino acid sequence of PhaP shows a 20 kDa extremely hydrophilic product with no obvious similarity to known sequences ( Figure 6).
  • Inclusion-body associated low molecular weight proteins (phasins) have been described in many bacteria (49), but where sequences were available no similarities of identifiable significance with PhaP of Bacillus megaterium were found.
  • PHA inclusion-body abundant proteins play an important role in PHA producing cells, since they are involved in determining inclusion-body size and shape, and are present in quantities up to 5% of total protein in the case of PHA producing A. eutrophus (48). It is an interesting observation that the amino acid sequences of phasin proteins are so dissimilar, even in closely related bacteria. Some similarity between such proteins would be expected in closely related bacteria, were they to have a role in inclusion-body biogenesis, however, conservation of sequence would be entirely unnecessary should they have a role as storage proteins.
  • PhaC proteins from these three bacterial strains respectively, have 355, 378, and 355 amino acids while PhaC from Bacillus megaterium has 362 amino acids. All other PhaC proteins studied are larger in size, and range from 559 amino acids for that of P. oleovorans (22) to 636 amino acids for that of Rhizobium etli (2).
  • the phaP, -Q, -R, -B and -C gene cluster can complement a deletion mutant of B. megaterium.
  • This mutant PHA05 was constructed by a gene substitution technique.
  • a plasmid (based on pGMlO) in which the pha genes were substituted by the erythromycin gene, was propagated in B. megaterium 11561. Selection on erythromycin allowed isolation of the PHA05 mutant that was negative for PHA synthesis.
  • Complementation with the phaP, -Q, -R, -B and -C gene cluster was obtained when pGM7H or pGM13 was introduced into the PHA05 strain.
  • the transcription start points were mapped in the region from the EcoRI restriction site in phaP to the Hindlll site in ykrM by primer extension analysis, using the Promega system for primer extension on RNA templates.
  • DNA oligonucleotide primers 17 to 20 nucleotides in length, were synthesized to match target sequences, initially at approximately 500 base pair intervals and subsequently at about 50 to 250 nucleotides down-stream from the predicted transcription start points.
  • the 32p 5' end-labeled primers were extended with reverse transcriptase using total RNA (10 ⁇ g per reaction) purified from Bacillus megaterium (31).
  • the primers used to identify the transcription start nucleotides for the phaP, phaQ, and phaRBC promoters were, respectively, CCCCTTTGTCCATTGTTCCC (SEQ ID NO: 16); CCATGTAGATTCCACCCTC (SEQ ID NO: 17); and CTCCATCTCCTTTCTTGTG (SEQ ID NO: 18).
  • Primer extension products showed a single band from each reaction, indicating one transcript, while control reactions in which RNA was omitted showed no bands.
  • the extension products run alongside sequencing reaction products obtained with the same primer ( Figure 2C), identified the 5' ends of the transcripts thus allowing the putative promoter sequences at approximately -10 and -35 -bp for phaP, -Q and -R to be identified.
  • the arrangement of genes in the pha cluster of Bacillus megaterium is unique among those already published and phaA is notably absent.
  • the phaP, -Q, -R, -B and -C genes were shown to be in a 4,104 -bp region, with phaP and -Q transcribed in one orientation, each from a separate promoter, while phaR, -B and - C were divergently transcribed from a promoter in front of phaR.
  • the putative promoters responsible for transcription of phaQ and phaR, phaB and phaC show strong similarity to both Bacillus subtilis Sigma A type (34) and Escherichia coli, Sigma 70 type promoters (14), which can express constitutively. This is in keeping with previous data for Alcaligenes eutrophus showing that phaC is constitutively synthesized, but PHA is not constitutively accumulated (19).
  • the third putative promoter in this region, the phaP promoter resembles a Sigma D (SigD) type promoter known to control the expression of a regulon of genes associated with flagellar assembly, chemotaxis and motility (13, 20, 46).
  • Bacillus subtilis Sigma D is expressed in the exponential phase and peaks in late exponential phase of growth. This parallels the pattern of PHA accumulation previously described for Bacillus megaterium 1 1561 (32). However, further experiments are required to test the hypothesis that PHA accumulation in regulated by sigma D or products of its resulting transcripts.
  • the phaP gene has 18 -bp duplicate sequences that could base-pair to form a r 70-independent terminator close to its translational stop codon ( Figure 2B).
  • Example 12 Expression of Bacillus megaterium pha genes in Escherichia coli and Pseudomonas putida
  • Escherichia coli which is naturally PHA negative, and Pseudomonas putida GPpl04, a phaC " mutant. Plasmids carrying one or more of these genes were introduced and the resulting transformants were tested for PHA accumulation following growth on LB or M9 medium with various carbon sources and the appropriate antibiotic for plasmid selection.
  • Triplicate 500 mL cultures were grown in 2 liter flasks at 30°C, rotating at 250, using 1 %> inocula of 16 hour cultures, which had been grown in LB, centrifuged and resuspended in equal volumes of 0.9%> saline. At 48 hours samples were removed for microscopy and cells were harvested, washed once in dH 2 O and lyophilized. For PHA extraction, lyophilized cells were suspended in 10 volumes of 5% (w/v) bleach, shaken at 65°C for 1 hour and centrifuged. The pellet was resuspended in 10 volumes of 5% bleach and centrifuged followed by sequentially washing in water and 95%> ethanol. The amount of PHA is expressed as percent PHA per mass of vacuum dried cells (w/w).
  • Escherichia coli carrying pGM7 or pGMlO accumulated low levels of PHA while Escherichia coli carrying pGMl or pGM6 accumulated no PHA.
  • Fluorescence microscopy of Nile Blue A stained cells showed approximately 1 cell in 20 had one or several inclusion-bodies and the quantity of PHA produced was approximately 5% of cell dry weight. Since Escherichia coli does not have PhaA, a low level or no PHA is the expected result.
  • Pseudomonas putida GPpl04 accumulated PHA on rich as well as minimal medium with various carbon sources to >50%> of cell dry weight, and 90 to 100%) of cells appeared full of PHA (Table 5).
  • the positive control P. oleovorans (equivalent to wild-type Pseudomonas putida) accumulated PHA only when grown on longer chain carbon sources, and not on LB. No PHA was accumulated by the negative control or by Pseudomonas putida carrying phaC alone (pDRl).
  • Proteins associated with purified PHA inclusion-bodies may not accurately reflect the localization of the these proteins within the growing cell.
  • Visualization ofphar.gfp gene product fusion proteins in living cells throughout culture growth is a useful method for determining both the localization of the pha gene products and their comparative levels in growing cells.
  • PhaP and PhaC, as fusion proteins ( Figure 4) localized to PHA inclusion-bodies at all time points tested throughout growth of Bacillus megaterium 11561.
  • the negative control (pHPS9) showed no fluorescence at any time point.
  • the localization control (pGM13C) showed non-localized green fluorescence at all time points.
  • PhaP monitored as a PhaP::GFP fusion protein in pGM16.2 ( Figures 5A and 5B), decreased significantly during the first half (2 hours) of lag phase growth, increased during late lag phase and early to mid-exponential phase, decreased in mid to late exponential phase and increased during stationary phase growth.
  • PhaP may be a storage protein that is degraded as a source of amino acids.
  • the profile of PHA accumulation in these cells (carrying pGM16.2) followed a similar pattern to that of PhaP except that PHA decreased only in the lag phase and continued to accumulate throughout other phases of culture growth.
  • PhaC, monitored as a PhaC:: GFP fusion protein in pGM13 showed a similar profile of expression to that of PhaP with one exception: PhaC did not reduce in level during lag phase growth. It did, however, reduce in level in mid to late exponential phase growth, as did PhaP.
  • the profile of PHA accumulation in these cells carrying PhaC::GFP was similar to that of cells carrying PhaP::GFP, except that the PHA level did not reduce during lag phase growth.
  • the increased quantity of PhaC in the cell is a likely explanation since PhaC remained functional in the fusion protein PhaC::GFP.
  • Stereospecificity assays were conducted on the Bacillus megaterium reductase using various chain length enoyl-CoA esters (C4-C8, Table 6). The assay was done using crotonase from Sigma (L-hydroxy acids) or hydratase from Rhodosprillum rubrum (D-hydroxy acids) to form the 3-hydroxyacyl-CoA compounds from the enoyl-CoA esters. Acetoacetyl-CoA reductase activity was monitored spectrophotometrically as the reduction of NADP + while 3-hydroxyacyl- CoAs were oxidized.
  • Bacillus megaterium reductase is a D-specific enzyme with a preference for C6 carbon chains. Enzyme reactions using NADH as electron donor for 3-ketoacyl-CoA reduction did not indicate significant enzyme activity with this cofactor. Table 6: Analysis for stereo-specificity of the Bacillus megaterium 3-ketoacyl-CoA reductase.
  • Clone Bl-30 contains pMON48213; clone B5-20 contains pMON48214.
  • Example 15 Verification of the Bacillus megaterium 3-ketoacyl-CoA reductase for PHA accumulation
  • Plasmid pMON48213 contains the same pha sequences as pMON48220, but was constructed with pS ⁇ 380 (Invitrogen, Carlsbad, CA), a high level expression vector. Plasmid pMON48221 contains the same pha sequences as pMON48220, but lacks a small fragment of the multicloning site between phaA Re and phaB Bm .
  • 3-Ketoacyl-CoA reductase was monitored in a total volume of 1 mL containing 100 mM potassium phosphate buffer pH 7.0, 50 ⁇ M acetoacetyl-CoA and 150 ⁇ M NADPH.
  • the reaction mixture contained between 5 and 50 ⁇ L cell extract.
  • Assays were monitored spectrophotometrically at 340 nm.
  • Table 7 Application of the Bacillus megaterium 3-ketoacyl-CoA reductase for PHA formation in
  • Table 8 Enzyme activity of the Bacillus megaterium 3-ketoacyl-CoA reductase using pMON48220 and pMON48213
  • the 7,916 base pair genomic fragment (SEQ ID NO:l) additionally contained three complete open reading frames and one incomplete open reading frame encoding proteins in addition to PhaP, PhaQ, PhaR, PhaB, and PhaC.
  • sequence comparisons suggest that ykoY (SEQ ID NO:22) encodes toxic anion resistance protein YkoY (SEQ ID NO:23), ykoZ (SEQ ID NO:24) encodes RNA polymerase sigma factor protein YkoZ (SEQ ID NO:25), and ykrM (SEQ ID NO:26) encodes a portion of the Na + -transporting ATP synthase protein YkrM (SEQ ID NO:27).
  • Sequence sspD matches the known Bacillus megaterium sequence (4, 10) encoding SspD (SEQ ID NO:29). While the activity of the proteins is identified by their similarity to other known proteins, it is possible that the proteins may have additional functionality involved in polyhydroxyalkanoate biosynthesis.
  • nucleic acid and amino acid sequences may be used in nucleic acid segments, recombinant vectors, transgenic host cells, and transgenic plants.
  • PHA synthases have been identified to be either one or two subunit enzymes (51). Single subunit enzymes have only the PhaC protein, while two subunit enzymes have PhaC and PhaE protein subunits. Nucleic acid sequences encoding PhaE subunits have been found to be located adjacent to the nucleic acid sequences encoding PhaC. Table 10: One and two subunit PHA synthases
  • the B. megaterium would be expected to be part of a two subunit synthase.
  • the nucleic acid sequences adjacent to phaC in the 7,916 base pair genomic fragment show no significant similarity to a phaE sequence.
  • Upstream of phaC is phaB, and downstream is ykrM, a suspected Na + transporting ATP synthase (Table 4).
  • the B. megaterium sequences were able to complement P. putida GPpl04 to accumulate PHA, this suggests that the B. megaterium phaC may encode a novel class of PHA synthase, i.e. a single subunit synthase with a molecular weight in the range of two subunit PhaC proteins.
  • Example 18 Pathway for the production of C4/C6/C8/C 10 PHA copolymers
  • Figure 10 outlines a proposed biosynthetic pathway for the production of PHA copolymers incorporating C4 and/or C6 monomer units.
  • Produced polymers may include C4-co- C6, C4-co-C8, C4-co-C6-co-C8, C6-co-C8, C6, and C8.
  • a recombinant host cell or plant may be constructed to contain the nucleic acid sequences encoding the required enzymes.
  • the ⁇ -ketothiolase is preferably BktB (53, WO 98/00557).
  • the ⁇ -ketothiolase can condense two molecules of acetyl-CoA to form acetoacetyl-CoA.
  • This product may be reduced to 3HB-CoA by the Bacillus megaterium 3-keto-acyl-CoA reductase protein.
  • 3HB-CoA may be converted to crotonyl-CoA by a hydratase such as that from Aeromonas caviae (54).
  • butyryl-CoA dehydrogenase such as that cloned from Clostridium acetobutylicum (55).
  • This product may be condensed with acetyl-CoA by the ⁇ -ketothiolase to afford 3-ketohexanoyl-CoA.
  • This is the preferred substrate of the Bacillus megaterium reductase, leading to the production of 3-hydroxyhexanoyl-CoA.
  • This product may be incorporated into C6 polymers or copolymers (e.g. C4-co-C6) by a PHA synthase having a broad substrate specificity (e.g. (56)).
  • An additional round of condensation may lead to production of the C8 monomer, allowing the introduction of C8 into PHA polymers or copolymers.
  • a further additional round of condensation may lead to production of the CIO monomer, allowing the introduction of CIO into PHA polymers or copolymers.
  • nucleic acid sequence encoding a protein may lead to mutant protein sequences that display equivalent or superior enzymatic characteristics when compared to the sequences disclosed herein.
  • This invention accordingly encompasses nucleic acid sequences which are similar to the sequences disclosed herein, protein sequences which are similar to the sequences disclosed herein, and the nucleic acid sequences that encode them. Mutations may include deletions, insertions, truncations, substitutions, fusions, shuffling of subunit sequences, and the like.
  • Mutations to a nucleic acid sequence may be introduced in either a specific or random manner, both of which are well known to those of skill in the art of molecular biology.
  • Random or non-specific mutations may be generated by chemical agents (for a general review, see Singer and Kusmierek, Ann. Rev. Biochem. 52: 655-693, 1982) such as nitrosoguanidine (Cerda-Olmedo et al., J. Mol. Biol. 33: 705-719, 1968; Guerola, et al. Nature New Biol. 230: 122-125, 1971) and 2- aminopurine (Rogan and Bessman, J. Bacteriol 103: 622-633, 1970), or by biological methods such as passage through mutator strains (Greener et al. Mol. Biotechnol. 7: 189-195, 1997).
  • Nucleic acid hybridization is a technique well known to those of skill in the art of DNA manipulation.
  • the hybridization properties of a given pair of nucleic acids is an indication of their similarity or identity.
  • Mutated nucleic acid sequences may be selected for their similarity to the disclosed nucleic acid sequences on the basis of their hybridization to the disclosed sequences.
  • Low stringency conditions may be used to select sequences with multiple mutations.
  • High stringency conditions may be used to select for nucleic acid sequences with higher degrees of identity to the disclosed sequences.
  • Conditions employed may include about 0.02 M to about 0.15 M sodium chloride, about 0.5%) to about 5%> casein, about 0.02%> SDS and/or about 0.1%> N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at temperatures between about 50°C and about 70°C. More preferably, high stringency conditions are 0.02 M sodium chloride, 0.5%> casein, 0.02%> SDS, 0.001 M sodium citrate, at a temperature of 50°C.
  • Example 20 Determination of homologous and degenerate nucleic acid sequences Modification and changes may be made in the sequence of the proteins of the present invention and the nucleic acid segments which encode them and still obtain a functional molecule that encodes a protein with desirable properties. The following is a discussion based upon changing the amino acid sequence of a protein to create an equivalent, or possibly an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the nucleic acid sequence, according to the codons given in Table 11.
  • Certain amino acids may be substituted for other amino acids in a protein sequence without appreciable loss of enzymatic activity. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed protein sequences, or their corresponding nucleic acid sequences without appreciable loss of the biological activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol. Biol, 157: 105-132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within +1 are more preferred, and those within ⁇ 0.5 are most preferred.
  • an amino acid may be substituted by another amino acid having a similar hydrophilicity score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein.
  • substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are more preferred, and those within ⁇ 0.5 are most preferred.
  • amino acid substitutions are therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Changes which are not expected to be advantageous may also be used if these resulted in functional fusion proteins. Plant Vectors
  • transformation vectors capable of introducing nucleic acid sequences encoding polyhydroxyalkanoate biosynthesis enzymes are easily designed, and generally contain one or more nucleic acid coding sequences of interest under the transcriptional control of 5' and 3' regulatory sequences.
  • Such vectors generally comprise, operatively linked in sequence in the 5' to 3 ' direction, a promoter sequence that directs the transcription of a downstream heterologous structural nucleic acid sequence in a plant; optionally, a 5' non-translated leader sequence; a nucleic acid sequence that encodes a protein of interest; and a 3 " non-translated region that encodes a polyadenylation signal which functions in plant cells to cause the termination of transcription and the addition of polyadenylate nucleotides to the 3 ' end of the mRNA encoding the protein.
  • Plant transformation vectors also generally contain a selectable marker.
  • Typical 5'- 3' regulatory sequences include a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • Vectors for plant transformation have been reviewed in Rodriguez et al. (Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston., 1988), Glick et al. (Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., 1993), and Croy (Plant Molecular Biology Labfax, Hames and Rickwood (Eds.), BIOS Scientific Publishers Limited, Oxford, UK., 1993).
  • Plant Promoters Plant promoter sequences can be constitutive or inducible, environmentally- or developmentally-regulated, or cell- or tissue-specific. Often-used constitutive promoters include the CaMV 35S promoter (Odell, J.T. et al., Nature 313: 810-812. 1985), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins et al.. Nucleic Acids Res. 20: 8451-8466, 1987), the mannopine synthase (mas) promoter, the nopaline synthase (nos) promoter, and the octopine synthase (ocs) promoter.
  • CaMV 35S promoter Odell, J.T. et al., Nature 313: 810-812. 1985
  • the enhanced CaMV 35S promoter the Figwort Mosaic Virus (FMV) promoter (Richins et al.. Nucleic Acids Res. 20:
  • Useful inducible promoters include promoters induced by salicylic acid or polyacrylic acids (PR-1. Williams , S. W. et al, Biotechnology 10: 540-543, 1992), induced by application of safeners (substituted benzenesulfonamide herbicides, Hershey, H.P. and Stoner, T.D.. Plant Mol Biol. 17: 679-690, 1991), heat-shock promoters (Ou-Lee et al., Proc. Natl. Acad. Sci U.S.A. 83: 6815-6819, 1986; Ainley et al., Plant Mol.
  • tissue-specific, developmentally-regulated promoters include the ⁇ - conglycinin 7S promoter (Doyle, J.J. et al., J. Biol. Chem.
  • Plant functional promoters useful for preferential expression in seed plastids include those from plant storage protein genes and from genes involved in fatty acid biosynthesis in oilseeds.
  • promoters examples include the 5' regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res. 1 : 209-219, 1991), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl- ACP desaturase, and oleosin. Seed-specific gene regulation is discussed in EP 0 255 378. Promoter hybrids can also be constructed to enhance transcriptional activity (Comai, L. and Moran, P.M., U.S. Patent No. 5,106,739, issued April 21, 1992), or to combine desired transcriptional activity and tissue specificity. A developing seed selective promoter may be obtained from the fatty acid hydroxy lase gene of Lesquerella (P-lh) (Broun, P. and C. Somerville. Plant Physiol. 113: 933-942, 1997).
  • a variety of different methods can be employed to introduce such vectors into plant protoplasts, cells, callus tissue, leaf discs, meristems, etcetera, to generate transgenic plants, including Agrobacterium-mediated transformation, particle gun delivery, microinjection, electroporation, polyethylene glycolmediated protoplast transformation, liposome-mediated transformation, etcetera (reviewed in Potrykus, I. Ann. Rev. Plant Physiol Plant Mol. Biol. 42: 205-225, 1991).
  • transgenic plants comprising cells containing and expressing DNAs encoding polyhydroxyalkanoate biosynthesis proteins can be produced by transforming plant cells with a DNA construct as described above via any of the foregoing methods; selecting plant cells that have been transformed on a selective medium; regenerating plant cells that have been transformed to produce differentiated plants; and selecting a transformed plant which expresses the protein-encoding nucleotide sequence.
  • Particularly useful plants for polyhydroxyalkanoate production include those that produce carbon substrates, including tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as co , soybean, canola, oil seed rape, sugarbeet, sunflower, flax, peanut, sugarcane, switchgrass, and alfalfa.
  • Example 21 Plastid transformation
  • polyhydroxyalkanoate biosynthesis enzymes facilitating the increase in oil content of plants and/or herbicide resistance discussed herein can be expressed in situ in plastids by direct transformation of these organelles with appropriate recombinant expression constructs.
  • Constructs and methods for stably transforming plastids of higher plants are well known in the art (Svab, Z. et al., Plant Mol. Biol. 14(2): 197-205, 1990; Svab et al., Proc. Natl. Acad. Sci. US A. 90(3): 913-917, 1993; Staub et al., EMBO J. 12(2): 601-606, 1993; Maliga et al., U.S. Patent No.
  • Nucleic acid constructs for plastid transformation generally comprise a targeting segement comprising flanking nucleic acid sequences substantially homologous to a predetermined sequence of a plastid genome, which targeting segment enables insertion of nucleic acid coding sequences of interest into the plastid genome by homologous recombination with the predetermined sequence; a selectable marker sequence, such as a sequence encoding a form of plastid 16S ribosomal RNA that is resistant to spectinomycin or streptomycin, or that encodes a protein which inactivates spectinomycin or streptomycin (such as the aadA gene), disposed within the targeting segment, wherein the selectable marker sequence confers a selectable phenotype upon plant cells, substantially all the plastids of which have been transformed with the nucleic acid construct; and one or more nucleic acid coding sequences of interest disposed within the targeting segment relative to the selectable marker sequence so as not to interfere with conferring of the
  • plastid expression constmcts also generally include a plastid promoter region and a transcription termination region capable of terminating transcription in a plant plastid, wherein the regions are operatively linked to the nucleic acid coding sequences of interest.
  • Transformation of plastids with nucleic acid constmcts comprising a viral single subunit RNA polymerase-specific promoter specific to the RNA polymerase expressed from the nuclear expression constructs operably linked to nucleic acid coding sequences of interest permits control of the plastid expression constructs in a tissue and/or developmental specific manner in plants comprising both the nuclear polymerase construct and the plastid expression constructs.
  • Expression of the nuclear RNA polymerase coding sequence can be placed under the control of either a constitutive promoter, or a tissue- or developmental stage-specific promoter, thereby extending this control to the plastid expression construct responsive to the plastid-targeted, nuclear-encoded viral RNA polymerase.
  • the introduced nucleic acid coding sequence can be a single encoding region, or may contain a number of consecutive encoding sequences to be expressed as an engineered or synthetic operon.
  • the latter is especially attractive where, as in the present invention, it is desired to introduce multigene biochemical pathways into plastids.
  • This approach is more complex using standard nuclear transformation techniques since each gene introduced therein must be engineered as a monocistron, including an encoded transit peptide and appropriate promoter and terminator signals. Individual gene expression levels may vary widely among different cistrons, thereby possibly adversely affecting the overall biosynthetic process. This can be avoided by the chloroplast transformation approach.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.
  • Bacillus megaterium spore protein C-3 nucleotide sequence of its gene and the amino acid sequence at its spore cleavage site. Gene, 30:
  • Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes. J. Bacteriol, 178: 4794-4800.

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Abstract

Cette invention concerne un fragment d'acide nucléique à 7916 paires de bases du Bacillus megaterium. Ce fragment code pour cinq protéines, PhaP, PhaQ, PhaR, PhaB, PhaC, intervenant ou étant présumées intervenir dans la biosynthèse de matières en polyhydroxyalcanoate.
PCT/US2000/000364 1999-01-07 2000-01-07 Proteines associees a la biosynthese de polyhydroxyalcanoate et regions de codage du $i(bacilius megaterium) WO2000040730A1 (fr)

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MXPA01006958A MXPA01006958A (es) 1999-01-07 2000-01-07 Proteinas asociadas a biosintesis de polihidroxialcanoato y region de codificacion en bacillus megaterium.
JP2000592426A JP2003523170A (ja) 1999-01-07 2000-01-07 Ｂａｃｉｌｌｕｓｍｅｇａｔｅｒｉｕｍのポリヒドロキシアルカノエート生合成関連蛋白質及びそのコード領域
CA002363803A CA2363803A1 (fr) 1999-01-07 2000-01-07 Proteines associees a la biosynthese de polyhydroxyalcanoate et regions de codage du $i(bacilius megaterium)
AU24949/00A AU771433B2 (en) 1999-01-07 2000-01-07 Polyhydroxyalkanoate biosynthesis associated proteins and coding region in bacillus megaterium
EP00903161A EP1141317A1 (fr) 1999-01-07 2000-01-07 Proteines associees a la biosynthese de polyhydroxyalcanoate et regions de codage du $i(bacilius megaterium)

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CN113699168A (zh) * 2021-04-23 2021-11-26 河北牧群生物科技有限公司 蜡样芽孢杆菌hbu-ai编码合成pha的基因序列及其应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113699168A (zh) * 2021-04-23 2021-11-26 河北牧群生物科技有限公司 蜡样芽孢杆菌hbu-ai编码合成pha的基因序列及其应用

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