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WO1999035278A1 - Biosynthese de polyhydroxyalcanoates a longueur de chaine moyenne - Google Patents

Biosynthese de polyhydroxyalcanoates a longueur de chaine moyenne Download PDF

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Publication number
WO1999035278A1
WO1999035278A1 PCT/US1998/000083 US9800083W WO9935278A1 WO 1999035278 A1 WO1999035278 A1 WO 1999035278A1 US 9800083 W US9800083 W US 9800083W WO 9935278 A1 WO9935278 A1 WO 9935278A1
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WIPO (PCT)
Prior art keywords
seq
nucleic acid
acid sequence
leu
ala
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PCT/US1998/000083
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English (en)
Inventor
Yves Poirier
Volker Mittendorf
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Monsanto Company
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Filing date
Publication date
Application filed by Monsanto Company filed Critical Monsanto Company
Priority to AU59071/98A priority Critical patent/AU5907198A/en
Priority to EP98902393A priority patent/EP1044278A1/fr
Priority to PCT/US1998/000083 priority patent/WO1999035278A1/fr
Publication of WO1999035278A1 publication Critical patent/WO1999035278A1/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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/06—Fusion polypeptide containing a localisation/targetting motif containing a lysosomal/endosomal localisation signal

Definitions

  • the invention relates to the biosynthesis of polymers and more specifically to the biosynthesis of polyhydroxyalkanoate polymers in plants.
  • a transgenic plant producing peroxisome- or glyoxysome-targeted polyhydroxyalkanoate synthase resulting in the production of polyhydroxyalkanoate materials.
  • PHAs are bacterial polyesters that accumulate in a wide variety of bacteria. These polymers have properties ranging from stiff and brittle plastics to rubber-like materials, and are biodegradable. Because of these properties, PHAs are an attractive source of nonpolluting plastics and elastomers.
  • BiopolTM a random copolymer of 3 -hydroxy butyrate (3HB) and 3 -hydroxy vai erate (3HV).
  • This bioplastic is used to produce biodegradable molded material (e.g., bottles), films, coatings, and in drug release applications.
  • BiopolTM is produced via a fermentation process employing the bacterium Alcaligenes eutrophus (Byrom, Trends Biotechnol. 5: 246 (1987)).
  • the current market price is $6-7/lb, and the annual production is 1,000 tons. By best estimates, this price can be reduced only about 2-fold via fermentation (Poirier et al., Bio/Technology 13:
  • Polyhydroxyalkanoate is a family of polymers composed primarily of R-3- hydroxyalkanoic acids (Anderson, A. J. & Dawes, E. A. Microbiol Rev. 54: 450-472. (1990); Steinb ⁇ chel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan, New York), pp. 123-213. (1991); Poirier, Y. Nawrath, C. & Somerville, C. Bio/Technology 13: 143-150 (1995)).
  • Polyhydroxybutyrate is the most well characterized PHA.
  • PHB high molecular weight PHB is found as intracellular inclusions in a wide variety of bacteria (Steinb ⁇ chel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan, New York), pp. 123-213. (1991)).
  • PHB typically accumulates to 80% dry weight with inclusions being typically 0.2-1 ⁇ m in diameter.
  • Small quantity of PHB oligomers of approximately 150 monomer units are also found associated with membranes of bacteria and eukaryotes, where they form channels permeable to calcium (Reusch, R. N., Can. J. Microbiol. 41 (Suppl. 1): 50-54 (1995)).
  • PHAs have the properties of thermoplastics and elastomers. Numerous bacteria and fungi can hydrolyze PHAs to monomers and oligomers, which are metabolized as a carbon source. PHAs have, thus, attracted attention as a potential source of renewable and biodegradable plastics and elastomers.
  • PHB is a highly crystalline polymer with rather poor physical properties, being relatively stiff and brittle (de Koning, G., Can. J. Microbiol. 41 (Suppl. 1): 303-309 (1995)).
  • PHA copolymers containing monomer units ranging from 3 to 5 carbons for short-chain-length PHA (SCL-PHA), or 6 to 14 carbons for medium-chain-length PHA (MCL-PHA), are less crystalline and more flexible polymers (de Koning, G., Can. J. Microbiol. 41 (Suppl. 1): 303-309 (1995)).
  • PHB has been produced in the plant Arabidopsis thaliana expressing the A. eutrophus PHB biosynthetic enzymes (Poirier, Y., et al., Science 256: 520-523 (1992);
  • PHB was also shown to be synthesized in insect cells expressing a mutant fatty acid synthase (Williams, M. D., et al., Appl. Environ. Microbiol. 62: 2540-2546 (1996)), and in yeast expressing the A. eutrophus PHB synthase (Leaf, T. A., et al. Microbiol. 142: 1169-1180 (1996)).
  • a number of pseudomonads including Pseudomonas putida and Pseudomonas aeruginosa, accumulate MCL-PHAs when cells are grown on alkanoic acids (Anderson, A. J. & Dawes, E. A. Microbiol. Rev. 54: 450-472. (1990); Steinb ⁇ chel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan, New York), pp. 123-213. (1991); Poirier, Y. Nawrath, C. & Somerville, C. Bio/Technology 13: 143-150 (1995)).
  • the nature of the PHA produced is related to the substrate used for growth and is typically composed of monomers which are 2n carbons shorter than the substrate.
  • MCL-PHAs are synthesized by the PHA synthase from 3-hydroxyacyl-CoA intermediates generated by the ⁇ -oxidation of alkanoic acids (Huijberts, G. N. M., et al. Appl Environ. Microbiol. 58: 536-544 (1992); Huijberts, G. N. M., et al., J. Bacterial 176: 1661-1666 (1994)).
  • this patent application discloses the materials and methods for the use of a peroxisome targeted polyhydroxyalkanoate synthase protein in the biosynthesis of polyhydroxyalkanoate polymers. Localization in the peroxisomes allow for the utilization of intermediates from the lipid ⁇ -oxidation pathway. Plants expressing a P. aeruginosa polyhydroxyalkanoate synthase modified for peroxisome targeting produce PHA containing saturated and unsaturated 3-hydroxyalkanoic acids ranging from 6 to 16 carbons. Polyhydroxyalkanoate granules are found within the glyoxysomes or leaf-type peroxisomes of dark-and light-grown plants, respectively, as well as in the vacuoles.
  • the invention is directed towards materials and methods for the biosynthesis of polyhydroxyalkanoate polymers. More particularly, a fusion protein comprising a polyhydroxyalkanoate synthase protein subunit and a peroxisome targeting protein subunit renders a host cell or plant capable of producing polyhydroxyalkanoate polymer materials.
  • the invention provides a non-naturally ocurring fusion protein comprising a peroxisome targeting protein subunit and a polyhydroxyalkanoate synthase protein subunit.
  • the peroxisome targeting protein subunit and the polyhydroxyalkanoate synthase protein subunit may be any subunit suitable for participation in the invention.
  • the peroxisome targeting subunit may be an N-terminal or C-terminal subunit.
  • the N-terminal subunit is preferably PTS2.
  • the C-terminal peroxisome targeting subunit preferably comprises a tripeptide.
  • the first amino acid in the N-terminus to C- terminus direction is preferably S, A, or P.
  • the second amino acid in the N-terminus to C- terminus direction is preferably K, R, S, or H.
  • the third amino acid in the N-terminus to C- terminus direction is L, M, I, or F.
  • the C-terminal peroxisome targeting subunit comprises ARM, SRM, SKL, ARL, SRL, PSI, or PRM.
  • the peroxisome targeting subunit is preferably at least 70% identical to SEQ ID NO: 14, more preferably at least 80% identical to SEQ ID NO: 14, even more preferably at least 90% identical to SEQ ID NO: 14, and most preferably is SEQ ID NO: 14.
  • the polyhydroxyalkanoate synthase protein subunit is preferably a Pseudomonas subunit, and more preferably a Pseudomonas aeruginosa subunit.
  • the polyhydroxyalkanoate synthase protein subunit may preferably be either a PHAC1 or PHAC2 subunit.
  • the PHAC1 subunit is preferably at least 70% identical to SEQ ID NO:2, more preferably at least 80% identical to SEQ ID NO:2, even more preferably at least 90% identical to SEQ ID NO:2, and most preferably is SEQ ID NO:2.
  • the PHAC2 subunit is preferably at least 70% identical to SEQ ID NO:4, more preferably at least 80% identical to SEQ ID NO:4, even more preferably at least 90% identical to SEQ ID NO:4, and most preferably is SEQ ID NO:4.
  • the fusion protein is preferably at least 70% identical to SEQ ID NO: 18 or SEQ ID NO:20, more preferably at least 80% identical to SEQ ID NO: 18 or SEQ ID NO:20, even more preferably at least 90% identical to SEQ ID NO: 18 or SEQ ID NO:20, and most preferably is SEQ ID NO: 18 or SEQ ID NO:20.
  • the invention encompasses a nucleic acid segment encoding a non-naturally occurring fusion protein.
  • the nucleic acid segment preferably comprises a nucleic acid sequence encoding a peroxisome targeting protein subunit, and a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit.
  • the nucleic acid sequence encoding a peroxisome targeting protein subunit preferably comprises at least a 6 contiguous nucleic acid sequence from SEQ ID NO: 13.
  • the length of the contiguous nucleic acid sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etcetera, 50, 51, 52, etcetera, 100, 101, 102, etcetera, up to and including the entire length of SEQ ID NO: 13.
  • the nucleic acid sequence encoding a peroxisome targeting protein subunit is preferably at least 70% identical to SEQ ID NO: 13, more preferably at least 80% identical to SEQ ID NO: 13, even more preferably at least 90% identical to SEQ ID NO: 13, and most preferably is SEQ ID NO: 13.
  • the nucleic acid sequence encoding a peroxisome targeting protein subunit preferably hybridizes to SEQ ID NO: 13.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit preferably comprises at least a 6 contiguous nucleic acid sequence from SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16.
  • the length of the contiguous nucleic acid sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etcetera, 50, 51, 52, etcetera, 100, 101, 102, etcetera, up to and including the entire length of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit is preferably at least 70% identical to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16, more preferably at least 80% identical to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16, even more preferably at least 90% identical to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16, further preferably is SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:15, or SEQ ID NO:16, and most preferably is SEQ ID NO: 15 or SEQ ID NO: 16.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit preferably hybridizes to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16.
  • the encoded peroxisome targeting protein subunit may be an N- terminal or C-terminal peroxisome targeting protein subunit.
  • the encoded N-terminal peroxisome targeting subunit is preferably PTS-2.
  • the encoded C-terminal peroxisome targeting protein subunit preferably comprises a tripeptide.
  • the tripeptide preferably comprises a first amino acid in the N-terminus to C-terminus direction being S, A, or P; a second amino acid in the N-terminus to C-terminus direction being K, R, S, or H; and a third amino acid in the N-terminus to C-terminus direction being L, M, I, or F.
  • the encoded tripeptide preferably is ARM, SRM, SKL, ARL, SRL, PSI, or PRM.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit preferably encodes at least a 5 contiguous amino acid sequence from SEQ ID NO:2 or SEQ ID NO:4.
  • the length of the contiguous nucleic acid sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etcetera, 50, 51, 52, etcetera, 100, 101, 102, etcetera, up to and including the entire length of SEQ ID NO:2 or SEQ ID NO:4.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit preferably encodes an amino acid sequence at least 70% identical to SEQ ID NO:2 or SEQ ID NO:4, more preferably at least 80% identical to SEQ ID NO:2 or SEQ ID NO:4, even more preferably at least 90% identical to SEQ ID NO:2 or SEQ ID NO:4, and most preferably is SEQ ID NO:2 or SEQ ID NO:4.
  • the invention discloses a recombinant vector comprising in the 5' to 3' direction a) a promoter that directs transcription of a structural nucleic acid sequence encoding a non-naturally occurring fusion protein, wherein the fusion protein comprises a peroxisome targeting protein subunit and a polyhydroxyalkanoate synthase protein subunit, b) a structural nucleic acid sequence encoding a non-naturally occurring fusion protein, wherein the fusion protein comprises a peroxisome targeting protein subunit and a polyhydroxyalkanoate synthase protein subunit, and c) a 3' transcription terminator.
  • the recombinant vector may further comprise a 3' polyadenylation signal sequence that directs the addition of polyadenylate nucleotides to the 3' end of R A transcribed from the structural nucleic acid coding sequence.
  • the recombinant vector may further comprise a selectable marker.
  • the selectable marker may generally be any selectable marker suitable for the intended host cell or plant, and preferably is a kanamycin resistance marker, a hygromycin resistance marker, or a herbicide resistance marker.
  • the promoter may be constitutive, inducible, tissue specific, or combinations thereof.
  • the constitutive promoter may generally any constitutive promoter suitable for the intended host cell or plant, and preferably is CaMV35S, enhanced CaMV35S, FMV, mas, nos, or ocs.
  • the inducible promoter may generally be any inducible promoter suitable for the intended host cell or plant, and preferably is tac, salicylic acid induced, polyacrylic acid induced, safener induced, heat shock promoter, nitrate induced, hormone induced, or light induced.
  • the tissue specific promoter may generally be any tissue specific promoter suitable for the intended host cell or plant, and preferably is the ⁇ -conglycinin 7S promoter, napin promoter, phaseolin promoter, zein promoter, soybean trypsin inhibitor promoter, ACP promoter, stearoyl-ACP desaturase promoter, or oleosin promoter.
  • the nucleic acid sequence encoding a peroxisome targeting protein subunit preferably comprises at least a 6 contiguous nucleic acid sequence from SEQ ID NO: 13.
  • the length of the contiguous nucleic acid sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etcetera, 50, 51, 52, etcetera, 100, 101, 102, etcetera, up to and including the entire length of SEQ ID NO: 13.
  • the nucleic acid sequence encoding a peroxisome targeting protein subunit is preferably at least 70% identical to SEQ ID NO: 13, more preferably at least 80% identical to SEQ ID NO: 13, even more preferably at least 90% identical to SEQ ID NO: 13, and most preferably is SEQ ID NO: 13.
  • the nucleic acid sequence encoding a peroxisome targeting protein subunit preferably hybridizes to SEQ ID NO: 13.
  • the encoded peroxisome targeting protein subunit may be an N-terminal or C-terminal peroxisome targeting protein subunit.
  • the encoded N-terminal peroxisome targeting subunit is preferably PTS-2.
  • the encoded C- terminal peroxisome targeting protein subunit preferably comprises a tripeptide.
  • the tripeptide preferably comprises a first amino acid in the N-terminus to C-terminus direction being S, A, or P; a second amino acid in the N-terminus to C-terminus direction being K, R, S, or H; and a third amino acid in the N-terminus to C-terminus direction being L, M, I, or F.
  • the encoded tripeptide preferably is ARM, SRM, SKL, ARL, SRL, PSI, or PRM.
  • the encoded polyhydroxyalkanoate synthase protein subunit is preferably a Pseudomonas subunit, and more preferably is a Pseudomonas aeruginosa subunit.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit preferably comprises at least a 6 contiguous nucleic acid sequence from SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:15, or SEQ ID NO:16.
  • the length of the contiguous nucleic acid sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etcetera, 50, 51, 52, etcetera, 100, 101, 102, etcetera, up to and including the entire length of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit is preferably at least 70% identical to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16, more preferably at least 80% identical to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16, even more preferably at least 90% identical to SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16, further preferably is SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16, and most preferably is SEQ ID NO: 15 or SEQ ID NO: 16.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit preferably hybridizes to SEQ ID NO: l , SEQ ID NO:3, SEQ ID NO: 15, or SEQ ID NO: 16.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit preferably encodes at least a 5 contiguous amino acid sequence from SEQ ID NO:2 or SEQ ID NO:4.
  • the length of the contiguous nucleic acid sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etcetera, 50, 51, 52, etcetera, 100, 101, 102, etcetera, up to and including the entire length of SEQ ID NO:2 or SEQ ID NO:4.
  • the nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit preferably encodes an amino acid sequence at least 70% identical to SEQ ID NO:2 or SEQ ID NO:4, more preferably at least 80% identical to SEQ ID NO:2 or SEQ ID NO:4, even more preferably at least 90% identical to SEQ ID NO:2 or SEQ ID NO:4, and most preferably is SEQ ID NO:2 or SEQ ID NO:4.
  • the structural nucleic acid sequence preferably comprises SEQ ID NO: 17 or SEQ ID NO: 19, and preferably encodes SEQ ID NO: 18 or SEQ ID NO:20.
  • the invention encompasses a recombinant host cell comprising a nucleic acid segment encoding a non-naturally occurring fusion protein, wherein the nucleic acid segment comprises a nucleic acid sequence encoding a peroxisome targeting protein subunit and a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit.
  • the recombinant host cell may generally be any type of host cell, and preferably is a fungal or plant host cell.
  • the fungal cell is generally any type of fungal cell, and preferably a Schizosaccharomyces pombe, Streptomyces rimofaciens, Fusarium, Aspergillus niger, or Saccharomyces cerevisiae cell.
  • the plant cell is generally any type of plant cell, and preferably an alfalfa, banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat cell.
  • the recombinant host cell may further comprise a nucleic acid segment encoding an acyl-ACP thioesterase, a fatty acyl hydroxylase, a yeast multifunctional protein (MFP), or an hydroxyacyl-CoA epimerase.
  • a nucleic acid segment encoding an acyl-ACP thioesterase, a fatty acyl hydroxylase, a yeast multifunctional protein (MFP), or an hydroxyacyl-CoA epimerase.
  • a further alternative embodiment describes a genetically transformed plant cell comprising in the 5' to 3' direction: a) a promoter to direct transcription of a structural nucleic acid sequence encoding a non-naturally occurring fusion protein, wherein the structural nucleic acid sequence comprises: i) a nucleic acid sequence encoding a peroxisome targeting protein subunit; and ii) a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit; b) a structural nucleic acid sequence encoding a non-naturally occurring fusion protein, wherein the structural nucleic acid sequence comprises: i) a nucleic acid sequence encoding a peroxisome targeting protein subunit; and ii) a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit; c) a 3' transcription terminator sequence; and d) a 3' polyadenylation signal sequence that directs the addition of polyadenylate nucleotides
  • the plant cell is generally any type of plant cell, and preferably an alfalfa, banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat cell.
  • the plant cell may further comprise a nucleic acid segment encoding an acyl-ACP thioesterase, a fatty acyl hydroxylase, a yeast multifunctional protein (MFP), or an hydroxyacyl-CoA epimerase.
  • An additional embodiment describes a genetically transformed plant comprising in the 5' to 3' direction: a) a promoter to direct transcription of a structural nucleic acid sequence encoding a non-naturally occurring fusion protein, wherein the structural nucleic acid sequence comprises: i) a nucleic acid sequence encoding a peroxisome targeting protein subunit; and ii) a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit; b) a structural nucleic acid sequence encoding a non-naturally occurring fusion protein, wherein the structural nucleic acid sequence comprises: i) a nucleic acid sequence encoding a peroxisome targeting protein subunit; and ii) a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit; c) a 3' transcription terminator sequence; and d) a 3' polyadenylation signal sequence that directs the addition of polyadenylate nucleotides to the
  • the plant may generally be any type of plant, and preferably an alfalfa, banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat plant.
  • the promoter may be constitutive, inducible, tissue specific, or combinations thereof.
  • the constitutive promoter may generally any constitutive promoter suitable for the intended plant, and preferably is CaMV35S, enhanced CaMV35S, FMV, mas, nos, or ocs.
  • the inducible promoter may generally be any inducible promoter suitable for the intended plant, and preferably is tac, salicylic acid induced, polyacrylic acid induced, safener induced, heat shock promoter, nitrate induced, hormone induced, or light induced.
  • the tissue specific promoter is generally any tissue specific promoter, and preferably is the ⁇ -conglycinin 7S promoter, napin promoter, phaseolin promoter, zein promoter, soybean trypsin inhibitor promoter, ACP promoter, stearoyl-ACP desaturase promoter, or oleosin promoter.
  • the plant may further comprise a nucleic acid segment encoding an acyl-ACP thioesterase, a fatty acyl hydroxylase, a yeast multifunctional protein (MFP), or an hydroxyacyl-CoA epimerase.
  • the invention describes a method for preparing host cells useful to produce a non- naturally occurring fusion protein comprising the steps of: a) selecting a host cell b) transforming the selected host cell with a recombinant vector having a structural nucleic acid sequence encoding a non-naturally occurring fusion protein, wherein the structural nucleic acid sequence comprises: i) a nucleic acid sequence encoding a peroxisome targeting protein subunit; and ii) a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit; and c) obtaining transformed host cells.
  • the vector may further comprise a selectable marker.
  • the selectable marker may generally be any selectable marker suitable for use in the intended host cell, and more preferably for plants is a kanamycin resistance marker, a hygromycin resistance marker, or a herbicide resistance marker.
  • the host cell may generally be any type of cell, and preferably is a fungal or plant cell.
  • the fungal cell may generally be any type of fungal cell, and more preferably is a Schizosaccharomyces pombe, Streptomyces rimofaciens, Fusarium, Aspergillus niger, or Saccharomyces cerevisiae cell.
  • the plant cell may generally be any type of plant cell, and more preferably is an alfalfa, banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat cell.
  • an alfalfa banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat cell.
  • the invention further describes a method of preparing a transformed plant useful to produce a non-naturally occurring fusion protein comprising the steps of: a) selecting a host plant cell b) transforming the selected host cell with a recombinant vector having a structural nucleic acid sequence encoding a non-naturally occurring fusion protein, wherein the structural nucleic acid sequence comprises: i) a nucleic acid sequence encoding a peroxisome targeting protein subunit; and ii) a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein subunit; c) obtaining transformed host plant cells; and d) regenerating the transformed host plant cells.
  • the vector may further comprise a selectable marker.
  • the selectable marker may generally be any selectable marker suitable for use in the intended host cell, and more preferably is a kanamycin resistance marker, a hygromycin resistance marker, or a herbicide resistance marker.
  • the host plant cell may generally be any type of plant cell, and more preferably is an alfalfa, banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat cell.
  • the invention also encompasses the plant made by the above described methods.
  • a preferred embodiment is a method for the preparation of a polyhydroxyalkanoate, comprising the steps of: a) obtaining a cell capable of producing a non-naturally occurring fusion protein, wherein the fusion protein comprises: i) a peroxisome targeting protein subunit; and ii) a polyhydroxyalkanoate synthase protein subunit; b) establishing a culture of the cell; and c) culturing the cell under conditions suitable for the production of the polyester.
  • the method may further comprise isolating the polyhydroxyalkanoate from the cultured cell.
  • the culture may further comprise fatty acids, and more preferably natural fatty acids, non-natural or synthetic fatty acids, or mixtures thereof.
  • the cell may generally be any type of cell, and preferably is a fungal or plant cell.
  • the fungal cell may generally be any type of fungal cell, and more preferably is a Schizosaccharomyces pombe, Streptomyces rimofaciens, Fusarium, Aspergillus niger, or Saccharomyces cerevisiae cell.
  • the plant cell may generally be any type of plant cell, and more preferably is an alfalfa, banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat cell.
  • an alfalfa banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat cell.
  • the polyhydroxyalkanoate isolated from the cell may generally be any type of polyhydroxyalkanoate, and preferably comprises 3-hydroxyhexanoic acid (H:6), 3- hydroxyoctanoic acid (H:8), 3 -hydroxy decanoic acid (H:10), 3 -hydroxy dodecanoic acid (H:12), 3-hydroxytetradecanoic acid (H:14), 3-hydroxyhexadecanoic acid (H:16), 3- hydroxyheptanoic acid (H:7), 3 -hydroxy nonanoic acid (H9), 3-hydroxyundecanoic acid (H:l l), 3-hydroxytridecanoic acid (H:13), 3-hydroxyhexadecatrienoic acid (H16:3), 3- hydroxyhexadecadienoic acid (H16:2), 3 -hydroxy hexadecenoic acid (H16:l), 3- hydroxytetradecatrienoic acid (H14:3), 3-hydroxytetradecadienoic acid (H14:
  • the invention presents a method for the preparation of a polyhydroxyalkanoate, comprising the steps of: a) obtaining a plant capable of producing a non-naturally occurring fusion protein, wherein the fusion protein comprises: i) a peroxisome targeting protein subunit; and ii) a polyhydroxyalkanoate synthase protein subunit; and c) growing the plant under conditions suitable for the production of the polyhydroxyalkanoate.
  • the method may further comprise the step of isolating the polyhydroxyalkanoate from the plant.
  • the method may further comprise supplementing the plant with natural fatty acids, non-natural fatty acids, or mixtures thereof.
  • the plant may generally be any type of plant, and preferably is an alfalfa, banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat plant.
  • alfalfa banana, barley, bean, cabbage, canola/oilseed rape, carrot, castorbean, celery, clover, coconut, corn, cotton, cucumber, linseed, melon, olive, palm, parsnip, pea, peanut, pepper, potato, potato, radish, rapeseed, rice, soybean, spinach, sunflower, tobacco, tomato, or wheat plant.
  • the polyhydroxyalkanoate isolated from the plant may generally be any type of polyhydroxyalkanoate, and preferably comprises 3-hydroxyhexanoic acid (H:6), 3- hydroxyoctanoic acid (H:8), 3 -hydroxy decanoic acid (H:10), 3-hydroxydodecanoic acid (H:12), 3-hydroxytetradecanoic acid (H:14), 3-hydroxyhexadecanoic acid (H:16), 3- hydroxyheptanoic acid (H:7), 3-hydroxynonanoic acid (H9), 3-hydroxyundecanoic acid (H:l l), 3-hydroxytridecanoic acid (H:13), 3-hydroxyhexadecatrienoic acid (HI 6:3), 3- hydroxyhexadecadienoic acid (HI 6:2), 3-hydroxyhexadecenoic acid (HI 6:1), 3- hydroxytetradecatrienoic acid (H14:3), 3-hydroxytetradecadienoic acid (H14:2), 3- hydroxy
  • the invention further encompasses plants containing polyhydroxyalkanoates, wherein the polyhydroxyalkanoate comprises 3-hydroxyhexanoic acid (H:6), 3- hydroxyoctanoic acid (H:8), 3 -hydroxy decanoic acid (H:10), 3 -hydroxy dodecanoic acid (H:12), 3-hydroxytetradecanoic acid (H:14), 3-hydroxyhexadecanoic acid (H:16), 3- hydroxyheptanoic acid (H:7), 3-hydroxynonanoic acid (H9), 3-hydroxyundecanoic acid (H:l l), 3-hydroxytridecanoic acid (H:13), 3 -hydroxy hexadecatrienoic acid (H16:3), 3- hydroxyhexadecadienoic acid (H16:2), 3-hydroxyhexadecenoic acid (H16:l), 3- hydroxytetradecatrienoic acid (H14:3), 3-hydroxytetradecadienoic acid (H14:2), 3-
  • the invention describes polyhydroxyalkanoates comprising 3 -hydroxy hexadecatrienoic acid (HI 6:3), 3 -hydroxy hexadecadienoic acid (H16:2), 3-hydroxytetradecatrienoic acid (H14:3), or 3-hydroxydodecadienoic acid (H12:2) monomers.
  • FIG. 1 GC-MS analysis of PHA in transgenic plants.
  • Trans-esterified chloroform extracts from phaC 1 -transformed line 3.3 (1 A. IB) and vector-transformed line 21 (IC, ID) were analyzed.
  • IC vector-transformed line 21
  • panels 1A and IC the total ion chromatogram is presented, while on panel IB and ID, only ions with a mass-to-charge ratio of 103 are shown.
  • SEQ ID NO: 1 Wild type PHA synthase C 1 nucleic acid sequence.
  • SEQ ID NO:2 Wild type PHA synthase CI protein sequence.
  • SEQ ID NO:3 Wild type PHA synthase C2 nucleic acid sequence
  • SEQ ID NO:4 Wild type PHA synthase C2 protein sequence.
  • SEQ ID NO:5 Forward PCR primer for PHA synthase CI fusion sequence.
  • SEQ ID NO:6 Reverse PCR primer for PHA synthase CI fusion sequence.
  • SEQ ID NO:7 Forward PCR primer for PHA synthase C2 fusion sequence.
  • SEQ ID NO: 8 Reverse PCR primer for PHA synthase C2 fusion sequence.
  • SEQ ID NO:9 Wild type isocitrate lyase nucleic acid sequence.
  • SEQ ID NO: 10 Wild type isocitrate lyase protein sequence.
  • SEQ ID NO: 11 Forward PCR primer for isocitrate lyase fusion sequence.
  • SEQ ID NO: 12 Reverse PCR primer for isocitrate lyase fusion sequence.
  • SEQ ID NO: 13 Nucleic acid sequence encoding the isocitrate lyase peroxisome targeting protein subunit.
  • SEQ ID NO: 14 Isocitrate lyase peroxisome targeting protein subunit.
  • SEQ ID NO: 15 PHA synthase CI nucleic acid sequence with plant preferred codon.
  • SEQ ID NO: 16 PHA synthase C2 nucleic acid sequence with plant preferred codon.
  • SEQ ID NO: 17 Nucleic acid sequence encoding PHA synthase CI and isocitrate lyase fusion protein.
  • SEQ ID NO: 18 PHA synthase CI and isocitrate lyase fusion protein.
  • SEQ ID NO: 19 Nucleic acid sequence encoding PHA synthase C2 and isocitrate lyase fusion protein.
  • SEQ ID NO:20 PHA synthase C2 and isocitrate lyase fusion protein.
  • SEQ ID NO:21 PCR amplified nucleic acid sequence encoding wild type Candida albicans MFP.
  • SEQ ID NO:22 Wild type Candida albicans MFP protein.
  • Candida albicans MFP SEQ ID NO:24 Candida albicans MFP protein with SKL substitution for AKI.
  • Candida albicans MFP lacking AKI sequence SEQ ID NO:26 Candida albicans MFP protein lacking AKI sequence.
  • Acyl-ACP thioesterase refers to proteins which catalyze the hydrolysis of acyl- ACP thioesters.
  • 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.
  • Fatty acyl hydroxylase refers to proteins which catalyze the conversion of fatty acids to hydroxylated fatty acids.
  • gene refers to chromosomal DNA, plasmid DNA, cDNA, synthetic
  • DNA or other DNA that encodes a peptide, polypeptide, protein, or RNA molecule, and regions flanking the coding sequence involved in the regulation of expression.
  • the term "genome” as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding DNAs 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. DNAs of the present invention introduced into plant cells can therefore be either chromosomally-integrated or organelle-localized.
  • Glyoxysome and “peroxisome” refer to the same organelle in a plant.
  • Glyoxysome refers to a type of peroxisome found in germinating seedlings, senescing tissues, or in dark-grown tissues. Glyoxysomes and peroxisomes contain enzymes responsible for the conversion of lipids to carbohydrates.
  • 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 CLUSTALW vl .6 (Thompson, et al. Nucl. Acids Res., 22: 4673-4680 (1994)).
  • 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.
  • 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 a amino group to the middle of the chain.
  • Nucleic acid refers to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
  • 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.
  • plastid refers to the class of plant cell organelles that includes amyloplasts, chloroplasts, chromoplasts, elaioplasts, eoplasts, etioplasts, leucoplasts, and proplastids. These organelles are self-replicating, and contain what is commonly referred to as the "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
  • 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 Hgation 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.
  • Protein subunit refers to a protein sequence that is part of a fusion protein.
  • Examples are ⁇ -galactosidase, FLAG, green fluorescent protein, and in the instant invention, polyhydroxyalkanoate synthase, and a peroxisome or glyoxysome targetting peptide.
  • PTS2 refers to an N-terminal protein subunit having the sequence (R/K)(L/Q/I)XXXXX(H/Q)L, wherein X is any amino acid.
  • 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 (e.g., a vector, recombinant nucleic acid molecule) into a cell or protoplast in which that exogenous nucleic acid is incorporated into a chromosome or is capable of autonomous replication.
  • exogenous nucleic acid sequence e.g., a vector, recombinant nucleic acid molecule
  • a “transformed cell” or “transgenic cell” is a cell whose DNA 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 DNA 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.
  • the phaCl and phaC2 genes were obtained from Steinb ⁇ chel (Timm, A. and Steinb ⁇ chel, A., Eur. J. Biochem. 209: 14-30 (1992), GenBank Accession Number X66592). PCR was used to amplify the genes and to modify their 5'- and 3'-termini as follows: At the 5 '-end the codons encoding the serine-2 and the arginine-2 residue of phaCl and phaC2, respectively, were modified to conform more closely with the general codon preferences of A. thaliana (Meyerowitz, E. M. in Methods in Arabidopsis research , eds. Koncz, C, Chua, N.-H. & Schell, J.
  • a PCR product encoding the ICL targeting sequence was cloned into the vector pART7 (Gleaves, A.P., Plant Mol. Biol. 20: 1202-1207 (1992), GenBank Accession Number X69707).
  • the PCR products containing the phaCl or phaC '2 genes were cloned 5 '-upstream of the ICL sequence to produce a contiguous open reading frame encoding the targeted fusion proteins.
  • the 5'- and 3'-ends of the genes in the resulting plasmids pART7_phaCl_ICL and pART7_phaC2_ICL were sequenced to verify the modifications.
  • the PHA accumulation-deficient mutant Pseudomonas putida KT2440 NK2:3 was obtained from Steinb ⁇ chel for complementation studies to verify the enzyme activities of the modified PHA synthases CI and C2.
  • the phaCI ICL and phaC2_ICL genes were cloned into the broad-host range plasmid pVLT35 behind the IPTG-inducible tac -promoter (Lorenzo, V. et al., Gene 123: 17-24 (1993)) and electroporated into the P. putida mutant. Streptomycin-resistant transformants were subcultured onto minimal medium containing either octanoate or gluconate as sole carbon source.
  • the Nile Blue A fluorescence stain (Page, W. J. and C. J. Tenove, Biotechnology Techniques 10: 215-220 (1996)) was used to visualize PHA accumulation.
  • IPTG induction PHA accumulation was observed with pVLT35_phaCl_ICL and pVLT35_phaC2_ICL, but not with pVLT35 alone, thus indicating that the modified genes were still active.
  • the Notl-cassettes of plasmids pART7_phaCl_ICL and pART7_phaC2_ICL containing the modified genes flanked by the Cauliflower mosaic virus 35S promoter (CaMV35S) and the octapine synthase (ocs) 3 '-terminator were cloned into the plant binary vector pART27 to obtain pART27_phaCl_ICL and pART27_phaC2_ICL.
  • These plasmids were transformed into A. thaliana ecotype Columbia by Agrobacterium GV3101 -mediated transfer utilizing an in planta vacuum-infiltration method (Bechtold, N. et al., C.R. Acad. Sci.
  • Transgenic Tl plants were selected for antibiotic resistance during germination of the seeds of infiltrated plants on plant growth medium containing mineral salts, sucrose and kanamycin. Negative control plants containing only the insert-less T-DNA of the vector pART27 were obtained in the same way.
  • Transgenic PHAC1 plants (Tl) expressing high amounts of PHA synthase CI were selected by Western analysis with an antiserum against the PHA synthase CI, which was obtained from Steinb ⁇ chel's laboratory. Unfortunately no antibodies against PHA synthase C2 were found to be suitable, so a different screening strategy was used, see below.
  • Six independent lines expressing varying quantities of PHA synthase CI were obtained from 12 originally infiltrated plants, which had been harvested individually (another 19 have not yet been investigated). Initially some problems with the western analysis were encountered, one of which was the precipitation of the PHA synthase in plant protein extracts upon freezing.
  • H6 which was from Beat Keller
  • D-3-hydroxy-hexanoic acid (3- OH-caproic acid, H6 monomer
  • DL-3-hydroxy-octanoic acid (3-OH-caprylic acid.
  • H8 monomer DL-3-hydroxy-capric acid (H10 monomer)
  • DL-3-hydroxy-lauric acid HI 2 monomer
  • DL-3-hydroxy-myristic acid HI 4 monomer
  • the transgenic plants expressing the PHA synthase CI showed a significant increase in the size of the peaks corresponding to the H6-H14 monomers compared to the negative control plants.
  • One novel peak was found only in PHAC1 plants and never in the negative controls.
  • GC-MS was used to confirm that the peaks observed in both the PHAC1 plants and the negative controls were really identical to the standards and the novel peak was determined as being due to 3-hydroxy-octenoyl-methyl-ester containing a single unsaturated bond (H8:l monomer).
  • the H6 monomer would then have to originate from the fatty acids C18:l, ⁇ 14-cis or C16:l, ⁇ 12-cis, while the H14 monomer would have to originate from C18:l, ⁇ 8-cis, or C16:l, ⁇ 4-cis or C14:l, ⁇ 2-cis, etcetera.
  • C18:l, ⁇ 14-cis or C16:l, ⁇ 12-cis while the H14 monomer would have to originate from C18:l, ⁇ 8-cis, or C16:l, ⁇ 4-cis or C14:l, ⁇ 2-cis, etcetera.
  • fatty acids C18:l, ⁇ 14-cis or C16:l, ⁇ 12-cis
  • H14 monomer would have to originate from C18:l, ⁇ 8-cis, or C16:l, ⁇ 4-cis or C14:l, ⁇ 2-cis, etcetera.
  • fatty acids C
  • negative control plants both A. thaliana wild type and pART27 transgenic plants
  • concentrations present in the negative controls were at least 1000 times smaller than in the positive plants, close to the detection limit of the methods at our availability. This was done by utilizing the GC-MS in the SIM mode (selected ion monitoring; ion 103 is characteristic for all of these 3-OH-fatty acid methyl esters) for which the detection limit was found to be approximately 4 pg/ ⁇ L of the various standards.
  • These compounds in the negative controls might also be intermediates of ⁇ -oxidation, i.e.
  • PHAC2 plants were screened directly for PHA production by analysis of dry leaves of T2 plants. Almost all of the T2 plants derived from 13 independently transformed plants were found to produce PHA in varying quantities, as judged by the presence of the novel peak due to the C8:l monomer and also the peaks of the other PHA monomers. The highest producing plants were analyzed further and homozygous T3 plants were obtained. Two homozygous single-locus T3 lines were selected, PHAC2#19.5 and PHAC2#8.6. In comparison to PHAC1#3.3 plants, these PHAC2 plants produced slightly smaller quantities of PHA in seedlings grown on plates containing MS salts, kanamycin and sucrose. The monomer composition of the respective transgenic plants was however identical. For that reason most of the further studies were only done with line PHACl #3.3.
  • T3 seedlings of lines PHAC1#3.3 and pART27#21 (negative control) were grown on plates containing MS salts, kanamycin and sucrose. Seedlings were grown for 7 days under continuous light or in the dark after one day of illumination, the latter was done to obtain etiolated seedlings in which glyoxysomes are more abundant. The seedlings were fixed and sent together with some anti-PHA synthase CI antiserum to Prof. Leech's laboratory at the University of York, where the immunolocalization was performed.
  • Glycolate oxidase was used as marker enzyme for peroxisomes of seedlings grown under light, while rubisco was used as chloroplastic marker. Antibodies against these two marker enzymes clearly identified the respective organelles in both PHACl seedlings and in the pART27 negative controls. Glycolate oxidase was found to be located in the organelles, i.e. the peroxisomes, containing PHA granules.
  • ICL isocitrate lyase
  • Line PHACl #3.3 was used to investigated if the total yield of PHA could be increased or if PHAs containing other monomers than the "native" PHA could be synthesized in PHACl transgenic plants.
  • seeds were sterilized and germinated in liquid medium containing mineral salts and 2% (w/v) sucrose supplemented with fatty acids or other compounds known to be degraded by ⁇ -oxidation.
  • experiment #1 the seedlings were grown for 3 days in the light before the substrates were added and the plant were moved into the dark. The material was harvested after 8 days and derivatized samples were analyzed by gas chromatography.
  • TWEEN-20 Sigma; 50% palmitic acid (C16) esterified with polyoxyethylenesorbitol, the remainder is made up by lauric acid (C12) and myristic acid (C14) also esterified)
  • TWEEN is a registered trademark of ICI Americas, Inc., Wilmington, DE
  • TWEEN- 20 The most pronounced effect of TWEEN- 20 on the monomer composition was the decrease in the content of the H8:l monomer from about 30% in native PHA to about 1%, which was most likely due to the lack of unsaturated fatty acid derivatives in the TWEEN-20.
  • the relative distribution of the other monomers could be explained by the step-by-step ⁇ -oxidation of the C16, C14 and C12 components in TWEEN-20.
  • a negative effect on seedling growth due to TWEEN-20 was observed, but it was small considering its high concentration (5% v/v) in the medium.
  • TWEEN-60 (Sigma; 50% stearic acid (C18) and some palmitic and myristic acid; all esterified to polyoxyethylenesorbitol) and TWEEN-80 (Sigma; 50% oleic acid (C18:l), esterified to polyoxyethylenesorbitol) had less impact on the PHA yield, the monomer composition and the seedling growth than TWEEN-20.
  • the relatively high level of the H8:l monomer might be due to a higher contamination of TWEEN-60 and -80 with unsaturated fatty acids like ⁇ -linolenic acid, see above.
  • the transesterified plant material (of specified weight) was in a volume of 1 mL chloroform, of which 1 ⁇ L was analyzed by GC.
  • 8-methyl-nonanoic acid (8M-C9) resulted in the incorporation of a whole range of novel monomers.
  • the identity of all these novel monomers was established by GC-MS. All of them had an uneven number of carbon atoms in their acyl chains and could be directly traced to the original fatty acid supplied in the medium or intermediates of its degradation by ⁇ -oxidation.
  • transgenic PHACl plants were found to contain a polymer having HI 3-, HI 1-, H9- and H7-3-hydroxy-alkanoic acid monomers.
  • D-3-hydroxy-acyl-CoA can be formed by the action of the enoyl-CoA hydratase (MFP) from 2-cis-enoyl-CoA (cis-unsaturated bond in even-numbered position), but the D-3-hydroxy-acyl-CoA cannot be utilized by the 3-hydroxy-acyl-CoA dehydrogenase (MFP), which can only act on the L-3-hydroxy-acyl-CoA.
  • MFP enoyl-CoA hydratase
  • MFP 3-hydroxy-acyl-CoA dehydrogenase
  • a dehydratase also called D-3-hydroxyacyl-CoA hydrolyase or D-specific 2- trans-enoyl-CoA hydratase II, see Engeland, K. and Kindl, H., EMr. J. Biochem. 200: 171- 178 (1991) converts the D-3-hydroxy-acyl-CoA to 2-trans-enoyl-CoA, which can then be reconverted to L-3-hydroxy-acyl-CoA by the enoyl-CoA hydratase I.
  • a 2,4-dienoyl- CoA reductase reduces the 2-trans-4-cis-acyl-CoA ⁇ -oxidation intermediate to the 3-cis- enoyl-CoA, which in turn will require the activity of an isomerase to form the 2-trans-enoyl- CoA ⁇ -oxidation intermediate.
  • the first two options would result in the generation of D-3- hydroxy-acyl-CoA intermediates which would be directly available to the PHA synthase.
  • a 8M-H9 and 6M-H7 refer to 8-methyl-3-D-hydroxy-nonanoic acid and 6-methyl-3-D- hydroxy-heptanoic acid, respectively.
  • 4-OH-H10 refers to D-4-hydroxy-decanoate.
  • the quantity of 4-OH-H10 was estimated by comparing peak sizes with H6 on a GC-MS chromatogram.
  • TWEEN-20-derived PHA approximately 16000 seeds (313 mg dry seeds) were germinated in 900 mL l/2xMS + 1% sucrose medium for 7 days under continuous illumination on a shaker, the medium was replaced with l/2xMS + 2% sucrose containing 5% TWEEN-20 and the seedlings were grown for another 9 days in the light.
  • the plant material was harvested, washed extensively with water to remove residual TWEEN-20, frozen and lyophilized.
  • the dry material was ground with a mortar and pestle, weighed, and lipids were extracted by a six-hour Soxhlet-extraction with methanol.
  • the methanol- insoluble PHA was extracted for 24 hours in the same manner with chloroform.
  • the chloroform extract was concentrated under reduced pressure and the PHA was precipitated by the addition of 10 volumes of cold methanol. This methanol precipitation was performed twice to ensure a high purity of the PHA. 27 mg of PHA was thus obtained from 5.35 g lyophilized and powdered seedling material, which related to 0.50% weight/dry weight.
  • the PHA was trans-esterified and analyzed by GC. It was found that 58% of the PHA present in the methanol-extracted plant powder was extracted by the chloroform. It has been established in previous experiments that this remaining PHA was recalcitrant to extraction. The chromatogram showed that the extracted PHA was adequately pure with the peaks of the six identified monomers constituting 93% of the total integrated area. The ratio of the integrated areas between the different monomers was very similar to the result shown in Table 1 for the sample containing TWEEN-20 and grown under light, see Table 4.
  • TWEEN-20 derived PHA produced by the transgenic plants is in the form of a high-M r polymer (about 200-250 monomers), although the molecular weight is only 20-25% of the bacterial polymers (about 1000 monomers).
  • This shorter polymer length can be explained by an overabundance of PHA synthase relative to its substrate concentration and similar results have also been obtained in in vitro polymerization assays with purified PHB synthase (Jun Sim, S. et al., Nature Biotechnology 15: 63-67 (1997)).
  • NMR analysis of the plant and bacterial PHAs revealed, that the TWEEN-20 derived plant PHA had the same structure as the bacterial PHA.
  • the NMR spectrum of the unmodified plant PHA showed the peaks characteristic for the PHA polymer backbone, as well as several other peaks which have not been properly assigned or identified at this stage, but which could be due to various unsaturated bonds in the side chains of the polymer.
  • EXAMPLE 10 The multifunctional protein (MFP) from the yeast Candida tropicalis
  • ⁇ -oxidation has been shown to proceed via the L- isomer of the 3-hydroxy-acyl-CoA intermediates and any D-isomers (which are predicted to arise in the degradation of fatty acids containing cis-unsaturated bonds at even-numbered carbons) have to be converted to the L-form in order to be oxidized further by the dehydrogenase activity of the multifunctional protein (MFP).
  • MFP multifunctional protein
  • yeast the ⁇ -oxidation was reported to proceed via the D-isomer (Nuttley, W. M. et al., Gene 69: 171-180 (1988); Hiltunen, J. K. et al., J. Biol. Chem.
  • the yeast multifunctional protein (MFP) was shown to contain enoyl-CoA hydratase II and D-3-hydroxyacyl-CoA dehydrogenase activities, which together converted trans-2-enoyl-CoA via D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA, i.e. the D- isomer was directly utilized by the dehydrogenase without prior conversion to the L-form.
  • the C. tropicalis MFP cDNA (Nuttley, W. M. et al., Gene 69: 171 -180 (1988), GenBank Accession Number M22765) was cloned via PCR amplification (SEQ ID NO:21 , encoding SEQ ID NO:22) into pART7 to obtain pART7_MFP.
  • the Notl-cassette containing the CAMV35S-promoter in front of the MFP gene and the ocs3'- terminator, was inserted into the plant binary vector pART27 to obtain pART27_MFP, which was transformed into Arabidopsis.
  • Transgenic plant were selected on kanamycin and screened for the expression of the MFP protein with an anti-MFP antiserum. Homozygous T2 plants were cross-fertilized with PHACl #3, PHACl #4 and PHACl #9 plants. Offspring from these crosses will be analyzed for their ability to biosynthesize PHA.
  • the COOH-terminal tripeptide -AKI was shown to be responsible for peroxisomal targeting of the MFP in yeast, but it has not yet been demonstrated to function in plant peroxisomal targeting.
  • SKL gene in which the 3'-terminal nucleotide sequence of the MFP gene encoding the -AKI tripeptide had been changed to -SKL by PCR site-directed mutagenesis (SEQ ID NO:23, encoding SEQ ID NO:24), was obtained from the laboratory of K. Hiltunen to ascertain that the MFP was properly targeted to the plant peroxisomes and to serve as a positive control in targeting studies with the yeast multifunctional protein (MFP) in plant cells.
  • MFP yeast multifunctional protein
  • the MFP.SKL gene was used to construct pART7_MFP.SKL.
  • the Notl-cassette of pART7_MFP.SKL containing the MFP-SKL gene flanked by the CaMV35S promoter and the ocs3'-terminator, was cloned into pART27 to obtain pART27_MFP.SKL, which was transformed into A. thaliana ecotype Columbia. Kanamycin resistant Tl plants were obtained.
  • the high-MFP.SKL-expressing lines will be selected by Western analysis of T2 plants, and the selected lines will be crossed with PHACl #3.3 plants.
  • the construct pART7_MFP ⁇ AKI was obtained by PCR amplification of the MFP gene such that the 3 '-terminal nucleotide sequence of the MFP gene encoding the -AKI tripeptide was deleted by the introduction of a stop codon (SEQ ID NO:25, encoding SEQ ID NO:26).
  • the "detargeted" MFP ⁇ AKI is expected to be localized in the cytoplasm and will be utilized as negative control in experiments to study the localization of MFP and MFP.SKL in plant cells.
  • pART27_MFP ⁇ AKI was transformed into A. thaliana ecotype Columbia and Kanamycin resistant Tl plants were obtained.
  • the high-MFP ⁇ AKI- expressing lines will be selected by Western analysis of T2 plants and these lines will be crossed with PHACl #3.3 plants.
  • EXAMPLE 11 Verification of enzyme activity of modified MFP constructs in Pichia
  • the modified MFP.SKL and MFP ⁇ AKI genes were subcloned from pART7_MFP.SKL and pART7_MFP ⁇ AKI into the yeast expression vector pHILD2.
  • the resulting plasmids pHILD2_MFP.SKL and pHILD2_MFP ⁇ AKI were transformed into Pichia and enzyme assays were performed in Hiltunen's laboratory. Results indicated that the modifications to the genes did not have an effect on the dehydrogenase and the hydratase enzymatic activities.
  • This increased flux of medium-chain fatty acids through ⁇ -oxidation may be exploited to improve the yield of PHA, as well as to modify the composition of the polymer towards saturated H6-H14 monomers in double transgenic plants expressing both acyl-ACP thioesterase and the PHACl synthase.
  • the plasmid pBJ49_FatB3 containing the Cuphea lancolata thioesterase FatB3 gene under control of a 200 bp minimal promoter derived from the 35S promoter was infiltrated into the A. thaliana PHACl #3.3 transgenic line which is homozygous for the PHACl gene.
  • Hygromycin resistant lines where obtained and the seed lipid content of Tl seeds was analysed for increased levels of medium chain length fatty acids and 1 1 separate lines expressing high levels of the acyl-ACP thioesterase were identified in this manner.
  • the increased polyhydroxyalkanoate yield was mainly due to a large increase in the content of the saturated polyhydroxyalkanoate monomers with an even number of carbons, namely 3-OH-octanoate (H8), 3-OH-decanoate (H10), 3-OH-dodecanoate (H12) and 3-OH-tetradecanoate (HI 4) (Table 8).
  • the recombinant FatB3 acyl-ACP thioesterase is naturally targeted to the chloroplast, where it removes medium chain-length acyl-ACP intermediates from the fatty acid biosynthesis.
  • These short chain fatty acids accumulate in the seed lipids, but not in the leaves of transgenic plants and it has been speculated, that they are immediately degraded by ⁇ -oxidation. Results with these double transgenic plants indicate that there is indeed an increase in the ⁇ -oxidation of medium chain length fatty acids in the leaves, which results in a higher yield of polyhydroxyalkanoate due to the incorporation of the ⁇ -oxidation intermediates into the PHA by the polyhydroxyalkanoate synthase.
  • EXAMPLE 13 Crossing PHACl #3.3 transgenic plants with fatty acyl hydroxylase LFahl2 transgenic plants
  • the amount of PHA present in plant tissues was influenced by the growth conditions .
  • the yield of PHA was approximately 0.6 mg/g dry weight (dwt). Removal of sucrose for the last week of growth in the light resulted in a 100% increase in PHA, while plants growing in 2% sucrose but shifted in the dark for the last week accumulated 22% more PHA (Table 9).
  • PTSl peroxisomal targeting sequence 1
  • S, A, or P small uncharged amino acid at position 1
  • K, R. S, or H positively-charged amino acids at position 2
  • L, M, I or F hydrophobic amino acid at position 3
  • the initial minimal PTSl sequence was defined as SKL.
  • a range of substition have been found to be effective PTSl signal, including ARM, SRM, SKL, ARL, SRL, PSI, or PRM.
  • Specific examples of targeting of foreign proteins in plants include: 6 amino acid PTSl (RAVARL, Volokita, M., Plant J ⁇ : 361-366 (1991)); 5 amino acids PTSl (AKSRM, Olsen, L. J. et al, Plant Cell 5: 941-952 (1993)); 4 amino acids PTSl (KSRM, Trelease, R. N.
  • the minimal peroxisomal targeting sequence 1 (PTSl) in plants has been found to be ARM, SRM, SKL, ARL, SRL, PSI, and PRM (Compilation from Volokita, M., Plant J., 1: 361-366 (1991); Olsen, L.J. et al., Plant Cell, 5: 941-952 (1993); Trelease, R.N. et al., Protoplasma, 195: 156-167 (1996); Gietl, C, Physiol Plant., 97: 599-608 (1996); Purdue, P.E. and Lazarow, P.B., J. Biol Chem., 269: 30065-30068 (1994); Subramani, Ann. Rev.
  • PTS2 peroxisome targeting sequence 2
  • a consensus sequnce of nine amino acids has been defined, being (R K)(L/Q/I)XXXXX(H/Q)L.
  • Foreign protein eg ⁇ - glucuronidase
  • PTS2 sequence can also be targeted in plants to the peroxisome by adding a PTS2 sequence at the N-terminal end of the protein (Kato et al, Plant Cell 8: 1601-1611 (1996)).
  • EXAMPLE 16 Co-expression of PHA with other sequences resulting in increased or novel PHA biosynthesis
  • PHA mcl synthesized in transgenic plants can include a large variety of monomers, with functional groups that can be used to modify and improve the characteristics of the polymer before or after extraction form the plant. For example, the presence of double bonds, epoxy groups, or acetylated groups within the PHA may be used to cross-link the polymer.
  • PHA polymers in plants that have a wide range of monomers, for example, higher proportion of short-chain monomers, unsaturated bonds at novel positions, monomers with hydroxylated groups, epoxy groups, acetylated groups, keto groups, cyclopentenyl groups, cyclopropanoid groups, furanoid groups or halogenated groups, branched chain, cyclic groups or any other novel monomers for which the equivalent functional groups exist in fatty acids in plants.
  • monomers for example, higher proportion of short-chain monomers, unsaturated bonds at novel positions, monomers with hydroxylated groups, epoxy groups, acetylated groups, keto groups, cyclopentenyl groups, cyclopropanoid groups, furanoid groups or halogenated groups, branched chain, cyclic groups or any other novel monomers for which the equivalent functional groups exist in fatty acids in plants.
  • the inco ⁇ oration of these novel monomers derived from fatty acids into plant PHAs could be accomplished by expressing a PHA synthase in a plant which synthesizes these unusual fatty acids either naturally or after expression of a transgene such as fatty-acyl-thioesterases, -hydroxylases, -desaturases, - epoxidases, or -acetylases. It is also conceivable that the substrate specificity of the PHA synthase could be modified to allow the inco ⁇ oration of a wider range of monomers into PHA.
  • acetyl-CoA is also found in the peroxisome, one can predict that co-expression of a PHA synthase with a substrate specificity for 3 -hydroxy acids ranging from H4 to H8 or higher in the peroxisome, and of the A. eutrophus acetoacetyl- CoA reductase, would lead to the biosynthesis of a copolymer containing hydroxybutyrate and hydroxyacids of H6 and higher. In this pathway, the expression of the 3-ketothiolase from A. eutrophus may not be required since the peroxisome already contains a 3- ketothiolase.
  • fatty acid modifying enzymes in conjunction with a PHA synthase in plants not only leads to an increase in the amount of PHA synthesized in plants, but also leads to a predictable changes in the PHA monomer composition, e.g. co-expression of a short-chain fatty acyl-ACP thioesterase would lead to an increase in the proportion of short-chain hydroxyacid monomers in plant PHA, co- expression of a long-chain fatty acyl-ACP thioesterase would lead to an increase in the proportion of long-chain hydroxyacid monomers in plant PHA, co-expression of a fatty acyl hydroxylase would lead to an increase in the proportion of hydroxylated hydroxyacid monomers in plant PHA, co-expression of a fatty acyl epoxidase would lead to an increase in the proportion of epoxidated monomers in plant PHA, co-expression of a fatty acyl acetylase would lead to an increase in the proportion of
  • Increase in flux of lipids through the ⁇ -oxidation cycle could also be accomplished by overexpressing the key regulators (i.e. transcriptional factors) involved in the up-regulation of the entire ⁇ -oxidation cycle pathway during germination or senescence. This last approach would have the advantage of turning-on the ⁇ -oxidation cycle in tissues which normally have only low activity, such as the developing seeds of oil crops.
  • the examples herein point out the impact of fatty acid modifying enzymes for the production of novel PHA in transgenic plants expressing a PHA synthase.
  • One key enzyme appears to be a 3-hydroxy-acyl-CoA epimerase.
  • the normal function of the epimerase is to convert D-3-hydroxy-acyl-CoAs to the L-form required for the action of the L-3-hydroxy-acyl-CoA dehydrogenase
  • the reverse reaction of the epimerase can be responsible for converting the L-form to the D-form, which is essential for the activity of the PHA synthase.
  • the epimerase is important for the supply of the substrates for the PHA synthase derived from ⁇ -oxidation in the peroxisomes.
  • Recombinant forms of such an epimerase activity expressed in peroxisomes or in other plant cell compartments like the cytoplasm or the plastids could play an important role in the production of PHA in transgenic plants. It is possible that the slow rate of the epimerase "reverse reaction" could be the major factor limiting the supply of substrates for the PHA synthase. The substrate limitation due to this could be the reason why PHA synthesis seemed to have reached a maximum in seedlings germinated both in the light and in the dark in liquid medium supplemented with TWEEN-20, which contains only saturated fatty acids.
  • the H8 and the H8:l monomer are predicted to originate from the unsaturated fatty acids linoleic acid (C18:2, 9,12-all cis) and linolenic acid (C18:3, 9,12,15-all cis). For that reason any plant containing high levels of fatty acids with unsaturated bonds starting at even-numbered carbons could be of interest for the production of PHA mcl , or the transgenic expression of suitable fatty acid desaturases producing such unsaturated fatty acids in plants containing the PHA synthase would be similarly attractive for PHA production and monomer manipulation.
  • Fatty acid biosynthesis occurs in the plastids in plant cells, and modifications of this pathway could turn the plastids into a suitable source of D-3-hydroxy-acyl-CoA intermediates, which could subsequently be used to produce PHA either in the plastid itself or in other cell compartments.
  • Leaves from transgenic plants were homogenized in 200 mM Tris-HCl (pH 7.5), 250 mM EDTA, 5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. The homogenate was clarified by centrifugation and protein analyzed by Western blot using the ECL detection system (Amersham, Arlington Heights, IL).
  • Transgenic plants were grown on media containing MS salts, 1% sucrose, 0.7% agar and 50 ⁇ g/mL kanamycin for either 7 days in the light or 1 day in the light followed by 6 days in the dark.
  • Whole plants were fixed for 2 hours at room temperature in 4% formaldehyde, 0.5% glutaraldehyde, 50 mM sodium cacodylate pH 7.3.
  • the tissue samples were dehydrated in an ethanol series and embedded in LR White resin.
  • Ultra thin sections were cut using a microtome, mounted on formvar-coated gold grids and blocked in 0.8% (w/v) bovine serum albumin, 0.1% (w/v) gelatine, 5% (w/v) normal goat serum and 2 mM sodium azide in PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4).
  • Grids were incubated for 1 hour at room temperature with antiserum against PHA synthase (1 :50), glycolate oxidase (1:2000) and isocitrate lyase (1 : 1000) in the blocking solution followed by a 4 hour incubation at room temperature with a 1 :50 dilution of gold-conjugated goat anti- rabbit antibodies (15 nm gold particles) in PBS. Immunolabeled sections were doubled- stained with uranyl acetate and lead citrate and viewed with a Jeol JEM transmission electron microscope.
  • Fresh or dried frozen plant material was ground in a mortar and lyophilized.
  • the powder was extracted with methanol in a Soxhlet apparatus for 24 hours followed by PHA extraction with chloroform for 24 hours, both at 85°C.
  • the PHA-containing chloroform was concentrated under reduced pressure and extracted once with water to remove residual solid particles.
  • PHA was precipitated by the addition of 10 volumes of cold methanol and subsequently washed by two cycles of chloroform solubilisation and methanol precipitation.
  • PHA dissolved in chloroform was transesterified by acid methanolysis (Huijberts, G. N. et al., Appl. Environ. Microbiol.
  • transformation vectors capable of introducing encoding DNAs involved in PHA biosynthesis are easily designed, and generally contain one or more DNA 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 DNA in a plant; optionally, a 5' non-translated leader sequence; a nucleotide 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 said 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 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 et al., Nature 313: 810 (1985)), the enhanced CaMV 35S promoter, the Fig wort Mosaic Virus (FMV) promoter (Richins et al., Nucleic Acids Res. 20: 8451 (1987)), the mannopine synthase (mas) promoter, the nopaline synthase (nos) promoter, and the octopine synthase (ocs) promoter.
  • CaMV 35S promoter Odell et al., Nature 313: 810 (1985)
  • the enhanced CaMV 35S promoter the Fig wort Mosaic Virus (FMV) promoter
  • FMV Fig wort Mosaic Virus
  • mannopine synthase mas
  • 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 (1986); Ainley et al., Plant Mol. Biol.
  • tissue-specific, developmentally-regulated promoters include the ⁇ -conglycinin 7S promoter (Doyle et al, J. Biol. Chem. 261 : 9228 (1986); Slighton and Beachy, Planta 172: 356 (1987)), and seed-specific promoters (Knutzon et al., Proc.
  • 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. Examples of such promoters include the 5' regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res.
  • 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 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, etc. (reviewed in Potrykus, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205 (1991)).
  • transgenic plants comprising cells containing and expressing DNAs encoding enzymes facilitating PHA biosynthesis 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 enzyme-encoding nucleotide sequence.
  • Particularly useful plants for PHA production include those that produce carbon substrates which can be employed for PHA biosynthesis, including tobacco, wheat, potato, Arabidopsis, and high oil seed plants such as corn, soybean, canola, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, and alfalfa.
  • the host plant of choice does not produce the requisite fatty acid substrates in sufficient quantities, it can be modified, for example by mutagenesis or genetic transformation, to block or modulate the glycerol ester and fatty acid biosynthesis or degradation pathways so that it accumulates the appropriate substrates for PHA production.
  • Expression of enzymes such as acyl-ACP thioesterase, fatty acyl hydroxylase, and yeast multifunctional protein (MFP) may serve to increase the flux of substrates in the peroxisome, leading to higher levels of PHA biosynthesis.
  • nucleic acid sequence encoding a fusion 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, 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.
  • 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 1 1.
  • 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.,
  • 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); s 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 (-3.2); glutamate/glutamine/aspartate/asparagine (-3.5); lysine (- 3.9); and arginine (-4.5).
  • amino acids may be substituted by other amino 0 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.
  • hydrophihcity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 ⁇ 1); serine (+0.3); 0 asparagine/glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (- 1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
  • amino acid may be substituted by another amino acid having a similar hydrophihcity score and still result in a protein with similar biological activity, i.e., 5 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, hydrophihcity, charge, size, and the like.
  • 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.
  • 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.
  • CAGAGCATCC TCAACCCACC GGGCAACCCC AAGGCACGCT TCATGACCAA TCCGGAACTG 1500
  • Gin Ser lie Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg Phe Met Thr 485 490 495
  • Lys Glu Leu Phe Asn Thr Gly Gly lie Ser Leu Leu Asn Gly Val Arg 145 150 155 160 His Leu Leu Glu Asp Leu Val His Asn Gly Gly Met Pro Ser Gin Val 165 170 175 ys Thr Ala Phe Glu lie Gly Arg Asn Leu Ala Thr Thr Gin Gly 180 185 190
  • Thr Ala Val Asp Leu Gly Lys Val Ala lie Asp Ser Phe His Val Ala 435 440 445 Gly lie Thr Asp His lie Thr Pro Trp Asp Ala Val Tyr Arg Ser Ala 450 455 460 Leu Leu Leu Gly Gly Gin Arg Arg Phe lie Leu Ser Asn Ser Gly His 465 470 475 480 lie Gin Ser lie Leu Asn Pro Pro Gly Asn Pro Lys Ala Cys Tyr Phe 485 490 495
  • CAGAGCATCC TCAACCCACC GGGCAACCCC AAGGCACGCT TCATGACCAA TCCGGAACTG 1500
  • CAGAGCATCC TCAACCCACC GGGCAACCCC AAGGCACGCT TCATGACCAA TCCGGAACTG 1500 CCCGCCGAGC CCAAGGCCTG GCTGGAACAG GCCGGCAAGC ACGCCGACTC GTGGTGGTTG 1560
  • Gin Ser lie Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg Phe Met Thr 485 490 495 Asn Pro Glu Leu Pro Ala Glu Pro Lys Ala Trp Leu Glu Gin Ala Gly
  • Leu Leu Leu Leu Gly Gly Gin Arg Arg Phe lie Leu Ser Asn Ser Gly His 465 470 475 480 He Gin Ser He Leu Asn Pro Pro Gly Asn Pro Lys Ala Cys Tyr Phe
  • GGTAAGTCAC AAAACTCCTT TGCCAAGTTG TTGCGTAACT TCAACCCAAT GTTGTTGTTG 2100
  • GACTCTAGGA AGCCAGAATA CTTGAAGAAC CAATACCCAT TCATGTTGAA CGACTACGCC 900
  • ACTTTGACCA ACGAAGCTAG AAAGTTGCCA GCTAACGATG CTTCTGGTGC TCCAACTGTC 960 TCCTTGAAGG ACAAGGTTGT TTTGATCACC GGTGCCGGTG CTGGTTTGGG TAAAGAATAC 1020 GCCAAGTGGT TCGCCAAGTA CGGTGCCAAG GTTGTTGTTA ACGACTTCAA GGATGCTACC 1080
  • ATCAGAAACT GTCAAGCCGA CAACAAGGTC TACGCTGACC GTCCAGCATT CGCCACCAAC 2340 CAATTCTTGG CACCAAAGAG AGCCCCAGAC TACCAAGTTG ACGTTCCAGT CAGTGAAGAC 2400
  • GACTCTAGGA AGCCAGAATA CTTGAAGAAC CAATACCCAT TCATGTTGAA CGACTACGCC 900
  • GGTAAGTCAC AAAACTCCTT TGCCAAGTTG TTGCGTAACT TCAACCCAAT GTTGTTGTTG 2100

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Abstract

La présente invention concerne des acides nucléiques, des protéines et des techniques utilisés dans la biosynthèse de polymères à base de polyhydroxyalcanoates. Dans une réalisation préférée, l'expression d'une protéine polyhydroxyalcanoate synthétase à l'aide d'un peptide de ciblage de peroxysome conduit à la biosynthèse de polyhydroxyalcanoates à longueur de chaîne moyenne. Dans une autre réalisation, l'addition exogène d'acides gras à une plante ou à une cellule contenant une protéine polyhydroxyalcanoate synthétase ayant fait l'objet d'un ciblage du peroxysome mène à la biosynthèse de polymères nouveaux.
PCT/US1998/000083 1998-01-05 1998-01-05 Biosynthese de polyhydroxyalcanoates a longueur de chaine moyenne WO1999035278A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU59071/98A AU5907198A (en) 1998-01-05 1998-01-05 Biosynthesis of medium chain length polyhydroxyalkanoates
EP98902393A EP1044278A1 (fr) 1998-01-05 1998-01-05 Biosynthese de polyhydroxyalcanoates a longueur de chaine moyenne
PCT/US1998/000083 WO1999035278A1 (fr) 1998-01-05 1998-01-05 Biosynthese de polyhydroxyalcanoates a longueur de chaine moyenne

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1998/000083 WO1999035278A1 (fr) 1998-01-05 1998-01-05 Biosynthese de polyhydroxyalcanoates a longueur de chaine moyenne

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WO2001009364A1 (fr) * 1999-08-03 2001-02-08 Oulun Yliopisto Procede de regulation des (3r)-hydroxyacyl-coa esters cellulaires, molecules precurseurs dans la synthese polyhydroxyalcanoate dans des organismes genetiquement modifies
WO2001023580A3 (fr) * 1999-09-29 2002-01-10 Pioneer Hi Bred Int Genes de la polyhydroxyalcanoate synthase
WO2001023596A3 (fr) * 1999-09-29 2002-01-31 Pioneer Hi Bred Int Production de polyhydroxyalcanoate dans des plantes
EP1369484A1 (fr) * 2001-03-14 2003-12-10 Riken Plante synthetisant du copolyester a partir d'un monomere derive d'un acide gras a chaine courte et procede de production d'un polyester
US6806401B2 (en) 2000-12-27 2004-10-19 Pioneer Hi-Bred International, Inc. OAR polynucleotides, polypeptides and their use in PHA production in plants
US7176349B1 (en) 1999-09-29 2007-02-13 Pioneer Hi-Bred International, Inc. Production of polyhydroxyalkanoate in plants
WO2007037706A3 (fr) * 2005-09-27 2007-06-28 Bernd Helmut Adam Rehm Particules polymeriques et leurs applications
US7429469B2 (en) * 2001-10-10 2008-09-30 Kaneka Corporation Enzyme gene participating in the synthesis of polyester and process for producing polyester using the same
US7622277B2 (en) 2002-08-30 2009-11-24 Massey University Process for the production of biodegradable, functionalised polymer particles, and use thereof as pharmaceutical supports
WO2009145840A2 (fr) * 2008-04-04 2009-12-03 Massachusetts Institute Of Technology Production cellulaire d'hydroxyvalérates à partir de levulinate
ITRM20080545A1 (it) * 2008-10-13 2010-04-14 Alberto Ballistreri Produzione di plastica biodegradabile da olio di brassica carinata ad alto contenuto di acido erucico e da acidi grassi a catena molto lunga.
WO2010057271A1 (fr) * 2008-11-21 2010-05-27 Sugar Industry Innovation Pty Ltd Production de polyhydroxyalcanoate dans des peroxysomes de plantes
CN111334445A (zh) * 2018-12-19 2020-06-26 中国科学院微生物研究所 长链二元酸生产菌株及其制备方法和应用
CN113528496A (zh) * 2021-07-15 2021-10-22 华中农业大学 Icl基因在调控番茄抗坏血酸积累中的应用
EP4344697A1 (fr) * 2022-09-28 2024-04-03 Chanel Parfums Beauté Composition cosmétique comprenant au moins un polyhydroxyalcanoate complexe
US12188068B2 (en) 2014-04-10 2025-01-07 Genomatica, Inc. Semisynthetic routes to organic compounds

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Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009364A1 (fr) * 1999-08-03 2001-02-08 Oulun Yliopisto Procede de regulation des (3r)-hydroxyacyl-coa esters cellulaires, molecules precurseurs dans la synthese polyhydroxyalcanoate dans des organismes genetiquement modifies
US7341856B2 (en) 1999-09-29 2008-03-11 Pioneer Hi-Bred International, Inc. Production of polyhydroxyalkanoate in plants
WO2001023580A3 (fr) * 1999-09-29 2002-01-10 Pioneer Hi Bred Int Genes de la polyhydroxyalcanoate synthase
WO2001023596A3 (fr) * 1999-09-29 2002-01-31 Pioneer Hi Bred Int Production de polyhydroxyalcanoate dans des plantes
US6475734B1 (en) 1999-09-29 2002-11-05 Pioneer Hi-Bred International, Inc. Polyhydroxyalkanoate synthase genes
US7176349B1 (en) 1999-09-29 2007-02-13 Pioneer Hi-Bred International, Inc. Production of polyhydroxyalkanoate in plants
US6806401B2 (en) 2000-12-27 2004-10-19 Pioneer Hi-Bred International, Inc. OAR polynucleotides, polypeptides and their use in PHA production in plants
US7361807B2 (en) 2000-12-27 2008-04-22 Pioneer Hi-Bred International. Inc. OAR polynucleotides, polypeptides and their use in PHA production in plants
US7129395B2 (en) 2000-12-27 2006-10-31 Pioneer Hi-Bred International, Inc. OAR polynucleotides, polypeptides and their use in PHA production in plants
EP1369484A4 (fr) * 2001-03-14 2005-12-28 Riken Plante synthetisant du copolyester a partir d'un monomere derive d'un acide gras a chaine courte et procede de production d'un polyester
EP1369484A1 (fr) * 2001-03-14 2003-12-10 Riken Plante synthetisant du copolyester a partir d'un monomere derive d'un acide gras a chaine courte et procede de production d'un polyester
US7429469B2 (en) * 2001-10-10 2008-09-30 Kaneka Corporation Enzyme gene participating in the synthesis of polyester and process for producing polyester using the same
US7622277B2 (en) 2002-08-30 2009-11-24 Massey University Process for the production of biodegradable, functionalised polymer particles, and use thereof as pharmaceutical supports
EP1929010A2 (fr) * 2005-09-27 2008-06-11 Bernd Helmut Adam Rehm Particules polymeriques et leurs applications
JP2009509530A (ja) * 2005-09-27 2009-03-12 ヘルムート アダム レフム，ベルン ポリマー粒子およびその使用
EP1929010A4 (fr) * 2005-09-27 2009-05-27 Polybatics Limited Particules polymeriques et leurs applications
WO2007037706A3 (fr) * 2005-09-27 2007-06-28 Bernd Helmut Adam Rehm Particules polymeriques et leurs applications
JP2015051002A (ja) * 2005-09-27 2015-03-19 ポリバティックス リミティド ポリマー粒子およびその使用
WO2009145840A2 (fr) * 2008-04-04 2009-12-03 Massachusetts Institute Of Technology Production cellulaire d'hydroxyvalérates à partir de levulinate
WO2009145840A3 (fr) * 2008-04-04 2010-01-21 Massachusetts Institute Of Technology Production cellulaire d'hydroxyvalérates à partir de levulinate
WO2010044118A1 (fr) * 2008-10-13 2010-04-22 Alberto Ballistreri Production de plastiques biodégradables à partir d'huile de brassica carinata à teneur élevée en acide érucique et à partir d'acides gras à chaîne très longue
ITRM20080545A1 (it) * 2008-10-13 2010-04-14 Alberto Ballistreri Produzione di plastica biodegradabile da olio di brassica carinata ad alto contenuto di acido erucico e da acidi grassi a catena molto lunga.
WO2010057271A1 (fr) * 2008-11-21 2010-05-27 Sugar Industry Innovation Pty Ltd Production de polyhydroxyalcanoate dans des peroxysomes de plantes
US12188068B2 (en) 2014-04-10 2025-01-07 Genomatica, Inc. Semisynthetic routes to organic compounds
CN111334445A (zh) * 2018-12-19 2020-06-26 中国科学院微生物研究所 长链二元酸生产菌株及其制备方法和应用
CN113528496A (zh) * 2021-07-15 2021-10-22 华中农业大学 Icl基因在调控番茄抗坏血酸积累中的应用
EP4344697A1 (fr) * 2022-09-28 2024-04-03 Chanel Parfums Beauté Composition cosmétique comprenant au moins un polyhydroxyalcanoate complexe

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