US20030182678A1

US20030182678A1 - Multigene expression vectors for the biosynthesis of products via multienzyme biological pathways

Info

Publication number: US20030182678A1
Application number: US10/215,328
Authority: US
Inventors: Timothy Mitsky; Steven Slater; Steven Reiser; Ming Hao; Kathryn Houmiel
Original assignee: Monsanto Technology LLC
Current assignee: Monsanto Technology LLC
Priority date: 1999-03-05
Filing date: 2002-08-08
Publication date: 2003-09-25
Also published as: AU3516100A; WO2000052183A1; US6448473B1; EP1159435A1

Abstract

The use of multigene vectors for the preparation of transformed host cells and plants is disclosed. Multigene vectors reduce the number of transformations required, and leads to increased production of polyhydroxyalkanoate polymer in the resulting transformed host cells and plants.

Description

This application is based on U.S. Provisional Application No. 60/123,015, filed Mar. 5, 1999.[0001]

FIELD OF THE INVENTION

The invention relates to the construction and use of multigene expression vectors useful to enhance production of materials by multienzyme pathways. In particular, the construction and use of multigene vectors encoding proteins in the polyhydroxyalkanoate biosynthetic pathway is disclosed.

BACKGROUND OF THE INVENTION

Metabolic engineering is a process by which the normal metabolism of an organism is altered to change the concentration of normal metabolites, or to create novel metabolites. This process often involves introduction or alteration of numerous enzymatic steps, and thus often requires introduction of multiple genes. An efficient system for introducing and expressing multiple genes is therefore desirable. In prokaryotes such as Escherichia coli, introduction of multiple genes is relatively straightforward in that operons can be constructed to express multiple open reading frames, or multiple complete genes can be expressed from a single plasmid. However, introduction of pathways into plants is more difficult due in part to the complexity of plant genes, the difficulty of constructing vectors harboring multiple genes for expression in plants, and the difficulty of introducing large vectors intact into plants.

Polyhydroxyalkanoates 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, polyhydroxyalkanoates are an attractive source of non-polluting plastics and elastomers.

Currently, there are approximately a dozen biodegradable plastics in commercial use that possess properties suitable for producing a number of specialty and commodity products (Lindsay, Modern Plastics 2: 62, 1992). One such biodegradable plastic in the polyhydroxyalkanoate (PHA) family that is commercially important is Biopol™, a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). This bioplastic is used to produce biodegradable molded material (e.g., bottles), films, coatings, and in drug release applications. Biopl™ is produced via a fermentation process employing the bacterium Ralstonia eutropha (Byrom, D. Trends Biotechnol. 5: 246-250, 1987). (R. eutropha was formerly designated Alcaligenes eutrophus [Yabuuchi et al., Microbiol. Immunol. 39:897-904, 1995]). 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, Y. et al., Bio/Technology 13: 142, 1995). Competitive synthetic plastics such as polypropylene and polyethylene cost about 35-45¢/lb (Layman, Chem. & Eng. News, p. 10 (Oct. 31, 1994). The annual global demand for polyethylene alone is about 37 million metric tons (Poirier, Y. et al., Int. J. Biol. Macromol. 17: 7-12, 1995). It is therefore likely that the cost of producing P(3HB-co-3HV) by microbial fermentation will restrict its use to low-volume specialty applications.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids (Anderson, A. J. and Dawes, E. A. Microbiol. Rev. 54: 450-472, 1990; Steinbüchel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan, N.Y.), pp. 123-213, 1991); Poirier, Y., Nawrath, C. & Somerville, C. Bio/Technology 13: 143-150, 1995). Polyhydroxybutyrate (PHB) is the most well-characterized PHA. High molecular weight PHB is found as intracellular inclusions in a wide variety of bacteria (Steinbüchel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan, N.Y.), pp. 123-213, 1991). In Ralstonia eutropha, PHB typically accumulates to 80% dry weight with inclusions being typically 0.2-1 μm 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). High molecular weight polyhydroxyalkanoates have the properties of thermoplastics and elastomers. Numerous bacteria and fungi can hydrolyze polyhydroxyalkanoates to monomers and oligomers, which are metabolized as a carbon source. Polyhydroxyalkanoates have accordingly 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). In contrast, 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 R. eutropha PHB biosynthetic enzymes (Poirier, Y. et al., Science 256: 520-523, 1992; Nawrath, C., et al., Proc. Natl. Acad. Sci. U.S.A. 91: 12760-12764, 1994). In plants expressing the PHB pathway in the plastids, leaves accumulated up to 14% PHB per gram dry weight (Nawrath, C., et al., Proc. Natl. Acad. Sci. U.S.A. 91: 12760-12764, 1994). High-level synthesis of PHB in plants opened the possibility of utilizing agricultural crops as a suitable system for the production of polyhydroxyalkanoates on a large scale and at low cost (Poirier, Y. et al., Bio/Technology 13: 143-150, 1995; Poirier, Y. et al., FEMS Microbiol. Rev. 103: 237-246, 1992; Nawrath, C., et al. Molecular Breeding 1: 105-22, 1995). 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 R. eutropha 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; Steinbüchel, A. in Novel Biomaterials from Biological Sources, ed. Byrom, D. (MacMillan, N.Y.), 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 2 n carbons shorter than the substrate. These studies indicate that MCL-PHAs are synthesized by the PHA synthase from 3-hydroxyacyl-CoA intermediates generated by the β-oxidation of alkanoic acids (Huijberts, G. N. M., et al. Appl. Environ. Microbiol. 58: 536-544, 1992; Huijberts, G. N. M., et al., J. Bacteriol. 176: 1661-1666, 1994).

Chen et al. ( Nature Biotech., 16: 1060-1064, 1998; reviewed by Gelvin, S. B., Nature Biotech., 16: 1009-1010, 1998) describes the cobombardment of embryogenic rice tissues with a mixture of 14 different pUC based plasmids. Integration of multiple transgenes was observed to occur at one or two genetic loci.

Creating a transgenic host cell or plant that produces multiple enzymes within a biosynthetic pathway is often a daunting task. Individual vectors must be created for each enzyme. Transformation of the host cell or plant is typically accomplished by one of three general methods: serial transformation, parallel transformation followed by crossing, or batch transformation. Each method has serious practical drawbacks.

Serial transformation involves transforming a host cell or plant with the first vector, selecting and characterizing the transformed cell or plant, transforming with the second vector, and so on. This process can become quite laborious and time consuming.

Parallel transformation followed by crossing involves separately transforming cells with each of the individual vectors, and subsequently mating or crossbreeding the transformed cells or plants to obtain a final cell or plant which contains all of the individual sequences. This is a lengthy process, especially for the crossbreeding of plant lines.

Batch transformation involves a single transformation event involving all of the individual vectors. A wide array of cells are produced, each containing between none and all of the vectors. While only a single transformation is required, extensive characterization of the resulting cells is necessary. As the number of vectors increases, it is increasingly likely that no cells will be obtained containing all of the vectors. If no desired transformed cells are identified, the transformation must be repeated.

An additional concern with all three of these methods is that they do not allow any control over the relative copy numbers of the individual vectors in the transformed cell or plant. It would be desirable to have a transformation method that permits control of the relative copy numbers of the individual sequences in the transformed cell or plant, and also coordinates the positional effect of the insertion locus.

There exists a need for improved materials and methods for the preparation of transgenic organisms transformed with multiple nucleic acid sequences encoding members of a multi-enzyme biosynthetic pathway.

SUMMARY OF THE INVENTION

The invention involves the construction and use of nucleic acid segments and vectors containing multiple sequences encoding members of a biosynthetic pathway. The resulting vector allows a single transformation event to produce a transformed cell or plant containing all of the nucleic acid sequences. Furthermore, the researcher has total control over the number of copies of each coding sequence within the constructed vector. Single or multiple copies of each coding sequence may easily be designed into the vector.

An unexpected beneficial result of the invention is that organisms transformed with a multi-enzyme coding vector produce the biosynthetic product in higher yield than organisms produced by serial transformation, parallel transformation with crossing, or batch transformation methods.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed generally towards the construction and use of nucleic acid segments comprising sequences encoding multiple enzymes in a multi-enzyme biosynthetic pathway. The biosynthetic pathway may generally be any biosynthetic pathway. Examples of such multi-enzyme biosynthetic pathways are the TCA cycle, polyketide synthesis pathway, carotenoid synthesis, glycolysis, gluconeogenesis, starch synthesis, lignins and related compounds, production of small molecules that serve as pesticides, fungicides, or antibiotics, and polymer synthesis pathways. Preferably, the biosynthetic pathway is a polyhydroxyalkanoate biosynthesis pathway.

This disclosure describes multigene vectors designed to produce polyhydroxyalkanoate (PHA) in plants. Some of these vectors are designed to produce poly(β-hydroxybutyrate), and some are designed to produce poly(β-hydroxybutyrate-co-βhydroxyvalerate) (Gruys et al., WO 98/00557, 1998). In general, the efficiency of PHA production was dramatically increased when all sequences necessay for a pathway were introduced on the same vector. Herein, construction of these multigene vectors, and their use for polyhydroxyalkanoate production in Arabidopsis thaliana and Brassica napus, and Zea mays is described.

An embodiment of the present invention is an isolated nucleic acid segment comprising multiple nucleic acid sequences, each encoding a different protein within the biosynthetic pathway. Preferably, the isolated nucleic acid segment comprises a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a second nucleic acid sequence encoding a β-ketoacyl reductase protein; and a third nucleic acid sequence encoding a β-ketothiolase protein. The nucleic acid segment may further comprise additional nucleic acid sequences encoding additional proteins such as a threonine deaminase protein or a deregulated threonine deaminase protein.

An alternative embodiment of the invention is a recombinant vector comprising multiple nucleic acid sequences, each encoding a different protein within the biosynthetic pathway. The recombinant vector may be arranged with a single promoter producing a polycistronic RNA transcript from the multiple nucleic acid sequences, or with each nucleic acid sequence being under the control of its own promoter. The multiple promoters may be the same or different. It is also possible to have one or more nucleic acid sequence under the control of its own promoter, while other nucleic acid sequences may be jointly under the control of a single promoter producing a polycistronic RNA transcript.

A recombinant vector placing the biosynthetic pathway nucleic acid sequences under the control of a single promoter preferably comprises operatively linked in the 5′ to 3′ direction: a promoter that directs transcription of the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence; a first nucleic acid sequence; a second nucleic acid sequence; a third nucleic acid sequence; a 3′ transcription terminator; and a 3′ polyadenylation signal sequence; wherein: the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins; and the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a β-ketoacyl reductase protein, and a nucleic acid sequence encoding a β-ketothiolase protein. The nucleic acid sequences encoding the biosynthetic pathway enzymes may be in any order relative to each other and the promoter. The promoter must be expressed in plastids. It may have either been derived from a plastid, or may have been derived from a bacterium or phage having promoters recognized by the plastid transcription enzymes, or be a synthetic promoter recognized by the plastid transcription enzymes.

A recombinant vector placing the biosynthetic pathway nucleic acid sequences under the control of multiple promoters preferably comprises a first element comprising operatively linked in the 5′ to 3′ direction: a first promoter that directs transcription of the first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3′ transcription terminator; a first 3′ polyadenylation signal sequence; a second element comprising operatively linked in the 5′ to 3′ direction: a second promoter that directs transcription of the second nucleic acid sequence; a second nucleic acid sequence encoding a β-ketoacyl reductase protein; a second 3′ transcription terminator; a second 3′ polyadenylation signal sequence; and a third element comprising operatively linked in the 5′ to 3′ direction: a third promoter that directs transcription of the third nucleic acid sequence; a third nucleic acid sequence encoding a β-ketothiolase protein; a third 3′ transcription terminator; and a third 3′ polyadenylation signal sequence. The β-ketothiolase protein preferably condenses two molecules of acetyl-CoA to produce acetoacetyl-CoA; and condenses acetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA. The β-ketoacyl reductase protein preferably reduces acetoacetyl-CoA to β-hydroxybutyryl-CoA; and reduces β-ketovaleryl-CoA to β-hydroxyvaleryl-CoA. The polyhydroxyalkanoate synthase protein is preferably selected from the group consisting of: a polyhydroxyalkanoate synthase protein that incorporates β-hydroxybutyryl-CoA into P(3HB) polymer; and a polyhydroxyalkanoate synthase protein that incorporates a β-hydroxybutyryl-CoA and a β-hydroxyvaleryl-CoA into P(3HB-co-3HV) copolymer. The β-ketothiolase protein may comprise a transit peptide sequence that directs transport of the β-ketothiolase protein to the plastid. The β-ketoacyl reductase protein may comprise a transit peptide sequence that directs transport of the β-ketoacyl reductase protein to the plastid. The polyhydroxyalkanoate synthase protein may comprise a transit peptide sequence that directs transport of the polyhydroxyalkanoate synthase protein to the plastid. The recombinant vector may further comprise a nucleic acid sequence encoding a threonine deaminase protein or a deregulated threonine deaminase protein. The first promoter, second promoter, and third promoter are preferably active in plants. The first promoter, second promoter, and third promoter are preferably viral promoters. The first promoter, second promoter, and third promoter are preferably independently selected from the group consisting of a CMV 35S promoter, an enhanced CMV 35S promoter, maize chlorophyll A/B binding protein promoter, and an FMV 35S promoter. More preferably, the first promoter, second promoter, and third promoter are the CMV 35S promoter. The first promoter, second promoter, and third promoter may be tissue specific promoters. The first promoter, second promoter, and third promoter may independently be the Lesquerella hydroxylase promoter or the 7S conglycinin promoter, and preferably each is the Lesquerella hydroxylase promoter.

An alternative embodiment is directed towards transformed host cells. Transformed host cells may contain a non-integrated recombinant vector or an integrated recombinant vector.

A transformed host cell may comprise a recombinant vector, wherein the recombinant vector comprises a first element comprising operatively linked in the 5′ to 3′ direction: a first promoter that directs transcription of the first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3′ transcription terminator; a first 3′ polyadenylation signal sequence; a second element comprising operatively linked in the 5′ to 3′ direction: a second promoter that directs transcription of the second nucleic acid sequence; a second nucleic acid sequence encoding a β-ketoacyl reductase protein; a second 3′ transcription terminator; a second 3′ polyadenylation signal sequence; and a third element comprising operatively linked in the 5′ to 3′ direction a third promoter that directs transcription of the third nucleic acid sequence; a third nucleic acid sequence encoding a β-ketothiolase protein; a third 3′ transcription terminator; and a third 3′ polyadenylation signal sequence.

The transformed host cell may alternatively contain an integrated nucleic acid segment. Preferably, the transformed host cell may comprise a first element comprising operatively linked in the 5′ to 3′ direction: a first promoter that directs transcription of a first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3′ transcription terminator; a first 3′ polyadenylation signal sequence; a second element comprising operatively linked in the 5′ to 3′ direction: a second promoter that directs transcription of a second nucleic acid sequence; a second nucleic acid sequence encoding a β-ketoacyl reductase protein; a second 3′ transcription terminator; a second 3′ polyadenylation signal sequence; and a third element comprising operatively linked in the 5′ to 3′ direction: a third promoter that directs transcription of a third nucleic acid sequence; a third nucleic acid sequence encoding a β-ketothiolase protein; a third 3′ transcription terminator; and a third 3′ polyadenylation signal sequence. The first element, second element, and third element may be cointegrated within a continuous 10 Mb segment of genomic DNA, more preferably within a continuous 5 Mb, 2.5 Mb, 2 Mb, 1.5 Mb, 1 Mb, 500 kb, 250 kb, 100 kb, 50 kb, or 20 kb segment of genomic DNA. Alternatively, the first element, second element, and third element may be cointegrated between a left Ti border sequence and a right Ti border sequence. While it is preferable that a recombinant vector contain a single left Ti border sequence and a single right Ti border sequence, the invention encompasses recombinant vectors containing multiple left and/or right Ti border sequences, and the use thereof.

Alternatively, the host cell may comprise a nucleic acid segment containing nucleic acid sequences encoding enzymes in a biosynthetic pathway, where a single promoter directs transcription of the nucleic acid sequences.

The transformed host cell may generally be any host cell, and preferably is a bacterial, fungal, or plant cell. The bacterial cell is preferably an Escherichia coli cell. The fungal cell is preferably a yeast, Saccharomyces cerevisiae, or Schizosaccharomyces pombe cell. The plant cell may be a monocot plant cell, a dicot plant cell, an algae cell, or a conifer plant cell. The plant cell is preferably a tobacco, wheat, potato, Arabidopsis, corn, soybean, canola, sugar beet, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.

The promoters may be any of the promoters discussed earlier. The transformed host cells preferably produce polyhydroxyalkanoate polymer.

The invention also encompasses transformed plants. The transformed plant may contain an integrated set of nucleic acid sequences, or may contain the same set of nucleic acid sequences on a non-integrated vector. A preferred embodiment is directed towards a transformed plant comprising a first element comprising operatively linked in the 5′ to 3′ direction: a first promoter that directs transcription of a first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3′ transcription terminator; a first 3′ polyadenylation signal sequence; a second element comprising operatively linked in the 5′ to 3′ direction: a second promoter that directs transcription of a second nucleic acid sequence; a second nucleic acid sequence encoding a β-ketoacyl reductase protein; a second 3′ transcription terminator; a second 3′ polyadenylation signal sequence; and a third element comprising operatively linked in the 5′ to 3′ direction: a third promoter that directs transcription of a third nucleic acid sequence; a third nucleic acid sequence encoding a β-ketothiolase protein; a third 3′ transcription terminator; and a third 3′ polyadenylation signal sequence. The first element, second element, and third element may be cointegrated within a continuous 10 Mb segment of genomic DNA, more preferably within a continuous 5 Mb, 2.5 Mb, 2 Mb, 1.5 Mb, 1 Mb, 500 kb, 250 kb, 100 kb, 50 kb, or 20 kb segment of genomic DNA. Alternatively, the first element, second element, and third element may be cointegrated between a left Ti border sequence and a right Ti border sequence.

Alternatively, the transformed plant may comprise a nucleic acid segment containing nucleic acid sequences encoding enzymes in a biosynthetic pathway, where a single promoter directs transcription of the nucleic acid sequences.

The transformed plant may generally be any type of plant, and preferably is a tobacco, wheat, potato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.

The promoters may be any of the promoters discussed earlier. The transformed plant preferably produces polyhydroxyalkanoate polymer.

The invention also encompasses methods of preparing transformed host cells. The methods may produce a transformed host cell having nucleic acid sequences under the control of multiple promoters or under the control of a single promoter. The method preferably comprises the steps of selecting a host cell; transforming the selected host cell with a recombinant vector comprising: a first element comprising operatively linked in the 5′ to 3′ direction: a first promoter that directs transcription of the first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3′ transcription terminator; a first 3′ polyadenylation signal sequence; a second element comprising operatively linked in the 5′ to 3′ direction: a second promoter that directs transcription of the second nucleic acid sequence; a second nucleic acid sequence encoding a β-ketoacyl reductase protein; a second 3′ transcription terminator; a second 3′ polyadenylation signal sequence; and a third element comprising operatively linked in the 5′ to 3′ direction: a third promoter that directs transcription of the third nucleic acid sequence; a third nucleic acid sequence encoding a β-ketothiolase protein; a third 3′ transcription terminator; and a third 3′ polyadenylation signal sequence; and obtaining transformed host cells; wherein the transformed host cells produce polyhydroxyalkanoate polymer.

Alternatively, the method of preparing transformed host cells may comprise the steps of selecting a host cell; transforming the selected host cell with a recombinant vector comprising operatively linked in the 5′ to 3′ direction: a promoter that directs transcription of a first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence; a first nucleic acid sequence; a second nucleic acid sequence; a third nucleic acid sequence; a 3′ transcription terminator; and a 3′ polyadenylation signal sequence; and obtaining transformed host cells; wherein: the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins; the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a β-ketoacyl reductase protein, and a nucleic acid sequence encoding a β-ketothiolase protein; and the transformed host cells produce polyhydroxyalkanoate polymer.

The promoters may be any of the promoters discussed earlier.

Also disclosed are methods for preparing transformed plants. The methods may produce a transformed plant having nucleic acid sequences under the control of multiple promoters or under the control of a single promoter. The method preferably comprises the steps of selecting a host plant cell; transforming the selected host plant cell with a recombinant vector comprising: a first element comprising operatively linked in the 5′ to 3′ direction: a first promoter that directs transcription of a first nucleic acid sequence; a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein; a first 3′ transcription terminator; and a first 3′ polyadenylation signal sequence; a second element comprising operatively linked in the 5′ to 3′ direction: a second promoter that directs transcription of a second nucleic acid sequence; a second nucleic acid sequence encoding a β-ketoacyl reductase protein; a second 3′ transcription terminator; and a second 3′ polyadenylation signal sequence; and a third element comprising operatively linked in the 5′ to 3′ direction: a third promoter that directs transcription of a third nucleic acid sequence; a third nucleic acid sequence encoding a β-ketothiolase protein; a third 3′ transcription terminator; and a third 3′ polyadenylation signal sequence; obtaining transformed host plant cells; and regenerating the transformed host plant cells to produce transformed plants, wherein the transformed plants produce polyhydroxyalkanoate polymer.

Alternatively, the method of preparing a transformed plant may comprise the steps of selecting a host plant cell; transforming the selected host plant cell with a recombinant vector comprising operatively linked in the 5′ to 3′ direction: a promoter that directs transcription of a first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence; a first nucleic acid sequence; a second nucleic acid sequence; a third nucleic acid sequence; a 3′ transcription terminator; and a 3′ polyadenylation signal sequence; obtaining transformed host plant cells; and regenerating the transformed host plant cells to produce transformed plants; wherein: the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins; the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a β-ketoacyl reductase protein, and a nucleic acid sequence encoding a β-ketothiolase protein; and the transformed plants produce polyhydroxyalkanoate polymer.

The promoters may be any of the promoters discussed earlier.

The invention is also directed towards methods of producing biomolecules of interest. The multiple enzymes in the biosynthetic pathway may lead to the production of materials of commercial and scientific interest. Preferably, the biomolecules are polymers, and more preferably are polyhydroxyalkanoate polymers. The methods may comprise obtaining any of the above described transformed host cells or transformed plants, culturing or growing the transformed host cells or transformed plants under conditions suitable for the production of polyhydroxyalkanoate polymer, and recovering polyhydroxyalkanoate polymer. The methods, may further comprise the addition of nutrients, substrates, or other chemical additives to the growth media or soil to facilitate production of polyhydroxyalkanoate polymer. In a preferred embodiment, it is possible to extract the polyhydroxyalkanoate from the transformed host cells or transformed plants without killing the host cells or plants. This may be accomplished, for example, by various solvent extraction methods or by engineering the host cells or plants to secrete the polyhydroxyalkanoate polymer, or by directing production to tissues such as leaves or seeds which may be removed without causing serious injury to the plant. The polyhydroxyalkanoate polymer produced is preferably poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(4-hydroxybutyrate), or poly(3-hydroxybutyrate-co-4-hydroxybutyrate).

If repetitive sequences are used in a multi-gene plasmid system, there exists the possibility for gene silencing in subsequent generations of plants. If expression levels are high gene silencing could also occur and would be independent of repetitive elements. Repetitive sequences may include the use of the same promoters, chloroplast peptide encoding sequences, and other genetic elements for each of the multi-gene coding sequences. Gene silencing often manifests itself as a gradual reduction in protein levels, mRNA levels, or biosynthesis product concentrations in subsequent generations of related plants. If gene silencing is observed, changing the repetitive sequences through the use of diverse genetic elements such as different promoters, leaders, introns, transit peptide sequences, etc., different designed nucleotide sequence, or through mutagenesis of the existing sequence, may be successful in reducing or eliminating the gene silencing effects.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0042]
FIG. 1: Biosynthesis of poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (poly(3HB-co-3HV), PHBV) in [0043] Ralstonia eutropha.
FIG. 2: Plant transformation strategies for multi-enzyme metabolic pathway engineering. [0044]
FIG. 3: Plasmid map of pMON25642. A list of the restriction enzyme cutting sites for pMON25642 is provided in Table 10. [0045]
FIG. 4: Plasmid map of pMON10098. A list of the restriction enzyme cutting sites for pMON10098 is provided in Table 11. [0046]
FIG. 5: Plasmid map of pMON969. A list of the restriction enzyme cutting sites for pMON969 is provided in Table 12. [0047]
FIG. 6: Plasmid map of pMON25661. A list of the restriction enzyme cutting sites for pMON25661 is provided in Table 13. [0048]
FIG. 7: Plasmid map of pMON25897. A list of the restriction enzyme cutting sites for pMON25897 is provided in Table 14. [0049]
FIG. 8: Plasmid map of pMON25662. A list of the restriction enzyme cutting sites for pMON25662 is provided in Table 15. [0050]
FIG. 9: Plasmid map of pMON25663. A list of the restriction enzyme cutting sites for pMON25663 is provided in Table 16. [0051]
FIG. 10: Plasmid map of pMON25943. A list of the restriction enzyme cutting sites for pMON25943 is provided in Table 17. [0052]
FIG. 11: Plasmid map of pMON25948. A list of the restriction enzyme cutting sites for pMON25948 is provided in Table 18. [0053]
FIG. 12: Plasmid map of pMON25949. A list of the restriction enzyme cutting sites for pMON25949 is provided in Table 19. [0054]
FIG. 13: Plasmid map of pMON25951. A list of the restriction enzyme cutting sites for pMON25951 is provided in Table 20. [0055]
FIG. 14: Plasmid map of pMON34545. A list of the restriction enzyme cutting sites for pMON34545 is provided in Table 21. [0056]
FIG. 15: Plasmid map of pMON34565. A list of the restriction enzyme cutting sites for pMON34565 is provided in Table 22. [0057]
FIG. 16: Plasmid map of pMON25995. A list of the restriction enzyme cutting sites for pMON25995 is provided in Table 23. [0058]
FIG. 17: Plasmid map of pMON25973. A list of the restriction enzyme cutting sites for pMON25973 is provided in Table 24. [0059]
FIG. 18: Plasmid map of pMON25987. A list of the restriction enzyme cutting sites for pMON25987 is provided in Table 25. [0060]
FIG. 19: Plasmid map of pMON25991. A list of the restriction enzyme cutting sites for pMON25991 is provided in Table 26. [0061]
FIG. 20: Plasmid map of pMON25992. A list of the restriction enzyme cutting sites for pMON25992 is provided in Table 27. [0062]
FIG. 21: Plasmid map of pMON25993. A list of the restriction enzyme cutting sites for pMON25993 is provided in Table 28. [0063]
FIG. 22: Plasmid map of pMON36805. A list of the restriction enzyme cutting sites for pMON36805 is provided in Table 29. [0064]
FIG. 23: Plasmid map of pMON36814. A list of the restriction enzyme cutting sites for pMON36814 is provided in Table 30. [0065]
FIG. 24: Plasmid map of pMON36816. A list of the restriction enzyme cutting sites for pMON36816 is provided in Table 31. [0066]
FIG. 25: Plasmid map of pMON36824. A list of the restriction enzyme cutting sites for pMON36824 is provided in Table 32. [0067]
FIG. 26: Plasmid map of pMON36843. A list of the restriction enzyme cutting sites for pMON36843 is provided in Table 33. [0068]
FIG. 27: Plasmid map of pMON34543. A list of the restriction enzyme cutting sites for pMON34543 is provided in Table 34. [0069]
FIG. 28: Plasmid map of pMON36850. A list of the restriction enzyme cutting sites for pMON36850 is provided in Table 35. [0070]
FIG. 29: Plasmid map of pMON25963. A list of the restriction enzyme cutting sites for pMON25963 is provided in Table 36. [0071]
FIG. 30: Plasmid map of pMON25965. A list of the restriction enzyme cutting sites for pMON25965 is provided in Table 37. [0072]
FIG. 31: Method for creating multi-gene vectors. [0073]
FIG. 32: PHB biosynthetic pathway. PHB production requires the condensation of two acetyl-CoA molecules using a β-ketothiolase, a D-isomer-specific reduction by acetoacetyl-CoA reductase, and PHB polymerization by PHB synthase. The genes encoding these enzymes are indicated in parentheses. [0074]
FIG. 33: Schematic diagram of multi-gene vector used to transform [0075] Brassica napus. Vectors were constructed using modular cassettes. Each cassette consists of the Lesquerella hydroxylase promoter (P-Lh), a chloroplast transit peptide (ctp) fused to an open reading frame encoding a PHB synthesis enzyme, and the E9 3′ terminator. The plasmid also expresses EPSP synthase to provide resistance to glyphosate, contains bacterial replication origins, and a bacterially-expressed gene encoding resistance to streptomycin and spectinomycin. In pMON36814, bktB was replaced with phbA. Otherwise, the vectors were identical. RB, right border of T-DNA; LB, left border of T-DNA.
FIG. 34: Electron micrographs of [0076] Brassica napus plastids. Panel A: Leukoplast from wild type Brassica napus seed. Panel B: Leukoplast from Brassica napus seed producing PHB. Polymer (PHB) and oil bodies (0) are indicated. Note the greatly expanded size of leukoplasts in the PHB-producing line.
FIG. 35: A pathway designed to produce poly(P-hydroxybutyrate-co-β-hydroxyvalerate) in the plastids of plants. Propionyl-CoA is derived from threonine via threonine deaminase and the pyruvate dehydrogenase complex. Acetyl-CoA is drawn from normal intermediary metabolism. The pathway requires transformation of the plant with four genes (ilvA, bktB, phbB, and phbC), and relies on endogenous pyruvate dehydrogenase. All enzymes encoded by transgenes are targeted to the plastid using chloroplast transit peptides. [0077]
FIG. 36: Concentrations of selected 2-keto acids and amino acids in control plants and in Arabidopsis expressing threonine deaminase. (A) Comparison of pyruvate and 2-ketobutyrate concentrations in Arabidopsis harboring either a control plasmid or a plasmid expressing wild type [0078] E. coli ilvA (threonine deaminase). (B) Comparison of threonine, isoleucine, and 2-ketobutyrate concentrations in Arabidopsis harboring either a control plasmid or a plasmid expressing wild type E. coli ilvA. Note the different scales used in parts (A) and (B).
FIG. 37: [0079] ³C NMR spectra demonstrating poly(P-hydroxybutyrate-co-β-hydroxyvalerate) copolymer production in transgenic Arabidopsis. Note the presence of signals indicating presence of both 3-hydroxybutyrate and 3-hydroxyvalerate side chains.
FIG. 38: Analyses of total polymer production, the 3-hydroxyvalerate fraction of the polymer, and the activity of threonine deaminase Brassica oilseeds synthesizing PHBV copolymer. Note the distinct negative correlation between polymer concentration and the 3-HV content of the polymer. Also note that increasing threonine deaminase activity does not lead to increased 3-HV content. [0080]
FIG. 39: Multiple potential routes to produce propionyl-CoA in planta. Most alternative pathways have the potential to produce propionyl-CoA in plants. However, production of propionyl-CoA from threonine provides the most direct route. [0081]
FIG. 40: Bar graph of average % PHA produced from Arabidopsis transformation methods. [0082]
FIG. 41: Bar graph of average % PHA produced from canola transformation methods. [0083]
FIG. 42: Bar graph of maximum % PHA produced from Arabidopsis transformation methods. [0084]
FIG. 43: Bar graph of maximum % PHA produced from canola transformation methods.[0085]

DEFINITIONS

The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention. [0086]
“Acyl-ACP thioesterase” refers to proteins which catalyze the hydrolysis of acyl-ACP thioesters. [0087]
“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). [0088]
“CoA” refers to coenzyme A. [0089]
The phrases “coding sequence”, “open reading frame”, and “structural sequence” refer to the region of continuous sequential nucleic acid triplets encoding a protein, polypeptide, or peptide sequence. [0090]
The term “encoding DNA” or “encoding nucleic acid” 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. [0091]
“Fatty acyl hydroxylase” refers to proteins which catalyze the conversion of fatty acids to hydroxylated fatty acids. [0092]
The term “genome” as it applies to bacteria encompasses both the chromosome and plasmids within a bacterial host cell. Encoding nucleic acids of the present invention introduced into bacterial host cells can therefore be either chromosomally-integrated or plasmid-localized. The term “genome” as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components of the cell. Nucleic acids of the present invention introduced into plant cells can therefore be either chromosomally-integrated or organelle-localized. [0093]
“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 v1.6 (Thompson, et al. [0094] 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. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acids) or 50 (for proteins); and multiplied by 100 to obtain a percent identity.
The terms “microbe” or “microorganism” refer to algae, bacteria, fungi, and protozoa. [0095]
“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. [0096]
“Nucleic acid” refers to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). [0097]
A “nucleic acid segment” is a nucleic acid molecule that has been isolated free of total genomic DNA of a particular species, or that has been synthesized. Included with the term “nucleic acid segment” are DNA segments, recombinant vectors, plasmids, cosmids, phagemids, phage, viruses, etcetera. [0098]
“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. [0099]
The term “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, Calif., p. 132, 1994). [0100]
“Polyadenylation signal” or “polyA signal” refers to a nucleic acid sequence located 3′ to a coding region that directs the addition of adenylate nuclecotides to the 3′ end of the mRNA transcribed from the coding region. [0101]
The term “polyhydroxyalkanoate (or PHA) synthase” refers to enzymes that convert hydroxyacyl-CoAs to polyhydroxyalkanoates and free CoA. [0102]
The term “promoter” or “promoter region” 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. As contemplated herein, a promoter or promoter region includes variations of promoters derived by means of ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences. The promoter region disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its ability to produce mRNA. [0103]
“Regeneration” refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant). [0104]
“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. [0105]
A “transformed cell” is a cell whose nucleic acid has been altered by the introduction of an exogenous nucleic acid molecule into that cell. [0106]
A “transformed plant” or “transgenic plant” is a plant whose nucleic acid has been altered by the introduction of an exogenous nucleic acid molecule into that plant, or by the introduction of an exogenous nucleic acid molecule into a plant cell from which the plant was regenerated or derived. [0107]
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. [0108]

EXAMPLES

Example 1

Sources of Nucleic Acid Sequences

Nucleic acid sequences encoding the polyhydroxyalkanoate biosynthetic pathway include: phbA and phbB (GenBank accession number J04987), phbC (GenBank accession number J05003), and bktB (GenBank accession number AF026544). Production of PHBV copolymer can be accomplished by also expressing [0109] E. coli ilvA (GenBank accession number U00096, overlapping base 3953951:Gruys et al. WO 98/00557). The Ti DNA left border sequence is described in Baker, R. F., et al. (Plant Mol. Biol., 2: 335-350, 1983). The Ti DNA right border sequence is described in Depicker et. al. (J. Mol. App. Genet. 1: 561, 1982).

Example 2

Analysis of Nawrath Arabidopsis plants

Polyhydroxyalkanoates are a form of polyester accumulated by numerous bacterial species as a carbon and energy repository. This class of polymer also has useful thermoplastic properties, and is therefore of interest as a biodegradable plastic. Poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (poly(3HB-co-3HV), PHBV), a form of PHA, is commercially produced via fermentation of Ralstonia eutropha (FIG. 1). However, it is expected that the cost of production could be dramatically decreased if PHA could be produced in transgenic plants. The first attempts at PHA production in plants utilized transgenic Arabidopsis expressing the three genes required for the homopolymer poly-β-hydroxybutyrate (PHB) (Nawrath, C. et al., Proc. Natl. Acad. Sci. U.S.A. 91: 12760-12764, 1994). In this work, the authors transformed Arabidopsis plants with three independent gene cassettes and crossed the plants using traditional breeding methods. They reported PHB production up to 14% of the cell dry weight. However, this method took a significant amount of time before the three gene pathway could be assembled. In addition, the plants did not maintain a stable phb⁺ phenotype, as determined by our analysis of the progeny of these original plants (Table 1). This problem may be due to co-suppression (Finnegan, J., and D. McElroy. Bio/Technology. 12: 883-888, 1994), or to segregation of high-producing insertions in the progeny. The plants produced by Nawrath et al. were not fully characterized genetically, although it is known that all contained multiple insertions of the transgenes.

TABLE 1


Enzyme activity and polymer data of progeny of Nawrath Arabidopsis lines.

Specific activities

Western results

%

plant line	[protein]	thiolase	reductase	PhbA	PhbB	PhbC	polymer
number	(mg/mL)	(u/mg)	(u/mg)	thiolase	reductase	synthase	(C4)

134	0.158	0.027	0.069	+	+	−	0.041%
140	0.189	0.026	0.019	+	+	−	0.068%
151	0.377	0.042	0.045	+	+	+	0.038%
159	0.127	0.025	0.009	−	−	+	0.053%
168	0.216	0.018	0.034	+	+	+	0.070%
175	0.186	0.010	0.028	+	−	−	0.043%
177	0.166	0.026	0.000	+	−	−	0.043%
203	0.144	0.030	0.043	−	+	+	0.034%
228	0.250	0.038	0.021	+	+	+	0.048%
240	0.192	0.023	0.010	NA	NA	NA	0.045%

Example 3

Use of Multiple Vectors to Introduce PHA Biosynthesis Sequences into Arabidopsis

One vector was constructed containing sequences encoding both acetoacetyl-CoA reductase and PHB synthase proteins. A second vector was constructed containing a sequence encoding a β-ketothiolase protein. Two independent transformation events were obtained corresponding to each of these vectors. The complete pathway was assembled into a single plant using traditional cross-breeding methods. In all cases, plants exhibiting Mendelian segregation consistent with transgene insertion at a single locus were chosen. The results of these experiments are shown in Table 2. [0111]

The second strategy pursued was to simultaneously co-transform both plasmids into a single plant (simultaneous co-transformation) and assay the primary transformant for polymer accumulation, or to re-transform plants that already harbored a single vector (serial co-transformation). The results of these experiments are summarized in Table 3. Although the activity of enzymes expressed from the encoding sequences was comparable to that reported by Nawrath et al., none of the plants generated reached the polymer levels reportedly achieved in their study. Neither their experiments nor these results correlate enzyme activity with the intracellular concentration of PHA polymer (Nawrath, C. et al., Proc. Natl. Acad. Sci. U.S.A. 91: 12760-12764, 1994).

TABLE 2


Polymer data for Arabidopsis crosses.

Vector	Plant construct	# of lines	# of lines	C4 polymer
Number	description	assayed	positive	(% cell dry wt.)

25640	e35s ctpl phbA			0.01-1.55%
25665	e35s ctpl phbC	11	10	AVE: 0.651%
	e35s ctpl phbB			SD: 0.596%
25640	e35s ctpl phbA			0.03-0.047%
25739	e35s ctpl phbB	20	12	AVE: 0.178%
	e35s ctpl nocC			SD: 0.163%
25785	e35s ctpl bktB			0.04-0.88%
25665	e35s ctpl phbC	11	11	AVE: 0.354%
	e35s ctpl phbB			SD: 0.199%
25785	e35s ctpl bktB			0.03-0.21%
25739	e35s ctpl phbB	24	9	AVE: 0.065%
	e35s ctpl nocC			SD: 0.053%
25801	e35s ctpl bktB			0.02-0.04%
	e35s ctpl ilvA466	8	3	AVE: 0.029%
25665	e35s ctpl phbC			SD: 0.0095%
	e35s ctpl phbB
25801	e35s ctpl bktB			0.03-0.091%
	e35s ctpl ilvA466	17	9	AVE: 0.044%
25739	e35s ctpl phbB			SD: 0.022%
	e35s ctpl nocC
25812	e35s ctpl bktB			0.03-0.102%
	e35s ctpl ilvA w.t.	3	3	AVE: 0.073%
25665	e35s ctpl phbC			SD: 0.035%
	e35s ctpl phbB
25812	e35s ctpl bktB			0.02-0.11%
	e35s ctpl ilvA w.t.	10	7	AVE: 0.064%
25739	e35s ctpl phbB			SD: 0.031%
	e35s ctpl nocC

TABLE 3


Polymer data for re-transformed and co-transformed Arabidopsis.

25665	e35s ctpl phbC			0.03-0.81%
	e35s ctpl phbB	14	6	AVE: 0.25%
RE/25880	e35s ctpl bktB			SD: 0.29%
	e35s ctpl ilvA w.t.
25665	e35s ctpl phbC
	e35s ctpl phbB	5	0	NA
RE/25881	e35s ctpl bktB
	e35s ctpl ilvA219
25665	e35s ctpl phbC			0.02-0.33%
	e35s ctpl phbB	23	4	AVE: 0.16%
RE/25882	e35s ctpl bktB			SD: 1.3%
	e35s ctpl ilvA466
25785	e35s ctpl bktB			0.02-1.67%
25678	e35s ctpl phbB	21	8	AVE: 0.50%
	e35s ctpl phbC			SD: 0.64%
25785	e35s ctpl bktB			0.01-0.72%
25740	e35s ctpl phbB	27	18	AVE: 0.11
	e35s ctpl nocC			SD: 0.15
25801	e35s ctpl bktB			0.646-0.715%
	e35s ctpl ilvA466	2	1	AVE: 0.681
25678	e35s ctpl phbB			SD: 0.049%
	e35s ctpl phbC
25801	e35s ctpl bktB			0.02-0.17
	e35s ctpl ilvA466	28	16	AVE: 0.083%
25740	e35s ctpl phbB			SD: 0.050%
	e35s ctpl nocC
25812	e35s ctpl bktB			0.63-1.65%
	e35s ctpl ilvA w.t.	3	3*	AVE: 1.191%
25678	e35s ctpl phbB			SD: 0.463%
	e35s ctpl phbC
25812	e35s ctpl bktB			0.02-0.20%
	e35s ctpl ilvA w.t.	30	9	AVE: 0.112%
25740	e35s ctpl phbB			SD: 0.053%
	e35s ctpl nocC

Example 4

Construction of Multigene Vectors for Transformation of Arabidopsis

In an attempt to increase the speed and simplicity of genetic analysis, multigene vectors were constructed containing the entire PHB biosynthetic pathway on a single plasmid. Multigene vectors for PHA production in Arabidopsis were constructed from a series of base vectors, each with the desired open reading frame under control of the e35s promoter (Odell, J. T., et al, [0114] Nature, 313: 810-812, 1985) and the E9 3′ region (Coruzzi, EMBO J. 3:1671-1679, 1984). The first vector in this series, pMON25642 (FIG. 3), harbors phbC under control of the e35s promoter in pMON10098 (FIG. 4), a vector designed for Agrobacterium-mediated transformation of plants. The remaining intermediate vectors are all derived from pMON969 (FIG. 5), a high copy-number vector harboring the e35s promoter and the E9 3′ region. Constructs derived from pMON969 include those encoding phbA (pMON25661; FIG. 6), bktB (pMON25897; FIG. 7), phbB (pMON25662; FIG. 8), and ilvA (pMON25663; FIG. 9). From these and similar vectors were derived the final plasmids for transformation of Arabidopsis; pMON25943 (FIG. 10) pMON25948 (FIG. 11), pMON25949 (FIG. 12), pMON25951 (FIG. 13), and pMON34545 (FIG. 14). All cloning procedures were performed using standard ligation techniques (Sambrook, J., et al, “Molecular cloning: A laboratory manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), except that ligation of NotI-cut pMON25949 with the ilvA-containing NotI restriction fragment of pMON25663 produced plasmid pMON34565 (FIG. 15), that serendipitously contained two copies of the ilvA fragment. Each copy of ilvA contains a SnaBI restriction site, so deletion of a 3155 bp SnaBI restriction fragment from pMON34565 produced plasmid pMON34545, a plasmid with a single copy of ilvA.

The final vectors, pMON25943, pMON25948, pMON25949, pMON25951, and pMON34545 were used for Agrobacterium-mediated transformation of Arabidopsis (Bechtold N., et al. Comptes Rendus Acad. Sci. Paris Sciences Serie III Sciences de la Vie. 94-1199, 1993). This approach has proven successful in generating lines with the highest levels of PHB obtained to date in our laboratory. PHA production in the planats resulting from the first four of these vectors is summarized in Table 4. Data from pMON34545 transformations will be obtained. All of the data in Table 4 were derived from heterozygous plants, and the polymer concentration may increase once the plants are brought to homozygosity. For example, one plant that produced about 7% PHB by dry weight when heterozygous produced polymer up to 13% when homozygous.

TABLE 4


Polymer results from Arabidopsis derived from multigene vectors.

	Plant construct	# of lines	# of lines	C4 polymer
Vector number	description	assayed	positive	(% cell dry wt.)

	e35s ctpl phbC			0.11-2.94%
25943	e35s ctp2 phbB	34	28	AVE: 1.13%
	e35s ctpl bktB			SD: 0.65%
	e35s ctpl phbC			0.01-7.63%
25948	e35s etpl phbA	53	46	AVE: 2.08%
	e35s etpl phbB			SD: 1.56%
	e35s ctpl phbC			0.02-7.74%
25949	e35s ctp2 bktB	35	30	AVE: 1.82%
	e35s ctpl phbB			SD: 1.39%
	e35s ctpl phbC			0.20-3.78%
25951	e35s ctpl bktB	12	11	AVE: 1.60%
	e35s ctpl phbB			SD: 1.04%

These results demonstrate that use of a multigene vector provides consistently higher levels of polymer production than were achieved using multiple vectors. The striking beneficial results in polymer production obtained from the use of multigene vectors are visually displayed in FIGS. [0116] 40 and 42.
There are several possible explanations for the increased levels of polymer present in the multigene vector transformants. One explanation derives from the fact that it was possible to generate more independent lines with the multigene vectors, and the screening of more plants allowed detection of the relatively rare high-producing lines. This is one clear advantage of having the entire pathway on a single vector, but the distribution of polymer production in plants produced by the various methods suggests that numbers alone do not account for the increased polymer production of multigene vectors. It is also possible that having a metabolic pathway genetically linked at a single integration locus is more metabolically favorable due to some level of concerted gene expression and/or mRNA metabolism. This phenomenon is common in bacteria, but there are not many examples of clustering genes in plants for concerted gene expression. Another possibility is that the high local concentration of promoters may lead to locally high levels of transcription factors. Still another possibility is that having the genes tightly linked may reduce gene silencing, or co-suppression, in certain cases. [0117]

Example 5

Extraction of Polymer from Arabidopsis and Analysis of Polymer

For isolation of polymer from Arabidopsis, stems and leaves were harvested and dehydrated by lyophilization for approximately 36 hours. The material was ground to a fine powder, and 100 mg of powder was treated with 10 mL CLOROX bleach (CLOROX is a registered trademark of The Clorox Company, Oakland, Calif.) for 1 hour with shaking at room temperature. The extract was subjected to centrifugation at 2700× g for 10 minutes at 4° C., and the supernatant solutions was carefully removed. Ten [0118] mL 100% methanol were added, the solution was mixed by vortexing, and then centrifuged again. After a second, identical methanol extraction, the material was allowed to dry overnight. Polymer was extracted from the dried material with 1 mL of chloroform containing 1 μmol/mL methyl-benzoate standard. The tube was heated to 100° C. for 2.5 hours, solid material was removed by centrifugation, and the supernatant material was subjected to methanolysis. Methanolysis of polymer and gas chromatographic characterization of the methyl-ester residues were performed as described by Slater et al. (J. Bacteriol. 180:1979-1987, 1998).

Example 6

Use of Multiple Vectors for Gene Expression in the Seeds of Canola

Production of polyhydroxyalkanoate has also been accomplished within the seed of canola (oil seed rape). Initial efforts followed essentially the same strategy as the initial Arabidopsis strategy. That is, one vector carried the sequences encoding acetoacetyl-CoA reductase and PHA synthase proteins, while another carried the sequence encoding a β-ketothiolase protein. However the 7s promoter, which is expressed primarily in the seed, replaced the 35s promoter that was used in the Arabidopsis constructs. These 7s promoter vectors were used to transform oilseed rape, homozygous lines were crossed, and PHB accumulation was assayed in the resulting lines (Table 5). A number of lines that produce PHB were identified, but all produced relatively low concentrations of polymer, with the best lines containing about 2% polymer by dry weight.

TABLE 5


Polymer results for canola crosses.

	Plant construct	# of plants	# of plants	C4 polymer
Vector number	description	assayed	positive	(% dry wt.)

25638	7s ctpl phbA			0.024-1.99%
25626	7s ctpl phbC	42	37	0.58%
	7s ctpl phbB			SD: 0.59%
25638	7s ctpl phbA			0.039-0.053
25741	7s tpss phbC	12	2	0.05%
	7s tpss phbB			SD: 0.01%
25818	7s ctpl bktB			0.04-1.67%
	7s ctpl ilvA w.t.	22	17	AVE: 0.61%
25626	7s ctpl phbC			SD: 0.43%
	7s ctpl phbB
25818	7s ctpl bktB
	7s ctpl ilvA w.t.	15	0	NA
25741	7s tpss phbC
	7s tpss phbB
25820	7s ctpl bktB			0.26-0.72%
	7s ctpl ilvA466	19	12	AVE: 0.51%
25626	7s ctpl phbC			SD: 0.16%
	7s ctpl phbB
25820	7s ctpl bktB
	7s ctpl ilvA466	7	0	NA
25741	7s tpss phbC
	7s tpss phbB

Example 7

Construction of Multigene Vectors for Transformation of Canola

Large vectors for expression of multiple genes have also been used to produce polyhydroxyalkanoate in the seeds of canola (oil seed rape). In this case, the promoter was derived from the fatty acid hydroxylase gene of Lesquerella (P-lh) (Broun, P. and C. Somerville. [0120] Plant Physiol. 113: 933-942, 1997), which is expressed primarily within the developing seed. A series of vectors, each expressing the entire PHA biosynthesis pathway, was used for transformation of oilseed rape. The multigene vectors were constructed from a series of base vectors, each with the desired open reading frame under control of the Lesquerella hydroxylase promoter (P-lh; Broun, P. and Somerville, C. R. Plant Physiol., 113: 933-942, 1987) and the E9 3′ region. The first vector in this series, pMON25995 (FIG. 16), harbors phbC under control of P-lh in pMON25973 (FIG. 17), a vector designed for Agrobacterium-mediated transformation of plants. The remaining intermediate vectors are all derived from pMON25987 (FIG. 18), a high copy-number vector harboring P-lh and the E9 3′ region. Constructs derived from pMON25987 (FIG. 16) include those encoding phbA (pMON25991; FIG. 19), bktB (pMON25992; FIG. 20), phbB (pMON25993; FIG. 21), and ilvA (pMON36805; FIG. 22). These intermediate vectors were used to construct the final vectors for oilseed rape transformation; pMON36814 (FIG. 23), pMON36816 (FIG. 24), and pMON36824 (FIG. 25).
Construction of the multigene vectors for oilseed rape was not as straightforward as was the construction of the Arabidopsis vectors. This was primarily due to the large size of the promoter (P-lh is about 2.2 kb), and the resulting larger size of the multigene vector intermediates. As the vectors increased in size, it was found to be most efficient to perform ligations of two similar sized fragments, rather than one large vector and one small incoming fragment. In addition, it was desirable to avoid partial digests of the large vectors, and to perform cloning in which opposite ends of an individual fragment were not compatible. A number of intermediate vectors were constructed specifically to allow cloning in this manner. Another advantage of this approach is that it often allowed restriction enzyme-mediated digestion of the parental plasmids prior to transformation of [0121] Escherichia coli with ligation products. This procedure significantly increased the frequency of correct constructs recovered. The final vectors were used for Agrobacterium-mediated transformation of oilseed rape (Fry, J. et al., Plant Cell Rep. 6: 321-325, 1987).

The results of oilseed rape transformation with the multigene vectors are shown in Table 6. There are two primary points of interest in these data. First, multigene vectors larger than 26 kb were successfully constructed and used to transform oilseed rape, with a very low percentage of the plants failing to produce polymer. Second, the distribution of polymer concentrations among multigene vector transformants is higher than that of the plants derived from two separate 7s vectors.

TABLE 6


Polymer results from canola transformed with multigene vectors.

36814	lhydrox ctpl phbC			0.19-4.11%
	lhydrox ctpl phbA	68	59	AVE: 1.43%
	lhydrox tpss phbB			SD: 1.01%
36816	lhydrox ctpl phbC	225	195	0.02-6.28%
	lhydrox ctpl bktB			AVE: 1.0%
	lhydrox tpss phbB			SD: 1.02%
36824	lhydrox ctpl phbC	185	152	0.10-2.74%
	lhydrox ctpl bktB			AVE: 0.6%
	lhydrox tpss phbB			SD: 0.5%
	lhydrox ctpl ilvA

The comparative results for PHA production in canola are graphically presented in FIGS. [0123] 41 and 43. The beneficial results obtained from the use of multigene vectors compared to results obtained from traditional methods is visually impressive.
Since the promoters used in these two vectors sets (those containing the 7s promoter and those containing the Lesquerella hydroxylase promoter) are different, it cannot be distinguished whether it was the Lesquerella promoter or the use of a single vector that led to the increased polymer concentration. However, it is clear that the single vector approach is viable for seed expression of enzymes, including those required for PHA biosynthesis. In addition, the increased speed of plant construction and analysis using a single vector is a clear benefit. [0124]

Example 8

Extraction of Polymer from Oilseed Rape and Analysis of Polymer

For isolation of polymer from canola seed, seeds were ground to a fine powder with a mortar and pestle. Approximately 200 mg of each sample were extracted two times with 10 mL each of hexane for 1 hour at 60° C., then two times with 10 mL each of 100% methanol for one hour at 60° C. This procedure removed oil from the seed. The material was allowed to dry completely overnight. Polymer was extracted from the dried material with 1 mL of chloroform containing 1 μmol/mL methylbenzoate standard. The tube was heated to 100° C. for 5 hours, solid material was removed by centrifugation, and the supernatant material was subjected to methanolysis. Methanolysis of polymer and gas chromatographic characterization of the methyl-ester residues were performed as described by Slater et al. ([0125] J. Bacteriol. 180: 1979-1987, 1998).

Example 9

Multigene Vectors for Gene Expression in Mmonocots

For reasons described above, multigene vectors will also be desirable for expression of multi-enzyme metabolic pathways in monocots. Therefore, vectors designed to produce PHA in the leaves of maize were constructed. These vectors use the e35s, eFMV, or maize chlorophyll A/B binding protein (P-ChlA/B) promoters, and include the HSP70 intron designed to enhance expression in monocots. All enzymes were fused to the Arabidopsis RuBisCo small subunit transit peptide. Other promoters might also be used. Examples of vectors designed for gene expression in monocots are pMON36843 (FIG. 26), pMON34543 (FIG. 27), and pMON36850 (FIG. 28). These vectors have been used to transform maize, and polymer was analyzed as described above for Arabidopsis. Polymer production is summarized in Table 7.

TABLE 7


Polymer production in maize using multigene vectors.

36843	P-e35S phbC			1.14-4.81%
	P-e35S phbA	93	11	AVE: 1.84%
	P-e35S phbB			SD: 1.04%
34543	P-eFMV phbC	34	34	0.15-2.95%
	P-eFMV phbA			AVE: 0.7%
	P-eFMV phbB			SD: 0.9%
36850	P-ChlA/B, phbC	132	78	0.1-5.66%
	P-ChlA/B, phbA			AVE: 1.72%
	P-ChlA/B, phbB			SD: 1.17%

Example 10

System for Construction of Large, Multigene Vectors

Since multigene vectors are optimal for producing high levels of PHB, and this strategy is potentially optimal for expression of other multiple step pathways, a simple method to produce very large, multigene vectors is preferred. FIGS. 29 and 30 show plasmids pMON25963 and pMON25965, respectively. These vectors, used together, provide a system for constructing very large vectors. Plasmid pMON25965 provides a shuttle vector by which a gene cassette can be cloned into the NotI restriction sites and thereby be flanked by a series of restriction sites. These restriction sites are relatively rare in many genomes, and thereby of utility for subcloning many genes. Plasmid pMON25963 is a binary vector designed for transformation of plants by Agrobacterium. It contains a polylinker with the same sites found flanking the NotI restriction sites of plasmid pMON25965. Using this system, a series of gene “cassettes” can be produced using plasmid pMON25965, and each can be sequentially ligated into plasmid pMON25963. [0127]
In practice, a series of vectors similar to pMON25965, but having smaller polylinkers, will be preferred. Specifically, this series of vectors would have a single NotI (or similar enzyme) restriction site flanked by one or several other restriction enzyme sites. By ligating cassettes flanked by large portions of the pMON25965 polylinker into pMON25963, relatively large inverted repeats of polylinker DNA are formed. These inverted repeats are unstable in [0128] Escherichia coli, and plasmids harboring them do not replicate efficiently. Thus, diminishing the size of the polylinker in the shuttle vector can increase the probability of recovering stable recombinants.
Another strategy for generating multigene vectors and reducing the levels of background caused by vector re-ligation is shown in FIG. 31. This strategy could be adapted to accommodate any number of enzymes, depending on the availability of unique restriction sites. One can easily design such a polylinker to accommodate one's cloning needs. As the vector becomes larger, one will want to have a larger homologous overlap for the ligation process or choose restriction endonucleases producing ends that are very easily ligated, and not self-compatible. By following the cloning procedure outlined in FIG. 31, one can also control the directionality of the clone. If directionality is not important than clones generated from the ligation into the “shuttle vector” in either orientation could be used. (A←C or A→C). [0129]
As with any multigene vector strategy, the starting plasmid used for constructing the large multigene plasmids should be taken into consideration. The common plant transformation plasmid pBIN19 (Frisch, D. et al., [0130] Plant Mol Biol 27: 405-409, 1995) has a starting size of 11,777 bp. In contrast plasmid pMON10098 (FIG. 4) has a starting size of 8431 bp. The major difference between the two plasmids is the loss of the trfA function which is encoded in trans in Agrobacterium strain ABI. Providing the trfA function in trans allows replication only in the specific strains of Agrobacterium engineered to harbor trfA. It has been shown by Figurski and Helinski (Proc. Natl. Acad. Sci. U.S.A. 76: 1648-1652, 1979) that replication factors can function in trans. By providing the minimal origins of replication required for maintenance in both Escherichia coli and Agrobacterium the starting size of the initial plasmid can be reduced significantly.
Other possibilities to reduce the size of the starting plasmid would be to delete oriT since this sequence is required for conjugational transfer only. If electroporation is used to introduce the plasmid into Agrobacterium, oriT is not an essential element. Another possibility would be to use selection that is functional in plants, Agrobacterium, and [0131] Escherichia coli. This could be accomplished by embedding into the plant promoter for the selectable marker a suitable bacterial promoter sequence and a ribosome binding site in proper context with the start codon on the selectable marker. One could also place this selectable marker on the plasmid flanked by its own right and left border sequences. This may allow for the selectable marker to be integrated into the plant chromosome unlinked to the genes of interest and potentially removed from subsequent generations. Alternatively, plants could be co-transformed by taking the multigene plasmid and cotransforming on a separate plasmid the selectable marker for plants. This would eliminate the cloning of the selectable marker on the multi gene plasmid. The selectable marker can be delivered by mixing two different Agrobacterium strains, one containing the multigene plasmid and the other containing the selectable marker, or by using the same Agrobacterium strain but having different isolates containing either the multi gene plasmid or the selectable marker, or by having the selectable marker coexisting in the same Agrobacterium cell with the multigene vector, but on a separate plasmid with a compatible origin of replication.
One can also envision reducing the size of the selectable marker being used by using a trans complementation strategy. For example, one could transform a plant with a portion of a NptII gene that expresses a partial protein. If the transformation plasmid carries the complementary portion of the NptII protein, both fragments of the NptII protein may interact to confer resistance to kanamycin. This is analogous to the α-complementation strategy used for creating functional β-galactosidase (reviewed by Zabin, I. [0132] Mol. Cell. Biochem. 49: 87-96, 1982).
An example of an optimal starting plasmid for engineering multiple genes in plants would contain only the minimal essential elements required for replication in [0133] Escherichia coli and in Agrobacterium (having all other required functions encoded in trans) as well as a selection scheme that (1) reduces the need for redundancy in the selectable marker, and/or (2) reduces the size of the selectable marker, or (3) removes the necessity of having the plant selectable marker on the multi gene plasmid. The promoter used for driving the gene of interest in the multi gene vector should consist of the minimal essential elements required for temporal and spatial expression. The termination and polyadenylation signals should also contain only those sequences required for essential function.

Example 11

Poly(β-hydroxybutyrate) Production in Oil Seed Leukoplasts of Brassica napus

Using plants as factories is attractive for the production of biodegradable plastics since current fermentation technology used for the commercial production of polyhydroxyalkanoates (PHA) is prohibitively expensive. The simplest PHA, poly-β-hydroxybutyrate (PHB), has previously been produced in leaves of [0134] Arabidopsis thaliana (Nawrath, C., et al., Proc. Natl. Acad. Sci., U.S.A., 91: 12760-12764, 1994). Brassica napus oilseed, however, may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize the PHB polymer: a β-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate- 12 hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform Brassica napus. PHB accumulated in leukoplasts to levels as high as 7.7% of seed dry weight. Electron microscopy analyses indicate that leukoplasts from these plants are distorted, yet intact, and appear to expand in response to polymer accumulation.
Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally-sustainable alternative to petroleum based plastics (reviewed by Poirier, Y., et al., [0135] Biotechnology, 13: 142-150, 1995). The homopolymer Poly(β-hydroxybutyrate) (PHB), a particularly well studied PHA, is normally synthesized by various species of bacteria under conditions where nutrients become limited. PHB is stored in granules which can later be mobilized to provide a carbon and energy resource for the bacteria.
One of the best-studied pathways for PHB synthesis is derived from the bacterium [0136] Ralstonia eutropha (Slater, S. C., et al, J. Bacteriol., 170: 4431-4436, 1988; Schubert, P., et al., J. Bact., 170: 5837-47, 1988; Peoples, O. P., and Sinskey, A. J., J. Biol. Chem., 264: 15298-15303, 1989; Peoples, O. P., and Sinskey, A. J., J. Biol. Chem., 264: 15293-15297, 1989). The pathway requires three enzymes: a β-ketothiolase, an acetoacetyl-CoA reductase, and a PHB synthase (FIG. 32). R. eutropha uses least two β-ketothiolases, PhbA and BktB (Slater, S. C., et al., J. Bact., 180: 1979-1987, 1998), and both of these enzymes were used in this study. The acetoacetyl-CoA reductase and PHB synthase are designated PhbB and PhbC, respectively (Peoples, O. P., and Sinskey, A. J., J. Biol. Chem, 264: 15298-15303, 1989; Peoples, O. P., and Sinskey, A. J., J. Biol. Chem., 264: 15293-15297, 1989).
[0137] R. eutropha is fermented commercially for PHA production, but the process is not economically competitive with polymers derived from petroleum. Therefore, novel commercial efforts to produce PHAs focus on using plants as polymer factories. In this respect, our laboratory is considering two model systems: production in leaves and production in seeds. Since acetyl-CoA is a central metabolite for both PHB and fatty acid biosynthesis, and Brassica napus seeds are extremely efficient in oil production, the Brassica seeds seem an optimal environment in which to produce PHB (U.S. Pat. No. 5,502,273). Production of PHB in Arabidopsis thaliana leaves has been achieved using R. eutropha enzymes (Poirier, Y., et al., Science, 256: 520-523, 1992), and additional work showed that polymer accumulation up to 14% of plant dry weight was achieved when the PHB biosynthetic enzymes were targeted to the plastid (Nawrath, C., et al., Proc. Nat. Acad. Sci., 91: 12760-12764, 1994).
The work presented here demonstrates polymer production in the seeds of [0138] Brassica napus using a multi-gene vector approach. A significant advantage to using these multi-gene vectors is that the entire PHA pathway is introduced simultaneously, thereby obviating the need for elaborate crossing strategies and eliminating the problems associated with insertional effects at multiple loci. Construction of these multi-gene vectors involved the generation of modular cassettes, each harboring an individual gene. The cassettes were then assembled into a single vector expressing the entire PHB biosynthetic pathway (FIG. 33). Each cassette consisted of the Lesquerella fendleri oleate-12 hydroxylase promoter (Broun, P., et al., Plant J., 13: 201-210, 1998), a chloroplast transit peptide fused to the open reading frame of interest (bktB, phbA, phbB, or phbC), and the 3′ termination region of the Pisum sativum rbcSE9 gene (Coruzzi, G., et al., EMBO J., 3: 1671-1679, 1984). The Lesquerella promoter contains 2.2 kb of DNA upstream of the coding region for the oleate-12 hydroxylase gene. This promoter was chosen because it is expressed concurrently with the accumulation of storage lipid (Broun, P., et al., Plant J., 13: 201-210, 1998).

Expression of the PHB pathway in B. napus was achieved using Agrobacterium-mediated transformation, and glyphosate selection was used to identify transgenic events (Fry, J., et al., Plant Cell Rep., 6: 321-325, 1987). The T-DNA transferred into the plants from these experiments exceeded 16 kilobases in size. The co-expression rate of genes from the multi-gene vectors in Brassica seeds was high, with 87% of the glyphosate resistant plants also producing polymer. Polymer levels ranged from 0.02-7.7% for the transgenic plants carrying pMON36814 (R. eutropha phbA, phbB, phbC) and 0.02-6.3% for those carrying pMON36816 (R. eutropha bktB, phbB and phbC). The vast majority of plants producing polymer fall within the 0-3.0% polymer range (Table 8) and all polymer-producing lines generated viable seed.

TABLE 8


Polymer results from canola multigene vector transformations.

	Genetic	# of plants	# of plants	C4 polymer
Vector	elements	assayed	positive	(% dry wt.)

	p-Lh, phbC			0.02%-7.68%
36814	p-Lh, phbA	208	180	Avg: 1.73%
	p-Lh, phbB			SD: 1.45%
	p-Lh, phbC			0.02%-6.28%
36816	p-Lh, bktB	225	195	Avg: 1.00%
	p-Lh, phbB			SD: 1.02%

The [0140] B. napus line displaying 7.7% polymer was further analyzed by electron microscopy. Micrographs revealed that polymer accumulated within the plastid (FIG. 34), and that essentially every plastid contained polymer. Polymer production in the plastids is seemingly well tolerated; the size of the plastid expands to accommodate polymer production (compare FIGS. 34A and 34B). This phenomenon is similar to the size changes observed when amyloplasts accumulate starch, and suggests that plastids will change size to accommodate accumulation of any granular product. Thus, the signal initiating an increase in plastid volume is not specifically linked to accumulation of normal metabolites; rather, the increase is probably initiated simply by physical pressure applied to the plastid membrane.
These results demonstrate that PHA accumulation is possible in an oilseed system. Commercial oilseed PHA production will require approximately twice the amount of PHA accumulation achieved here. Moreover, commercial success will rely on the development of an integrated processing system to extract PHA, oil, and meal from the seeds. We believe that increases in PHA accumulation can be obtained using alternative promoters that are stronger and expressed for a longer duration during seed development. Other concerns regarding the feasibility of PHA production in planta largely revolve around the metabolic effects of PHA production in oilseeds. Specifically, analysis of the effect of PHA production on oil yield will be of particular interest, since both are derived from acetyl-CoA and produced simultaneously. Any untoward effect of PHA production on oil yield or seed quality will impact negatively on the economic feasibility of using [0141] B. napus as a commercial system.
Vector Construction and Plant Transformation [0142]
A single vector encoding the entire PHB biosynthetic pathway was used for Agrobacterium-mediated transformation of Brassica. This vector, pMON36814, encodes bktB, phbB, and phbC (FIG. 33). Each gene of interest was fused to a chloroplast transit peptide (ctp), so each protein is transported to the seed leukoplast. All enzymes were fused to the Arabidopsis RuBisCo small subunit la transit peptide that was previously used for PHB production (Nawrath, C, et al., [0143] Proc. Nat. Acad. Sci., 91: 12760-12764, 1994) except PhbB was fused to the transit peptide from pea RuBisCo small subunit (Cashmore, A. R., eds. Kosuge, T., Meredith C. P., Hollaender, A., (Plenum, N.Y.), 29-38, 1983). Each gene is controlled by the promoter from the fatty acid hydroxylase gene of Lesquerella (P-Lh; Broun, P., et al., Plant J., 13: 201-210, 1998), and terminated with the E9 3′ region of the Pisum rbcSE9 gene (Coruzzi, G., et al., EMBO J., 3: 1671-1679, 1984). P-Lh directs expression of these genes within the developing seed. The selection cassette for pMON36812 and 36814 consisted of the Figwort Mosaic Virus promoter followed by the Petunia RuBisCo small subunit 1a transit peptide, the Petunia EPSP synthase gene (CP4) and nopaline synthase 3′ termination/polyadenylation region (nos3′).
Transformation of [0144] Brassica napus was done as described in Fry, J. et al. (Plant Cell Rep., 6: 321-325, 1987) using glyphosate for selection.
Polymer Analysis [0145]
For isolation of polymer from canola seed, seeds were ground to a fine powder with a mortar and pestle. Approximately 200 mg of each sample were extracted two times in a glass tube with 10 mL each of hexane for 1 hour at 60° C., then two times with 10 mL each of 100% methanol for one hour at 60° C. This procedure removes oil from the seed. The material was allowed to dry completely overnight. Polymer was extracted from the dried material with 1 mL of chloroform containing 3 μmol/mL methyl-benzoate standard. The tube was heated to 100° C. for 5 hours and the samples were cooled. One mL methanol/sulphuric acid (85:15, v/v) was added, and the mixture was heated to 100° C. for exactly 2.5 hours. The solution was cooled, extracted with water and subjected to gas chromatography. Gas chromatographic characterization of the methyl-ester residues was performed as described by (Slater, S., et al., [0146] J. Bact., 180: 1979-1987, 1998) except that the temperature gradient was performed as follows: the initial temperature of 70° C. was held for 6 minutes, then the temperature was increased by 30° C. per minute to 130° C. Finally, the temperature was increased by 50° C. per minute to 300° C. and held at 300° C. for 5 minutes.
Electron Microscopy [0147]
Partial imbibition of Brassica seeds was achieved by the slight abrasion of the seed coats, followed by placement for 2 hours onto filter paper moistened with distilled water. The cotyledons of these seeds were then cut into 1 mm[0148] ³pieces and fixed in 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 for three hours, with the first 30 minutes under vacuum. The tissue was post-fixed in 1% osmium tetroxide in the above buffer, dehydrated in ethanol and propylene oxide and infiltrated with a 1:1 mixture of Spurr's: EMbed 812 resin. The resin was polymerized at 60° C. for 48 hours. The resulting blocks were sectioned on an Leica Ultracut E microtome. Sections 80 nm thick were picked up on formvar/carbon coated copper slot grids. The grids were post-stained with uranyl acetate and lead citrate in an LKB ultrastainer and examined with a JEOL 1200 transmission electron microscope. (All reagents were obtained from Electron Microscopy Sciences, Fort Washington, Pa.).

Example 12

Metabolic Engineering of Arabidopsis and Brassica for Poly(β-hydroxybulyrate-co-β-hydroxyvalerate) Copolymer Production

Poly(hydroxyalkanoates) are natural polymers with thermoplastic properties. One polymer of this class, poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) is currently produced by bacterial fermentation, but the process is not economically competitive with polymer production from petrochemicals. PHA production in green plants promises much lower costs, but producing polymer with the appropriate monomer composition is problematic. By redirecting metabolic pools of both short-chain fatty acids and amino acids, Arabidopsis and Brassica have now been engineered to produce PHBV, a copolymer with commercial applicability. In this Example, polymer production, metabolic intermediate analyses, and pathway dynamics for PHBV synthesis in planta are described. [0149]
Poly(hydroxyalkanoates) (PHAs) are a class of polymers accumulated by numerous bacterial species as carbon and energy reserves. These polymers have thermoplastic properties, and have received much attention as biodegradable alternatives to petrochemical plastics (Anderson, A. J., and Dawes, E. A. [0150] Microbiol. Rev. 54: 450-472, 1990). While the homopolymer poly(β-hydroxybutyrate) (PHB) is somewhat brittle, many copolymers such as poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) are more flexible due to reduced crystallinity, and suitable for many commercial applications.
The biochemical pathways for PHB and PHBV production are essentially identical, differing only in the initial metabolites. PHB synthesis is initiated by condensation of two acetyl-CoA molecules, whereas PHBV synthesis requires the additional condensation of acetyl-CoA with propionyl-CoA. Following condensation, the products are reduced by a D-isomer specific acetoacetyl-CoA reductase, and the resulting β-hydroxy products are polymerized by PHB synthase (Anderson, A. J., and Dawes, E. A. [0151] Microbiol. Rev. 54: 450-472, 1990; Steinbüchel and Schlegel, Mol. Microbiol. 5(3):535-42, 1991).
PHBV is produced commercially by growing [0152] Ralstonia eutropha on glucose and propionate (Byrum, D. FEMS Microbiol. Rev. 102: 247-250, 1992), but the cost of this process prohibits large-scale fermentation. Production of PHAs via genetic engineering of green plants is expected to reduce costs to economical levels (van der Leij, F. R., and Witholt, B. Can. J. Microbiol. 41(Suppl.1): 222-238, 1995), and production of PHB homopolymer in plants has been demonstrated (Poirier, Y., et al. Science 256: 520-523, 1992; Nawrath, C.; et al. Proc. Natl. Acad Sci. 91: 12760-12764, 1994). However copolymer production has been problematic, primarily due to the requirement for metabolic precursors other than acetyl-CoA.
Here we report metabolic engineering of plants to produce PHBV copolymer. By expressing four distinct transgenes and diverting metabolic pools of acetyl-CoA and threonine, copolymer was produced in [0153] Arabidopsis thaliana, and in the seeds of Brassica napus (oilseed rape). PHBV copolymer production opens the use of green plants as factories for commercial, environmentally-sustainable production of biodegradable plastics.
Results: A Pathway for Poly(β-hydroxybutyrate-co-β-hydroxyvalerate) Production in Plants [0154]
A pathway designed to engineer PHBV production in the plastids of plants is diagrammed in FIG. 35. Acetyl-CoA is drawn from plastid intermediary metabolism, whereas propionyl-CoA is generated from threonine via 2-ketobutyrate (Gruys et al WO 98/00557; Eschenlauer, A. C., et al. [0155] Int. J. Biol. Macromol. 19: 121-130, 1996). This pathway requires transformation of the plant with four separate genes: ilvA, bktB, phbB, and phbC. It also relies on the endogenous plastid pyruvate dehydrogenase complex (PDC). The threonine deaminase used in these studies is the biosynthetic enzyme IlvA from E. coli (Taillon, B. E., et al. Gene 63: 245-252, 1988). The acetoacetyl-CoA reductase (PhbB) and PHB synthase (PhbC) are the same R. eutropha enzymes used in earlier in planta studies (Poirier, Y., et al. Science 256: 520-523, 1992; Nawrath, C.; et al. Proc. Natl. Acad. Sci. 91: 12760-12764, 1994). The β-ketothiolase is BktB from R. eutropha (Slater, S., et al. J. Bacteriol. 180: 1979-1987, 1998). Previous work on PHB production in plants used the R. eutropha PhbA β-ketothiolase. However, PhbA cannot efficiently synthesize β-ketovaleryl-CoA, whereas BktB produces both β-ketovaleryl-CoA and acetoacetyl-CoA.
Metabolic Engineering of Arabidopsis and Brassica [0156]
Polymer production was studied in both [0157] Arabidopsis thaliana leaves and Brassica napus seeds. For PHBV production in Arabidopsis, two separate vectors were constructed. Plasmid pMON25678 encodes phbB and phbC, and plasmid pMON25812 encodes bktB and ilvA. Transgenic Arabidopsis were generated by simultaneous Agrobacterium-mediated transformation with both vectors, and subsequent selection on both glyphosate and kanamycin. All genes were controlled by the e35S promoter (Odell, J. T., et al. Nature 313: 810-812, 1985), leading to polymer production throughout the plant. In Brassica, all four genes in the transgenic pathway were expressed from a single vector, pMON36824, and polymer production was directed to the seeds by the Lesquerella hydroxylase promoter (Broun, P., et al. Plant J. 13: 201-210, 1998).
Previous work on PHA production in plants has shown that polymer is produced efficiently and that phenotypic effects on the plant are minimized when PHA production occurs in the chloroplasts (Nawrath, C. et al. [0158] Proc. Natl. Acad. Sci. 91: 12760-12764, 1994). The plastids are the site for synthesis of both oil, which is derived from acetyl-CoA, and threonine which is used to produce propionyl-CoA. In both Arabidopsis and Brassica, the PHA biosynthesis enzymes were targeted to the plastids using chloroplast transit peptides. In photosynthetic tissues of Arabidopsis the proteins are targeted to the chloroplasts, whereas in Brassica seeds the enzymes are targeted to the leucoplasts.
Generation of Propionyl-CoA from Threonine [0159]
Conversion of threonine to 2-ketobutyrate by IlvA is the first reaction catalyzed by one of the recombinantly-encoded enzymes. IlvA normally catalyzes the initial step in the conversion of threonine to isoleucine, and the enzyme is feedback-inhibited by isoleucine (Umbarger, H. E. Biosynthesis of branched-chain amino acids, pp. 442-457 in [0160] Escherichia coli and Salmonella: Cellular and Molecular Biology, Neidhart, F. C., Curtiss, R., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., and Umbarger, H. E. (eds.).ASM Press, Washington, D.C., 1996). However, ilvA mutants with diminished sensitivity to isoleucine have been described and two such mutants, ilvA466 (Pledger, W. J., and Umbarger, H. E. J. Bacteriol. 114: 183-194, 1973; Taillon, B. E., et al. Gene 63: 245-252, 1988) and ilvA219 (Burns, R. O., et al. J. Biol. Chem. 254: 1074-1079, 1979; Eisenstein, E., et al. Biochemistry 34: 9403-9412, 1995), were used along with wild-type ilvA in these studies. IlvA466 is partially sensitive to feedback inhibition by isoleucine, and IlvA219 is essentially insensitive (Pledger, W. J., and Umbarger, H. E. J. Bacteriol. 114: 195-207, 1973; LaRossa, R. A., et al. J. Bacteriol. 169: 1372-1378, 1987).
Both Arabidopsis and Brassica were initially transformed with separate vectors expressing wild-type ilvA, ilvA466, and ilvA219. In both organisms, no fertile transformants expressing ilvA219 were recovered, indicating that expression of completely isoleucine-insensitive IlvA is lethal. In Arabidopsis, plants expressing ilvA466 were recovered at a very low frequency, whereas Brassica tolerated ilvA466 rather well. This result may be due to the seed-specific nature of the Lesquerella promoter. Transformants expressing wild-type ilvA were efficiently recovered in both Arabidopsis and Brassica. [0161]
In order to monitor the metabolic effects of IlvA in transgenic plants, metabolites likely to be effected by this enzyme were analyzed. FIG. 36 shows profiles of selected 2-ketoacids and amino acids in a control plant, and in transgenic Arabidopsis expressing wild-type ilvA. As expected, the transgenic plant had elevated levels of both 2-ketobutyrate and isoleucine. In addition, a high concentration of 2-aminobutyrate was present. Formation of 2-aminobutyrate from 2-ketobutyrate is a freely-reversible reaction, probably catalyzed by the same branched-chain amino acid transaminase that catalyzes the final step in isoleucine biosynthesis (Singh, B. K. (1999) Biosynthesis of Valine, Leucine and Isoleucine. In: Singh, B. K. (ed.) Plant Amino Acids: Biochemistry and Biotechnology. Marcel Dekker, Inc., New York, pp.227-247, 1998). Although transgenic plants expressing ilvA contained more 2-ketobutyrate than did wild-type plants, the 2-ketobutyrate concentration was still below that of pyruvate. Most 2-ketobutyrate was apparently diverted to produce 2-aminobutyrate and isoleucine. The concentration of free threonine in a plant expressing ilvA decreased by only about 15%, suggesting that threonine synthesis was sufficiently robust to compensate for the diversion of threonine through 2-ketobutyrate. Similar analyses were performed on the seeds from control and transgenic Brassica, and essentially the same results were obtained. In plants expressing ilvA, isoleucine, 2-ketobutyrate, and 2-aminobutyrate concentrations were elevated, and free threonine was only marginally decreased (K. Gruys et al., unpublished data). [0162]
The second step in the formation of propionyl-CoA is catalyzed by the plastid pyruvate dehydrogenase complex, which is the sole endogenous enzyme required for PHBV production. This enzyme complex normally plays a central role in metabolism by converting pyruvate to acetyl-CoA. We found that PDC from isolated Brassica leukoplasts was also capable of converting 2-ketobutyrate to propionyl-CoA. However, PDC was approximately 10-fold less efficient when utilizing 2-ketobutyrate than when utilizing pyruvate; the specific activities were 0.4 units/mg and 3.6 units/mg for 2-ketobutyrate and pyruvate, respectively. [0163]
Synthesis of PHBV Copolymer [0164]
Once propionyl-CoA has been produced, the pathway is identical to that shown to produce PHBV copolymer in recombinant [0165] E. Coli (Slater, S., et al. J. Bacteriol. 180: 1979-1987, 1998). Propionyl-CoA is converted to D-β-hydroxyvaleryl-CoA by BktB and PhbB, and then is polymerized with D-β-hydroxybutyryl-CoA to form PHBV copolymer. The functionality of the entire pathway in plants is shown in FIG. 37, which shows ¹H-NMR spectra demonstrating the presence of PHBV copolymer in Arabidopsis. We also obtained ¹³C-NMR demonstrating PHBV copolymer production in Brassica, and all these data have been corroborated by coupled gas chromatography-mass spectrometry (data not shown). The molecular weight of PHBV isolated from Brassica seeds was approximately 1×10⁶, with a polydispersity index of 2.4. These parameters are suitable for commercial applications.
Although copolymer was made in both Arabidopsis and Brassica, the 3-hydroxyvalerate component varied with the in vivo polymer concentration. The polymer composition in Brassica seeds distinctly showed a negative correlation between the 3-hydroxyvalerate content of the polymer and total polymer production (FIG. 38). Threonine deaminase activity also negatively correlated with 3-HV content (FIG. 38), a somewhat surprising result considering the role of IlvA in the production of 3-HV. However, we have consistently found that introduction of vectors encoding multiple genes leads to a general, concerted expression of all encoded enzymes. Thus, elevated IlvA activity is consistent with elevated polymer production. [0166]
Discussion [0167]
The use of green plants as industrial factories will often require significant changes in plant metabolism, so metabolic engineering of multi-step pathways will become an important technology in “green chemistry” efforts. In this study, production of the PHA copolymer PHBV has been accomplished using a combination of endogenous and transgene-encoded enzymes. The pathway consists of five separate enzymes, four being encoded as transgenes. In the case of Brassica, all four genes were successfully introduced on a single vector. [0168]
Commercial application of this technology will rest on two primary metabolic issues: 1) can polymer be produced in planta to concentrations amenable to economical polymer extraction? and 2) as the polymer concentration increases, can the appropriate monomer composition be maintained? We expect that polymer concentrations in planta will need to reach at least 15% of dry weight for economical production to be feasible. PHB homopolymer concentrations near 15% have been reported (Nawrath, C. et al. [0169] Proc. Natl. Acad. Sci. 91: 12760-12764, 1994) and have also been achieved in our laboratory (data not shown). Thus, high-level PHB production appears technically attainable.
Production of PHBV copolymer has been accomplished in this study, although all plants produced copolymer at levels below 3% of plant tissue dry weight. The next challenge is high-level production of copolymer, and the data in FIG. 38 show that additional work is required to maintain the 3-hydroxyvalerate composition at high polymer concentrations. Specifically, as polymer production increased, the 3-hydroxyvalerate fraction of the polymer decreased, and increasing threonine deaminase expression did not effect this correlation. These data suggest a metabolic bottleneck in the provision of 3-hydroxyvalerate to PHA synthase. The BktB, PhbB, PhbC pathway efficiently synthesizes PHBV copolymer (Slater, S., et al. [0170] J. Bacteriol. 180: 1979-1987, 1998), and production of 2-ketobutyrate in planta is efficient, as estimated from the elevated levels of 2-ketobutyrate, 2-aminobutyrate and isoleucine (FIG. 36). Thus, the metabolic bottleneck must exist at the conversion of 2-ketobutyrate to propionyl-CoA by the pyruvate dehydrogenase complex. As noted above, the PDC strongly prefers pyruvate as a substrate, and this difference is compounded in vivo by the concentration ratio of pyruvate to 2-ketobutyrate (FIG. 36). Pyruvate dehydrogenase apparently cannot effectively compete for 2-ketobutyrate so propionyl-CoA synthesis is limited..
Production of copolymer to high internal concentrations may require a supplementary route for conversion of 2-ketobutyrate to propionyl-CoA. There are several ways to bypass the PDC or supplement its activity, but all will require additional transgenes. These routes include modifying the α-ketoacid dehydrogenase to more readily accept propionyl-CoA (Inoue H, et al. [0171] J. Bacteriol. 179: 3956-3962, 1997; Gruys et al WO 98/00557), expression of an alternative enzyme complex capable of forming propionyl-CoA from 2-ketobutyrate (Kerscher, L. and Oesterhelt, D., Eur. J. Biochem. 116: 587-594, 1981), or co-expression of a propionyl-CoA dehydrogenase (Horswill et al; Mitsky et al., unpublished data) with a propionyl-CoA synthetase or CoA transferase (Gruys et al WO 98/00557; Valentin et al, manuscript in preparation). Thus, a commercially viable transgenic plant producing PHA polymer from threonine may contain up to six separate transgenes.
Synthesis of propionyl-CoA can also be achieved through other metabolic pathways, although none presents a straightforward alternative to the threonine derived pathway (FIG. 39). For instance, propionyl-CoA may be generated from acetyl-CoA using a 5-step pathway, part of which is involved in propionyl-CoA degradation in plants (Goodwin, T. W. and Mercer, E. I. Introduction to Plant Biochemistry. Second Edition. Pergamon Press, Oxford, 1985; Eisenreich, W., et al. [0172] Eur. J. Biochem. 215: 619-632, 1993; Preifert, H., and Steinbüchel, A. J. Bacteriol. 174: 6590-6599, 1992; Podkowinski, J., et al. Proc. Natl. Acad. Sci. USA 93: 1870-1874, 1996; Sun, J., et al. Plant Physiol. 115: 1371-1383, 1997; Horswill A. R., and Escalante-Semerena J. C. J. Bacteriol. 179: 928-940, 1997; Gruys et al, unpublished data). Conversion of acrylyl-CoA to propionyl-CoA is potentially problematic, but an appropriate enzyme may be available from Chroroflexus aurantiacus (Eisenreich, W., et al. Eur. J. Biochem. 215: 619-632, 1993). Propionyl-CoA can also be derived from succinyl-CoA using a pathway present in both Rhodococcus ruber and Nocardia corallina (Williams, D. R.,et al. Appl. Microbiol. Biotechnol. 40: 717-723, 1994; Valentin, H. E., and Dennis, D. Appl. Environ. Microbiol. 62: 372-379, 1996). This pathway is initiated by methylmalonyl-CoA mutase, an enzyme that requires vitamin B₁₂as a cofactor. However, vitamin B₁₂is not synthesized in plants (Goodwin, T. W. and Mercer, E. I. Introduction to Plant Biochemistry. Second Edition. Pergamon Press, Oxford, 1985). Rhodococcus and Nocardia also produce minor amounts of 3-hydroxyvaleryl-CoA via a different, uncharacterized route. This route may be a link to amino acid metabolism, such as the pathways used by other bacteria and animals to degrade valine and isoleucine (FIG. 39). These pathways might also be engineered in plants, but a large number of genes are required.
Several other amino acids can be used to produce propionyl-CoA. Methionine, like threonine, generates 2-ketobutyrate during catabolism. This conversion is catalyzed by L-methionine γ-lyase in a reaction that also produces ammonia and methanethiol (Tanaka, H., et al. [0173] Enzyme Microb. Technol. 7: 530-537, 1985). The effect of methanethiol production on plants is unknown, and supplementation of PDC activity would still be required to efficiently produce propionyl-CoA. Another pathway, present in Clostridium propionicum, converts alanine to propionyl-CoA via lactic acid, lactyl-CoA and acrylyl-CoA (Schweiger, G., and Buckel, W. FEBS Lett. 171: 79-84, 1984; Cardon, B. P., and Barker, H. A. Arch. Biochem. Biophys. 12: 165-180, 1947). However, none of the required genes has been cloned, and some of the necessary enzymes are oxygen sensitive (Hofmeister, A. E. M., and Buckel, W. Eur. J. Biochem. 206: 547-552, 1992; Kuchta, R. D., and Abeles, R. H. J. Biol. Chem. 260: 13181-13189, 1985). β-alanine is another potential starting metabolite for the production of propionyl-CoA (Arst, H. N. Jr. Mol. Gen. Genet. 163: 23-27, 1978; Roberts, E., and Bregoff, H. M. J. Biol. Chem. 201: 393-398, 1953; Kupiecki, R. P., and Coon, M. J. J. Biol. Chem. 229: 743-754, 1957). β-alanine normally plays a critical role as a precursor to Coenzyme-A and acyl carrier protein. However, little is known about the concentration and compartmentalization of β-alanine in plants, and propionyl-CoA may actually be required for its synthesis.
In summary, poly(β-hydroxybutyrate-co-β-hydroxyvalerate) copolymer was produced in both Arabidopsis and Brassica by simultaneously accessing amino acid and short-chain fatty acid metabolite pools. In Brassica, all four required transgenes were introduced on a single vector, eliminating the plant crossing normally necessary to assemble a pathway of this size. The polymer molecular mass was adequate for commercial purposes, but an apparent metabolic bottleneck in conversion of 2-ketobutyrate to propionyl-CoA suggests that additional engineering may be required to achieve high-level production of polymer with the necessary P-hydroxyvalerate composition. [0174]
Generation of IlvA Mutants [0175]
All ilvA alleles used herein are derived from the [0176] E. coli ilvA gene (Lawther, R. P. et al., Nucl. Acids Res. 11: 2137-2155, 1987) that is harbored in pMON25659 (Gruys et al WO 98/00557). The ilvA219 mutation (Eisenstein, E., et al. Biochemistry. 34: 9403-9412, 1995) and ilvA466 mutation (Taillon, B. E., et al. Gene. 63: 245-252, 1988), both originally isolated in Salmonella typhimurium, were introduced into the E. coli gene by oligonucleotide-directed mutagenesis as previously described (Gruys et al. WO 98/00557).
Plasmid Construction and Transformation of [0177] Arabidopsis Thaliana and Brassica Napus
All transformation vectors are derived from pMON10098, a vector designed for Agrobacterium-mediated transformation of plants that encodes the nptII selectable marker. The trfA function is provided in trans by the host bacterium, [0178] Agrobacterium tumefaciens ABI. A. tumefaciens ABI is Agrobacterium strain GV3101 (Van Larebeke, N., et al. Nature. 252: 169-170, 1974) harboring the helper plasmid pMP9ORK (Koncz, C., and Schell, J. Mol. Gen. Genet. 204: 383-396, 1986).
All PHA production genes used in this study were initially constructed in intermediate vectors as cassettes including a promoter, a chloroplast transit peptide fused to the gene of interest, and a 3′ control region. In every case, the gene cassette is flanked by Not I restriction sites, plus several additional unique restriction sites. Each cassette was excised from it's intermediate vector using appropriate restriction enzymes, and sequentially ligated into the recombinant vector for plant transformation. [0179]
For metabolite analysis, Arabidopsis was transformed with either pMON15715, an ilvA-negative control vector, or pMON25668, which expresses both phbA and wild-type ilvA from e35S promoters. [0180]
For production of PHBV in Arabidopsis, two separate plasmids were used. [0181]
The first vector encoded both phbB and phbC (pMON25678), and the second vector encoded both bktB and ilvA (pMON25812). All genes were controlled by the e35S promoter (Odell, J. T., et al. [0182] Nature. 313: 810-812, 1995) and the E9 3′ region (Coruzzi, G., et al. EMBO J. 3: 1671-1679, 1984). All enzymes were fused to the Arabidopsis RuBisCo small subunit la transit peptide that was previously used for PHB production (Nawrath, C., et al. Proc. Natl. Acad. Sci. 91: 12760-12764, 1994). Plasmid pMON25678 encodes resistance to glyphosate, whereas pMON25812 encodes resistance to kanamycin. Both plasmids were simultaneously used for Agrobacterium-mediated Arabidopsis transformation (Bechtold N., et al. Comptes Rendus Acad. Sci. Paris Sciences Serie III Sciences de la Vie. 316: 1194-1199, 1993), and transformants were selected on both glyphosate and kanamycin as follows.
[0183] Arabidopsis thaliana Columbia plants were grown in Metro Mix 200 in 2.5 in. pots covered with a mesh screen. Sown seed was vernalized for 5 days and germinated under conditions of 16 hours light/8 hours dark at 20° C. to 22° C., 75% humidity. Plants were watered and fertilized twice weekly with ½X Peters 20-20-20 until infiltration.
A 1:50 dilution of an overnight culture of [0184] Agrobacterium tumefaciens ABI strain was grown at 28° C. in YEP containing Spectinomycin 100 mg/L, Streptomycin, 100 mg/L, Chloramphenicol 25 mg/L, and Kanamycin 50 mg/L. Each culture contained a different ABI construct. After 16-20 hours the Agrobacterium cultures were concentrated by centrifugation. The supernatant was discarded and the cell pellets were dried and resuspended in infiltration medium (MS Basal Salts 0.5%, Gamborg's B-5 Vitamins 1%, Sucrose 5%, MES 0.5 g/L, pH 5.7) with 0.44 nM benzylaminopurine (10 μL of a 1.0 mg/L stock in DMSO per liter) and 0.02% Silwet L-77 to an OD₆₀₀of 0.8. For co-infiltrations each culture was resuspended as described above and 150 mL each of two cultures were combined for a total of 300 mL.
Plants were soaked in water 30 minutes prior to infiltration. Inverted plants were placed into the cultures and vacuum infiltrated at 27 in. Hg for 10 minutes. The plants were placed on their sides in a diaper-lined tray and covered with a germination dome for one day. The pots were then turned upright and were not watered for five days. Infiltrated plants were grown to maturity as described above. Ripe seeds were harvested and sterilized. Harvested seed was placed in a 15 mL Corning tube and sterilized. The tubes containing seed were placed on their sides with lids loosened in a vacuum dessicator containing a beaker of Clorox and 1:100 hydrochloric acid. The dessicator was then sealed with a vacuum and the seed remained in the dessicator overnight. Sterilized seeds from co-infiltrated plants were placed on media containing MS Basal Salts 4.3 g/L, Gamborg's B-5 (500×) 2.0 g/L, glucose 10 g/L, MES 0.5 g/L, and 8 g/L phytagar with carbenicillin 250 mg/L, [0185] cefotaxime 100 mg/L, kanamycin 60 mg/L and 4 mM glyphosate. The seed was germinated at 26° C., 20 hours light/4 hours dark. Transformants were transferred to soil and covered with a germination dome for one week. The plants were grown in plant growth conditions described above.
For transformation of [0186] Brassica napus, a single vector encoding the entire PHBV biosynthesis pathway was used. This vector, pMON36824, encodes bktB, phbB, phbC, and ilvA466 (FIG. 3). As with the Arabidopsis vectors, each gene of interest was fused to a chloroplast transit peptide, so each protein is transported to the seed leukoplast. All enzymes were fused to the Arabidopsis RuBisCo small subunit 1 a transit peptide that was previously used for PHB production (Nawrath, C. et al. Proc. Natl. Acad. Sci. 91: 12760-12764, 1994), except PhbB was fused to the transit peptide from pea RuBisCo small subunit (Cashmore, A. R. Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. pp. 29-38 in Genetic Engineering of Plants, Kosuge, T., Meredith, C. P., Hollaender, A. (eds.). Plenum, N.Y., 1983). Each gene is controlled by the promoter from the fatty acid hydroxylase gene of Lesquerella (P-Lh; Broun, P., et al. Plant J. 13: 201-210, 1998), and the E9 3′ region (Coruzzi, G., et al. EMBO J. 3: 1671-1679, 1984). P-Lh directs expression of these genes within the developing seed. Transformation of Brassica was performed as described by Fry et al. (Plant Cell Rep. 6: 321-325, 1987), and transformants were selected on glyphosate.
Isolation of Brassica Seed Leukoplasts and Analysis of Pyruvate Dehydrogenase Complex Activity [0187]
Leukoplasts were isolated essentially as described by Kang and Rawsthome ([0188] Plant J. 6: 795-805, 1994). Isolated leucoplasts were lysed by sonication and debris removed by centrifugation at 10,000× g for 10 minutes. The crude extract was desalted using Pharmacia NAP-5 columns and the protein concentrations determined by the Bradford method (Bradford, M. Anal. Biochem. 72: 248-254, 1976). Five to 50 μL were added to assay mix which contained final concentrations of: 100 mM EPPS, pH 8.0; 5 mM MgCl₂; 2.4 mM coenzyme-A; 1.5 mM NAD⁺; and 0.2 mM TPP (cocarboxylase). The reaction was initiated with addition of either pyruvate or 2-ketobutyrate substrates to final concentrations of 1.5 mM and 30 mM, respectively. To aid in analysis and ensure peak identities, ¹⁴C labeled pyruvate and 2-ketobutyrate were spiked into both substrates. The reactions were quenched with 30 μL of 10% formic acid after 2 to 30 minutes. 100 μL of the reaction was injected onto a Beckman Ultrasphere HPLC column (5 μM, 4.6 mm×15 cm) and eluted with 1 mL/minute gradient of solvent A (50 mM ammonium acetate buffer pH 6.0 containing 5% acetonitrile) going from 0 to 40 % solvent B (acetonitrile) in 15 minutes. The reaction was followed by monitoring absorbance of CoA-derived products at 230 and 260 nm using a photodiode array detector. Use of radioisotope flow detector allowed confirmation of both substrate and product peak identities. The percent conversion of added substrates was used to determine the specific activities of the extracts. One unit equals one nmol product produced per minute per mg protein in extract.
Amino Acid and 2-Ketoacid Analysis [0189]
Amino Acid analysis was performed by Dr. Donald Willis at Ralston Analytical Laboratories, essentially as described by Willis ([0190] J. Chromatog. 408: 217-225, 1987).
Extraction and Gas Chromatography Analysis of Polymer from Arabidopsis [0191]
For isolation of polymer from Arabidopsis, stems and leaves were harvested and dehydrated by lyophilization for approximately 36 hours. The material was ground to a fine powder, and 100 mg of powder was treated with 10 mL Clorox bleach for 1 hour with shaking at room temperature. The extract was subjected to centrifugation at 1,600× g for ten minutes, and the supernatant solutions was carefully removed. Ten [0192] mL 100% methanol were added, the solution was mixed by vortex, and then centrifuged again. After a second, identical, methanol extraction, the material was allowed to dry overnight. Polymer was extracted from the dried material with 1 mL of chloroform containing 3 μmol/mL methyl-benzoate standard and 1 mL of methanol/sulphuric acid (85:15, v/v). The tube was heated to 100° C. for exactly 2.5 hours, and the solid material was removed by centrifugation. The solution was cooled, 1 mL water was added, and the liquid was mixed using a vortex mixer. The organic and aqueous phases were separated by centrifugation at 1,600× g for ten minutes. The chloroform layer was transferred to a clean test tube and vigorously mixed with approximately 200 mg of silica gel. Solid material was removed by centrifugation, and the supernatant material was subjected to gas chromatography. Gas chromatographic characterization of the methyl-ester residues was performed as described by Slater et al. (J. Bacteriol. 180: 1979-1987, 1998), except that the temperature gradient was performed as follows. The initial temperature of 70° C. was held for 6 minutes, then the temperature was increased by 30° C. per minute to 130° C. Finally, the temperature was increased by 50° C. per minute to 300° C. and held at 300° C. for 5 minutes.
Extraction and Gas Chromatography Analysis of Polymer from Brassica seeds [0193]
For isolation of polymer from canola seed, seeds were ground to a fine powder with a mortar and pestle. Approximately 200 mg of each sample were extracted two times in a glass tube with 10 mL each of hexane for 1 hour at 60° C., then two times with 10 mL each of 100% methanol for one hour at 60° C. This procedure removes oil from the seed. The material was allowed to dry to completion overnight. Polymer was extracted from the dried material with 1 mL of chloroform containing 3 μmol/mL methyl-benzoate standard. The tube was heated to 100° C. for 5 hours and the samples were cooled. One mL methanol/sulphuric acid (85:15, v/v) was added, and the mixture was heated to 100° C. for exactly 2.5 hours. The solution was cooled, extracted with water and subjected to gas chromatography as described above. [0194]
Characterization of Polymer by Nuclear Magnetic Resonance Spectroscopy and Gel Permeation Chromatography [0195]
Nuclear magnetic resonance (NMR) studies were done using a Varian Unity 500 MHz spectrometer. Proton spectra were obtained on a Varian pfg 5 mm probe at 30° C. from PHA samples of approximately 20 mg dissolved in 1 mL deuterochloroform. Acquisitions were taken at a 90° pulse, 2.3 s acquisition time, 30 s delay, collecting 65 k data points and 16 accumulations. Chemical shifts were referenced to CHCl[0196] ₃(δ=7.24 ppm). The 13C{1H} spectra (125 MHz) were taken at 30° C. on a Nalorac 3 mm ¹³C probe containing a solution of approximately 10 mg PHA in 200 μL deuterochloroform. The spectra were obtained using 30° pulses, 1.5 s acquisition time, zero delay, 131 k data points and 55,296 accumulations. Chemical shifts were measured relative to CHCl₃(δ=77.0 ppm).
Gel permeation chromatography was performed according to Koizumi et al. ([0197] J. M. S. Pure Appl. Chem. A32: 759-774,1995).

Example 13

Plant Promoters

Plant promoter sequences can be constitutive or inducible, environmentally- or developmentally-regulated, or cell- or tissue-specific. Often-used constitutive promoters include the [0198] CaMV 35S promoter (Odell et al., Nature 313: 810-812, 1985), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins, R. D. et al., Nucleic Acids Res. 20: 8451-8466, 1987), the mannopine synthase (mas) promoter, the nopaline synthase (nos) promoter, and the octopine synthase (ocs) promoter. Useful inducible promoters include promoters induced by salicylic acid or polyacrylic acids (PR-1, Willians , S. W. et al, Biotechnology 10: 540-543, 1992), induced by application of safeners (substituted benzenesulfonamide herbicides, Hershey, H. P. and Stoner, T. D., Plant Mol. Biol. 17: 679-690, 1991), heat-shock promoters (Ou-Lee et al., Proc. Natl. Acad. Sci. U.S.A. 83: 6815-6819, 1986; Ainley, W. M. et al., Plant Mol. Biol. 14: 949-967, 1990), a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back, E. et al., Plant Mol. Biol. 17: 9-18, 1991), hormone-inducible promoters (Yamaguchi-Shinozaki, K. et al., Plant Mol. Biol. 15: 905-912, 1990; Kares et al., Plant Mol. Biol. 15: 905-912, 1990), and light-inducible promoters associated with the small subunit of RuBP carboxylase and LHCP gene families (Kuhlemeier et al., Plant Cell 1: 471-478, 1989; Feinbaum, R. L. et al., Mol. Gen. Genet. 226: 449-456, 1991; Weisshaar, B. et al., EMBO J. 10: 1777-1786, 1991; Lam, E. and Chua, N. H., J. BioL Chem. 266: 17131-17135, 1990; Castresana, C. et al., EMBO J. 7: 1929-1936, 1988; Schulze-Lefert, P. et al., EMBO J. 8: 651-656, 1989). Examples of useful tissue-specific, developmentally-regulated promoters include the β-conglycinin 7S promoter (Doyle, J. J. et al., J. Biol. Chem. 261: 9228-9238, 1986; Slighton and Beachy, Planta 172: 356, 1987), and seed-specific promoters (Knutzon, D. S. et al., Proc. Natl. Acad. Sci. U.S.A. 89: 2624-2628, 1992; Bustos, M. M. et al., EMBO J. 10: 1469-1479, 1991; Lam, E. and Chua, N. H., Science 248: 471-474, 1991; Stayton et al., Aust. J. Plant. Physiol. 18: 507, 1991). 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. 1: 209-219, 1991), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, and oleosin. Seed-specific gene regulation is discussed in EP 0 255 378. Promoter hybrids can also be constructed to enhance transcriptional activity (Comai, L. and Moran, P. M., U.S. Pat. No. 5,106,739, issued Apr. 21, 1992), or to combine desired transcriptional activity and tissue specificity.

Example 14

Plant Transformation and Regeneration [0199]
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 glycol mediated protoplast transformation, liposome-mediated transformation, etc. (reviewed in Potrykus, [0200] Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205-225, 1991). In general, 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.
Specific methods for transforming a wide variety of dicots and obtaining transgenic plants are well documented in the literature (Gasser and Fraley, [0201] Science 244: 1293-1299, 1989; Fisk and Dandekar, Scientia Horticulturae 55: 5-36, 1993; Christou, Agro Food Industry Hi Tech, p.17 (1994); and the references cited therein).
Successful transformation and plant regeneration have been reported in the monocots as follows: asparagus ([0202] Asparagus officinalis; Bytebier et al., Proc. Natl. Acad. Sci. U.S.A. 84: 5345-5349, 1987); barley (Hordeum vulgarae; Wan and Lemaux, Plant Physiol. 104: 37-48, 1994); maize (Zea mays; Rhodes, C. A. et al., Science 240: 204-207, 1988; Gordon-Kamm et al., Plant Cell 2: 603-618, 1990; Fromm, M. E. et al., Bio/Technology 8: 833-839, 1990; Koziel et al., Bio/Technology 11: 194-200, 1993); oats (Avena sativa; Somers et al., Bio/Technology 10: 1589-1594, 1992); orchardgrass (Dactylis glomerata; Horn et al., Plant Cell Rep. 7: 469-472, 1988); rice (Oryza sativa, including indica and japonica varieties; Toriyama et al., Bio/Technology 6: 10, 1988; Zhang et al., Plant Cell Rep. 7: 379-384, 1988; Luo and Wu, Plant Mol. Biol. Rep. 6: 165, 1988; Zhang and Wu, Theor. Appl. Genet. 76: 835, 1988; Christou et al., Bio/Technology 9: 957-962, 1991); rye (Secale cereale; De la Pena et al., Nature 325: 274-276, 1987); sorghum (Sorghum bicolor; Casas, A. M. et al., Proc. Natl. Acad. Sci. U.S.A. 90: 11212-11216, 1993); sugar cane (Saccharum spp.; Bower and Birch, Plant J. 2: 409-416, 1992); tall fescue (Festuca arundinacea; Wang, Z. Y. et al., Bio/Technology 10: 691-696, 1992); turfgrass (Agrostis palustris; Zhong et al., Plant Cell Rep. 13: 1-6, 1993); wheat (Triticum aestivum; Vasil et al., Bio/Technology 10: 667-674, 1992; Weeks, T. et al., Plant Physiol. 102: 1077-1084, 1993; Becker et al., Plant J. 5: 299-307, 1994), and alfalfa (Masoud, S. A. et al., Transgen. Res. 5: 313, 1996).

Example 15

Host Plants

Particularly useful plants for polyhydroxyalkanoate production include those that produce carbon substrates which can be employed for polyhydroxyalkanoate 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. [0203]
If 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 polyhydroxyalkanoate production. Expression of enzymes such as acyl-ACP thioesterase, fatty acyl hydroxylase, and yeast MFP may serve to increase the flux of substrates in the peroxysome, leading to higher levels of polyhydroxyalkanoate biosynthesis. [0204]

EXAMPLE 16

Nucleic Acid Mutation and Hybridization

Variations in the 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. [0205]
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. A myriad of site-directed mutagenesis techniques exist, typically using oligonucleotides to introduce mutations at specific locations in a nucleic acid sequence. Examples include single strand rescue (Kunkel, T. [0206] Proc. Natl. Acad Sci. U.S.A., 82: 488-492, 1985), unique site elimination (Deng and Nickloff, Anal. Biochem. 200: 81, 1992), nick protection (Vandeyar, et al. Gene 65: 129-133, 1988), and PCR (Costa, et al. Methods Mol. Biol. 57: 31-44, 1996). 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. One may wish to employ conditions such as about 0.15 M to about 0.9 M sodium chloride, at temperatures ranging from about 20° C. to about 55° C. 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. [0207]

Example 17

Determination of Homologous and Degenerate Nucleic Acid Ssequences

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 9.

TABLE 9


Codon degeneracies of amino acids

		Three
Amino acid	One letter	letter	Codons

Alanine	A	Ala	GCA GCC GCG GCT
Cysteine	C	Cys	TGC TGT
Aspartic acid	D	Asp	GAC GAT
Glutamic acid	E	Glu	GAA GAG
Phenylalanine	F	Phe	TTC TTT
Glycine	G	Gly	GGA GGC GGG GGT
Histidine	H	His	CAC CAT
Isoleucine	I	Ile	ATA ATC ATT
Lysine	K	Lys	AAA AAG
Leucine	L	Leu	TTA TTG CTA CTC CTG CTT
Methionine	M	Met	ATG
Asparagine	N	Asn	AAC AAT
Proline	P	Pro	CCA CCC CCG CCT
Glutamine	Q	Gln	CAA CAG
Arginine	R	Arg	AGA AGG CGA CGC CGG CGT
Serine	S	Ser	AGC AGT TCA TCC TCG TCT
Threonine	T	Thr	ACA ACC ACG ACT
Valine	V	Val	GTA GTC GTG GTT
Tryptophan	W	Trp	TGG
Tyrosine	Y	Tyr	TAC TAT

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. [0209]
In making such changes, 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, [0210] J. Mol. Biol., 157: 105-132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate/glutamine/aspartate/asparagine (−3.5); lysine (−3.9); and arginine (−4.5). [0211]
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein. In making such changes, the 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. [0212]
It is also understood in the art that the substitution of like amino acids may be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (Hopp, T. P., issued Nov. 19, 1985) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0±1); serine (+0.3); 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). [0213]
It is understood that an amino acid may be substituted by another amino acid having a similar hydrophilicity score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein. In making such changes, the 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. [0214]
As outlined above, amino acid substitutions are therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Changes which are not expected to be advantageous may also be used if these resulted in functional fusion proteins. [0215]

All of the 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.

TABLE 10


RESTRICTION SITES FROM FIG. 3

	ENZYME	CUT SITE

Notl

	693
	Xhol	702
	BsaAl	1510
	Rsrll	1722
	Xhol	2170
	Dral	2817
	BsaAl	4975
	Dral	5980
	Dral	5999
	BsaAl	7195
	Dral	7677
	Dral	7754
	Bglll	8440
	Rsrll	8998
	Bglll	9296
	Ascl	9851
	SexAl	9917
	BsaAl	9933
	Sfil	10387
	Sbfl	10535
	EcoRl	10594

TABLE 11


RESTRICTION SITES FROM FIG. 4

	ENZYME	CUT SITE

Notl

	678
	Xhol	687
	BsaAl	1497
	Rsrll	1709
	Xhol	2157
	Dral	2804
	BsaAl	4924
	Dral	5929
	Dral	5948
	BsaAl	7144
	Dral	7626
	Dral	7703
	Bglll	8389
	EcoRl	8413

[0218]

TABLE 12

RESTRICTION SITES FROM FIG. 5

ENZYME CUT SITE

BsaAl 411

Notl

878

Bglll 1541

EcoRl 1555

Smal 1573

Smal 2240

Srfl 2240

Notl 2244

Dral 3368

Dral 3387

Dral 4079
[0219]

TABLE 13

RESTRICTION SITES FROM FIG. 6

ENZYME CUT SITE

BsaAl 411

Notl

878

Bglll 1541

BsaAl 2185

EcoRl 3094

EcoRl 3126

Smal 3144

Smal 3811

Srfl 3811

Notl 3815

Dral 4939

Dral 4958

Dral 5650

TABLE 14


RESTRICTION SITES FROM FIG. 7

	ENZYME	CUT SITE

	BsaAl	411
	Notl

	878
	Bglll	1541
	BsaAl	2019
	Sbfl	2150
	BsaAl	2523
	Sbfl	2789
	EcoRl	3083
	Smal	3101
	Smal	3768
	Srfl	3768
	Notl	3772
	Dral	4896
	Dral	4915
	Dral	5607

[0221]

TABLE 15

RESTRICTION SITES FROM FIG. 8

ENZYME CUT SITE

BsaAl 411

Notl

878

Bglll 1541

Sfil 2259

EcoRl 2603

EcoRl 2635

Smal 2653

Smal 3320

Srfl 3320

Notl 3324

Dral 4448

Dral 4467

Dral 5159
[0222]

TABLE 16

RESTRICTION SITES FROM FIG. 9

ENZYME CUT SITE

BsaAl 411

Notl

878

Bglll 1541

BsaAl 3070

EcoRl 3131

BsaAl 3183

Smal 4029

Srfl 4029

Notl 4033

Dral 5157

Dral 5176

Dral 5868

TABLE 17


RESTRICTION SITES FROM FIG. 10

	ENZYME	CUT SITE

Notl

	693
	Hindlll	704
	EcoRV	1241
	Bglll	1356
	Hindlll	1362
	Hindlll	1374
	Sphl	1678
	Sfil	2118
	Ncol	2166
	EcoRl	2462
	Smal	2480
	BamHl	2486
	Smal	3147
	Notl	3151
	Hindlll	3162
	EcoRV	3699
	Bglll	3814
	Hindlll	3820
	Hindlll	3832
	Sphl	4136
	BspHl	4138
	Ncol	5005
	EcoRl	5356
	Smal	5374
	BamHl	5380
	Smal	6041
	Notl	6045
	Xhol	6054
	Sphl	6963
	Ncol	6990
	Xhol	7522
	BspHl	11293
	BspHl	11793
	Sphl	12986
	Hindlll	13143
	EcoRV	13677
	Bglll	13792
	Sphl	13971
	Sphl	14061
	Ncol	14066
	EcoRV	14277
	Ncol	14321
	Bglll	14648
	SexAl	15269
	Sfil	15739
	EcoRl	15946
	BamHl	15964

TABLE 18


RESTRICTION SITES FROM FIG. 11

	ENZYME	CUT SITE

Notl

	693
	Hindlll	704
	EcoRV	1241
	Bglll	1356
	Sphl	1535
	Sphl	1625
	Ncol	1630
	Apal	2508
	EcoRl	2909
	EcoRl	2941
	Smal	2959
	BamHl	2965
	Smal	3626
	Notl	3630
	Hindlll	3641
	EcoRV	4178
	Bglll	4293
	Sphl	4472
	Sphl	4562
	Ncol	4567
	Sfil	5011
	Ncol	5059
	EcoRl	5355
	EcoRl	5387
	Smal	5405
	BamHl	5411
	Smal	6072
	Notl	6076
	Xhol	6085
	Sphl	6994
	Ncol	7021
	Xhol	7553
	BspHl	11324
	BspHl	11824
	Sphl	13017
	Hindll	13174
	EcoRV	13708
	Bglll	13823
	Sphl	14002
	Sphl	14092
	Ncol	14097
	EcoRV	14308
	Ncol	14352
	Bglll	14679
	SexAl	15300
	Sfil	15770
	EcoRl	15977
	BamHl	15995

TABLE 19


RESTRICTION SITES FROM FIG. 12

	ENZYME	CUT SITE

Notl

	693
	Bglll	1356
	BsaAl	1834
	Sbfl	1965
	BsaAl	2338
	Sbfl	2604
	EcoRl	2898
	Smal	2916
	Smal	3583
	Srfl	3583
	Notl	3587
	Bglll	4250
	Sfil	4968
	EcoRl	5312
	EcoRl	5344
	Smal	5362
	Smal	6029
	Srfl	6029
	Notl	6033
	Xhol	6042
	BsaAl	6850
	Rsrll	7062
	Xhol	7510
	Dral	8157
	BsaAl	10315
	Dral	11320
	Dral	11339
	BsaAl	12535
	Dral	13017
	Dral	13094
	Bglll	13780
	Rsrll	14338
	Bglll	14636
	Ascl	15191
	SexAl	15257
	BsaAl	15273
	Sfil	15727
	Sbfl	15875
	EcoRl	15934

TABLE 20


RESTRICTION SITES FROM FIG. 13

	ENZYME	CUT SITE

Notl

	693
	Hindlll	704
	EcoRV	1241
	Bglll	1356
	Sphl	1535
	Sphl	1625
	Ncol	2497
	Hindlll	2938
	EcoRV	2946
	EcoRl	2950
	EcoRl	2982
	Smal	3000
	BamHl	3006
	Smal	3667
	Notl	3671
	Hindlll	3682
	EcoRV	4219
	Bglll	4334
	Sphl	4513
	Sphl	4603
	Ncol	4608
	Sfil	5052
	Ncol	5100
	EcoRl	5396
	EcoRl	5428
	Smal	5446
	BamHl	5452
	Smal	6113
	Notl	6117
	Xhol	6126
	Sphl	7035
	Ncol	7062
	Xhol	7594
	BspHl	11365
	BspHl	11865
	Sphl	13058
	Hindlll	13215
	EcoRV	13749
	Bglll	13864
	Sphl	14043
	Sphl	14133
	Ncol	14138
	EcoRV	14349
	Ncol	14393
	Bglll	14720
	SexAl	15341
	Sfil	15811
	EcoRl	16018
	BamHl	16036

TABLE 21


RESTRICTION SITES FROM FIG. 14

	ENZYME	CUT SITE

Notl

	693
	Hindlll	704
	EcoRV	1241
	Bglll	1356
	Sphl	1535
	Sphl	1625
	Ncol	1630
	EcoRl	2946
	SnaBl	2998
	Ncol	3032
	EcoRV	3179
	BamHl	3183
	Smal	3844
	Notl	3848
	Hindlll	3859
	EcoRV	4396
	Bglll	4511
	Hindlll	4517
	Hindlll	4529
	Sphl	4833
	BspHl	4835
	Ncol	5702
	EcoRl	6053
	Smal	6071
	BamHl	6077
	Smal	6738
	Notl	6742
	Hindlll	6753
	EcoRV	7290
	Bgll	7405
	Sphl	7584
	Sphl	7674
	Ncol	7679
	Sfil	8123
	Ncol	8171
	EcoRl	8467
	EcoRl	8499
	Smal	8517
	BamHl	8523
	Smal	9184
	Notl	9188
	Xhol	9197
	Sphl	10106
	Ncol	10133
	Xhol	10665
	BspHl	14436
	BspHl	14936
	Sphl	16129
	Hindll	16286
	EcoRV	16820
	Bglll	16935
	Sphl	17114
	Sphl	17204
	Ncol	17209
	EcoRV	17420
	Ncol	17464
	Bglll	17791
	SexAl	18412
	Sfil	18882
	EcoRl	19089
	BamHl	19107

TABLE 22


RESTRICTION SITES FROM FIG. 15

	ENZYME	CUT SITE

Notl

	693
	Hindlll	704
	EcoRV	1241
	Bglll	1356
	Sphl	1535
	Sphl	1625
	Ncol	1630
	EcoRl	2946
	SnaBl	2998
	Ncol	3032
	EcoRV	3179
	BamHl	3183
	Smal	3844
	Notl	3848
	Hindlll	3859
	EcoRV	4396
	Bglll	4511
	Sphl	4690
	Sphl	4780
	Ncol	4785
	EcoRl	6101
	SnaBl	6153
	Ncol	6187
	EcoRV	6334
	BamHl	6338
	Smal	6999
	Notl	7003
	Hindlll	7014
	EcoRV	7551
	Bglll	7666
	Hindlll	7672
	Hindlll	7684
	Sphl	7988
	BspHl	7990
	Ncol	8857
	EcoRl	9208
	Smal	9226
	BamHl	9232
	Smal	9893
	Notl	9897
	Hindlll	9908
	EcoRV	10445
	Bglll	10560
	Sphl	10739
	Sphl	10829
	Ncol	10834
	Sfil	11278
	Ncol	11326
	EcoRl	11622
	EcoRl	11654
	Smal	11672
	BamHl	11678
	Smal	12339
	Notl	12343
	Xhol	12352
	Sphl	13261
	Ncol	13288
	Xhol	13820
	BspHl	17591
	BspHl	18091
	Sphl	19284
	Hindlll	19441
	EcoRV	19975
	Bglll	20090
	Sphl	20269
	Sphl	20359
	Ncol	20364
	EcoRV	20575
	Ncol	20619
	Bglll	20946
	SexAl	21567
	Sfil	22037
	EcoRl	22244
	BamHl	22262

TABLE 23


RESTRICTION SITES FROM FIG. 16

	ENZYME	CUT SITE

Notl

	678
	Spel	685
	BsaAl	693
	SanDl	698
	Rsrll	705
	SexAl	711
	Pacl	722
	Sgfl	730
	Sfil	741
	Ascl	748
	Sbfl	760
	Smal	766
	Srfl	766
	Dral	774
	Swal	774
	Xhol	779
	Dral	1426
	BsaAl	3546
	Dral	4551
	Dral	4570
	BsaAl	5766
	Dral	6248
	Dral	6325
	Dral	6424
	Pacl	7426
	Dral	7887
	BsaAl	8144
	BsaAl	8164
	Dral	8394
	Bglll	8582
	Rsrll	9140
	Bglll	9438
	Ascl	9993
	SexAl	10059
	BsaAl	10075
	Sfil	10529
	Sbfl	10677
	EcoRl	10736

TABLE 24


RESTRICTION SITES FROM FIG. 17

	ENZYME	CUT SITE

Notl

	678
	Spel	685
	BsaAl	693
	SanDl	698
	Rsrll	705
	SexAl	711
	Pacl	722
	Sgfl	730
	Sfil	741
	Ascl	748
	Sbfl	760
	Smal	766
	Srfl	766
	Dral	774
	Swal	774
	Xhol	779
	Dral	1426
	BsaAl	3546
	Dral	4551
	Dral	4570
	BsaAl	5766
	Dral	6248
	Dral	6325
	Dral	6424
	Pacl	7426
	Dral	7887
	BsaAl	8144
	BsaAl	8164
	Dral	8394
	Bglll	8582
	EcoRl	8606

TABLE 25


RESTRICTION SITES FROM FIG. 18

	ENZYME	CUT SITE

	BsaAl	411
	Notl

	878
	Dral	951
	Pacl	1953
	Dral	2414
	BsaAl	2671
	BsaAl	2691
	Dral	2921
	Bglll	3109
	EcoRl	3123
	Smal	3141
	Smal	3808
	Srfl	3808
	Notl	3812
	Dral	4936
	Dral	4955
	Dral	5647

TABLE 26


RESTRICTION SITES FROM FIG. 19

	ENZYME	CUT SITE

	BsaAl	411
	Notl

	878
	Dral	951
	Pacl	1953
	Dral	2414
	BsaAl	2671
	BsaAl	2691
	Dral	2921
	Bglll	3109
	BsaAl	3753
	EcoRl	4662
	Smal	4680
	Smal	5347
	Srfl	5347
	Notl	5351
	Dral	6475
	Dral	6494
	Dral	7186

TABLE 27


RESTRICTION SITES FROM FIG. 20

	ENZYME	CUT SITE

Notl

	878
	Hindlll	889
	Sphl	1041
	Pacl	1953
	BspHl	2613
	BspHl	2736
	Bglll	3109
	Sphl	3288
	Sphl	3378
	Ncol	4250
	Hindlll	4691
	EcoRV	4699
	EcoRl	4703
	Smal	4721
	BamHl	4727
	Smal	5388
	Notl	5392
	BspHl	6477
	BspHl	7485
	BspHl	7590

TABLE 28


RESTRICTION SITES FROM FIG. 21

	ENZYME	CUT SITE

Xhol

	271
	Dral	280
	Swal	280
	Smal	288
	Srfl	288
	Sbfl	298
	Ascl	302
	Sfil	316
	Sgfl	326
	Pacl	334
	SexAl	338
	Rsrll	346
	SanDl	353
	BsaAl	361
	Spel	365
	Notl	372
	Dral	445
	Pacl	1447
	Dral	1908
	BsaAl	2165
	BsaAl	2185
	Dral	2415
	Spel	2609
	Smal	2867
	Sfil	3315
	EcoRl	3608
	Smal	3626
	Smal	4293
	Srfl	4293
	Notl	4297
	Spel	4304
	BsaAl	4312
	SanDl	4317
	Rsrll	4324
	SexAl	4330
	Pacl	4341
	Sgfl	4349
	Sfil	4361
	Ascl	4368
	Sbfl	4380
	Smal	4386
	Srfl	4386
	Dral	4394
	Swal	4394
	Dral	5388
	Dral	5407
	Dral	6099

TABLE 29


RESTRICTION SITES FROM FIG. 22

	ENZYME	CUT SITE

	BsaAl	411
	Notl

	878
	Dral	951
	Pacl	1953
	Dral	2414
	BsaAl	2671
	BsaAl	2691
	Dral	2921
	Bglll	3109
	BsaAl	4638
	EcoRl	4699
	BsaAl	4751
	Smal	5597
	Srfl	5597
	Notl	5601
	Dral	6725
	Dral	6744
	Dral	7436

TABLE 30


RESTRICTION SITES FROM FIG. 23

ENZYME	CUT SITE	ENZYME	CUT SITE	ENZYME	CUT SITE

Notl

	678	Spel	7388	Notl	11596
Hindlll	689	EcoRV	7413	BspHl	15339
Sphl	841	Sphl	7573	BspHl	15839
Pacl	1753	Smal	7646	Sphl	17032
BspHl	2413	Sfil	8094	Hindlll	17189
BspHl	2536	Ncol	8142	Sphl	17341
Bglll	2909	EcoRl	8387	Pacl	18253
Sphl	3088	Smal	8405	BspHl	18913
Sphl	3178	BamHl	8411	BspHl	19036
Ncol	3183	BamHl	9358	Bglll	19409
Apal	4061	EcoRl	9376	Sphl	19588
EcoRl	4462	BspHl	10162	Sphl	19678
Smal	4480	Ncol	10435	Ncol	19683
BamHl	4486	BamHl	10546	EcoRV	19894
Smal	5147	Ncol	10558	Ncol	19938
Notl	5151	Sfil	10569	Bglll	20265
Hindlll	5162	Sphl	10757	SexAl	20886
Sphl	5314	Bglll	10980	Sfil	21356
Pacl	6226	EcoRl	11052	EcoRl	21563
BspHl	6886	EcoRl	11455	BamHl	21581
BspHl	7009	Hindlll	11585

TABLE 31


RESTRICTION SITES FROM FIG. 24

ENZYME	CUT SITE	ENZYME	CUT SITE	ENZYME	CUT SITE

Notl

	678	BspHl	7050	Hindlll	11626
Hindlll	689	Spel	7429	Notl	11637
Sphl	841	EcoRV	7454	BspHl	15380
Pacl	1753	Sphl	7614	BspHl	15880
BspHl	2413	Smal	7687	Sphl	17073
BspHl	2536	Sfil	8135	Hindlll	17230
Bglll	2909	Ncol	8183	Sphl	17382
Sphl	3088	EcoRl	8428	Pacl	18294
Sphl	3178	Smal	8446	BspHl	18954
Ncol	4050	BamHl	8452	BspHl	19077
Hindlll	4491	BamHl	9399	Bglll	19450
EcoRV	4499	EcoRl	9417	Sphl	19629
EcoRI	4503	BspHl	10203	Sphl	19719
Smal	4521	Ncol	10476	Ncol	19724
BamHl	4527	BamHl	10587	EcoRV	19935
Smal	5188	Ncol	10599	Ncol	19979
Notl	5192	Sfil	10610	Bglll	20306
Hindlll	5203	Sphl	10798	SexAl	20927
Sphl	5355	Bglll	11021	Sfil	21397
Pacl	6267	EcoRl	11093	EcoRl	21604
BspHl	6927	EcoRl	11496	BamHl	21622

TABLE 32


RESTRICTION SITES FROM FIG. 25

ENZYME	CUT SITE	ENZYME	CUT SITE	ENZYME	CUT SITE

Notl

	678	EcoRl	9013	Sphl	15521
Hindlll	689	EcoRV	9246	Bglll	15744
Sphl	841	BamHl	9250	EcoRl	15816
Pacl	1753	Smal	9911	EcoRl	16219
BspHl	2413	Notl	9915	Hindlll	16349
BspHl	2536	Hindlll	9926	Notl	16360
Bglll	2909	Sphl	10078	BspHl	20103
Sphl	3088	Pacl	10990	BspHl	20603
Sphl	3178	BspHl	11650	Sphl	21796
Ncol	4050	BspHl	11773	Hindlll	21953
Hindlll	4491	Spel	12152	Sphl	22105
EcoRV	4499	EcoRV	12177	Pacl	23017
EcoRl	4503	Sphl	12337	BspHl	23677
Smal	4521	Smal	12410	BspHl	23800
BamHl	4527	Sfil	12858	Bglll	24173
Smal	5188	Ncol	12906	Sphl	24352
Notl	5192	EcoRl	13151	Sphl	24442
Hindlll	5203	Smal	13169	Ncol	24447
Sphl	5355	BamHl	13175	EcoRV	24658
Pacl	6267	BamHl	14122	Ncol	24702
BspHl	6927	EcoRl	14140	Bglll	25029
BspHl	7050	BspHl	14926	SexAl	25650
Bglll	7423	Ncol	15199	Sfil	26120
Sphl	7602	BamHl	15310	EcoRl	26327
Sphl	7692	Ncol	15322	BamHl	26345
Ncol	7697	Sfil	15333

TABLE 33


RESTRICTION SITES FROM FIG. 26

ENZYME	CUT SITE	ENZYME	CUT SITE	ENZYME	CUT SITE

Bglll

	649	EcoRl	6440	Dral	8098
Dral	1202	Smal	6712	BsaAl	8190
Dral	1278	Notl	6717	BsaAl	8731
BsaAl	1370	Spel	6724	Rsrll	8943
Sfil	2185	BsaAl	6732	EcoRl	9280
EcoRl	2529	SanDl	6737	Dral	10201
Smal	2801	Rsrll	6744	BsaAl	12321
Notl	2806	SexAl	6750	Dral	13326
Bglll	3468	Pacl	6761	Dral	13345
Dral	4021	Sgfl	6769	BsaAl	14541
Dral	4097	Sfil	6780	Dral	15023
BsaAl	4189	Ascl	6787	Dral	15100
Rsrll	4844	Sbfl	6799	Bglll	15786
Bglll	5142	Smal	6805	Dral	16339
Ascl	5697	Srfl	6805	Dral	16415
SexAl	5763	Dral	6813	BsaAl	16507
BsaAl	5779	Swal	6813	BsaAl	17248
Sfil	6233	Bglll	7469	EcoRl	18157
Sbfl	6381	Dral	8022

TABLE 34


RESTRICTION SITES FROM FIG. 27

	ENZYME	CUT SITE

EcoRV

	637
	BglII	752
	EcoRV	2829
	HindIII	8420
	BglII	9445
	EcoRV	12082
	HindIII	12086
	BglIII	13111
	EcoRV	15257
	NotI	15268
	BglII	16310
	EcoRV	17613
	BglII	17984
	EcoRV	19548
	NotI	19559

[0241]

TABLE 35

RESTRICTION SITES FROM FIG. 28

ENZYME CUT SITE ENZYME CUT SITE

EcoRV

637 EcoRV 14937

EcoRV 2829 NotI 14948

HindIII 8420 EcoRV 17133

EcoRV 11922 EcoRV 19068

HindIII 11926 NotI 19079

TABLE 36


RESTRICTION SITES FROM FIG. 29

ENZYME	CUT SITE	ENZYME	CUT SITE	ENZYME	CUT SITE

Notl

	678	Ascl	748	BsaAl	3546
Spel	685	Sbfl	760	Dral	4551
BsaAl	693	Smal	766	Dral	4570
SanDl	698	Srfl	766	BsaAl	5766
Rsrll	705	Dral	774	Dral	6248
SexAl	711	Swal	774	Dral	6325
Pacl	722	Xhol	779	Bglll	7011
Sgfl	730	Dral	1426	EcoRl	7035
Sfil	741

TABLE 37


RESTRICTION SITES FROM FIG. 30

ENZYME	CUT SITE	ENZYME	CUT SITE	ENZYME	CUT SITE

Xhol

	271	BsaAl	361	SexAl	1771
Dral	280	Spel	365	Pacl	1782
Swal	280	Notl	372	Sgfl	1790
Smal	288	Smal	380	Sfil	1802
Srfl	288	Srfl	380	Ascl	1809
Sbfl	298	Smal	1047	Sbfl	1821
Ascl	302	EcoRl	1061	Smal	1827
Sfil	316	Bglll	1075	Srfl	1827
Sgfl	326	Notl	1738	Dral	1835
Pacl	334	Spel	1745	Swal	1835
SexAl	338	BsaAl	1753	Dral	2829
Rsrll	346	SanDl	1758	Dral	2848
SanDl	353	Rsrll	1765	Dral	3540

Claims

What is claimed is:

1. An isolated nucleic acid segment comprising:

a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;

a second nucleic acid sequence encoding a β-ketoacyl reductase protein; and

a third nucleic acid sequence encoding a β-ketothiolase protein.

2. The isolated nucleic acid segment of claim 1, further comprising a fourth nucleic acid sequence encoding a threonine deaminase protein.

3. The isolated nucleic acid segment of claim 1, further comprising a fourth nucleic acid sequence encoding a deregulated threonine deaminase protein.

4. The isolated nucleic acid segment of claim 1, wherein:

the first nucleic acid sequence further encodes a chloroplast transit peptide;

the second nucleic acid sequence further encodes a chloroplast transit peptide; and

the third nucleic acid sequence further encodes a chloroplast transit peptide.

5. A recombinant vector comprising operatively linked in the 5′ to 3′ direction:

a promoter that directs transcription of a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence;

a first nucleic acid sequence;

a second nucleic acid sequence;

a third nucleic acid sequence;

a 3′ transcription terminator; and

a 3′ polyadenylation signal sequence; wherein:

the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins; and

the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a β-ketoacyl reductase protein, and a nucleic acid sequence encoding a β-ketothiolase protein.

6. The recombinant vector of claim 5, wherein the promoter directs transcription of the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence in plants.

7. The recombinant vector of claim 5, wherein the promoter is a viral promoter.

8. The recombinant vector of claim 5, wherein the promoter is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S promoter.

9. The recombinant vector of claim 5, wherein the promoter is an enhanced CMV 35S promoter.

10. The recombinant vector of claim 5, wherein the promoter is a tissue specific promoter.

11. The recombinant vector of claim 5, wherein the promoter is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter.

12. The recombinant vector of claim 5, wherein the promoter is a Lesquerella hydroxylase promoter.

13. The recombinant vector of claim 5, wherein:

the first nucleic acid sequence further encodes a chloroplast transit peptide;

the third nucleic acid sequence further encodes a chloroplast transit peptide.

14. A recombinant vector comprising:

a first element comprising operatively linked in the 5′ to 3′ direction:

a first promoter that directs transcription of a first nucleic acid sequence;

a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;

a first 3′ transcription terminator; and

a first 3′ polyadenylation signal sequence;

a second element comprising operatively linked in the 5′ to 3′ direction:

a second promoter that directs transcription of a second nucleic acid sequence;

a second nucleic acid sequence encoding a β-ketoacyl reductase protein;

a second 3′ transcription terminator; and

a second 3′ polyadenylation signal sequence; and

a third element comprising operatively linked in the 5′ to 3′ direction:

a third promoter that directs transcription of a third nucleic acid sequence;

a third nucleic acid sequence encoding a β-ketothiolase protein;

a third 3′ transcription terminator; and

a third 3′ polyadenylation signal sequence.

15. The recombinant vector of claim 14, wherein the β-ketothiolase protein:

catalyzes the condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA; and

catalyzes the condensation of acetyl-CoA and propionyl-CoA to produce β-ketovaleryl-CoA.

16. The recombinant vector of claim 14, wherein the β-ketoacyl reductase protein:

catalyzes the reduction of acetoacetyl-CoA to β-hydroxybutyryl-CoA; and

catalyzes the reduction of β-ketovaleryl-CoA to β-hydroxyvaleryl-CoA.

17. The recombinant vector of claim 14, wherein the polyhydroxyalkanoate synthase protein is selected from the group consisting of:

a polyhydroxyalkanoate synthase protein that catalyzes the incorporation of β-hydroxybutyryl-CoA into P(3HB) polymer; and

a polyhydroxyalkanoate synthase protein that catalyzes the incorporation of β-hydroxybutyryl-CoA and β-hydroxyvaleryl-CoA into P(3HB-co-3HV) copolymer.

18. The recombinant vector of claim 14, wherein:

the β-ketothiolase protein comprises a transit peptide sequence that directs transport of the β-ketothiolase protein to the plastid;

the β-ketoacyl reductase protein comprises a transit peptide sequence that directs transport of the β-ketoacyl reductase protein to the plastid; and

the polyhydroxyalkanoate synthase protein comprises a transit peptide sequence that directs transport of the polyhydroxyalkanoate synthase protein to the plastid.

19. The recombinant vector of claim 14, further comprising a nucleic acid sequence encoding a threonine deaminase protein.

20. The recombinant vector of claim 14, further comprising a nucleic acid sequence encoding a deregulated threonine deaminase protein.

21. The recombinant vector of claim 14, wherein:

the first promoter directs transcription of the first nucleic acid sequence in plants;

the second promoter directs transcription of the second nucleic acid sequence in plants; and

the third promoter directs transcription of the third nucleic acid sequence in plants.

22. The recombinant vector of claim 14, wherein the first promoter, second promoter, and third promoter are viral promoters.

23. The recombinant vector of claim 14, wherein:

the first promoter is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S promoter;

the second promoter is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S promoter; and

the third promoter is a CMV 35S promoter, an enhanced CMV 35S promoter, or an FMV 35S promoter.

24. The recombinant vector of claim 14, wherein:

the first promoter is an enhanced CMV 35S promoter;

the second promoter is an enhanced CMV 35S promoter; and

the third promoter is an enhanced CMV 35S promoter.

25. The recombinant vector of claim 14, wherein:

the first promoter is a tissue specific promoter;

the second promoter is a tissue specific promoter; and

the third promoter is a tissue specific promoter.

26. The recombinant vector of claim 14, wherein:

the first promoter is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter;

the second promoter is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter; and

the third promoter is a Lesquerella hydroxylase promoter or a 7S conglycinin promoter.

27. The recombinant vector of claim 14, wherein:

the first promoter is a Lesquerella hydroxylase promoter;

the second promoter is a Lesquerella hydroxylase promoter; and

the third promoter is a Lesquerella hydroxylase promoter.

28. The recombinant vector of claim 14, wherein:

the first nucleic acid sequence further encodes a chloroplast transit peptide;

the third nucleic acid sequence further encodes a chloroplast transit peptide.

29. A transformed host cell comprising a recombinant vector, wherein the recombinant vector comprises:

a first element comprising operatively linked in the 5′ to 3′ direction:

a first promoter that directs transcription of a first nucleic acid sequence;

a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;

a first 3′ transcription terminator; and

a first 3′ polyadenylation signal sequence;

a second element comprising operatively linked in the 5′ to 3′ direction:

a second promoter that directs transcription of a second nucleic acid sequence;

a second nucleic acid sequence encoding a β-ketoacyl reductase protein;

a second 3′ transcription terminator; and

a second 3′ polyadenylation signal sequence; and

a third element comprising operatively linked in the 5′ to 3′ direction:

a third promoter that directs transcription of a third nucleic acid sequence;

a third nucleic acid sequence encoding a β-ketothiolase protein;

a third 3′ transcription terminator; and

a third 3′ polyadenylation signal sequence.

30. The transformed host cell of claim 29, wherein the transformed host cell is a bacterial cell.

31. The transformed host cell of claim 29, wherein the transformed host cell is a fungal cell.

32. The transformed host cell of claim 29, wherein the transformed host cell is a plant cell.

33. The transformed host cell of claim 29, wherein:

the first nucleic acid sequence further encodes a chloroplast transit peptide;

the third nucleic acid sequence further encodes a chloroplast transit peptide.

34. A transformed host cell comprising:

a first element comprising operatively linked in the 5′ to 3′ direction:

a first promoter that directs transcription of a first nucleic acid sequence;

a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;

a first 3′ transcription terminator; and

a first 3′ polyadenylation signal sequence;

a second element comprising operatively linked in the 5′ to 3′ direction:

a second promoter that directs transcription of a second nucleic acid sequence;

a second nucleic acid sequence encoding a β-ketoacyl reductase protein;

a second 3′ transcription terminator; and

a second 3′ polyadenylation signal sequence; and

a third element comprising operatively linked in the 5′ to 3′ direction:

a third promoter that directs transcription of a third nucleic acid sequence;

a third nucleic acid sequence encoding a β-ketothiolase protein;

a third 3′ transcription terminator; and

a third 3′ polyadenylation signal sequence;

wherein the first element, second element, and third element are cointegrated between a single left Ti border sequence and a single right Ti border sequence.

35. The transformed host cell of claim 34, wherein the transformed host cell is a fungal cell.

36. The transformed host cell of claim 34, wherein the transformed host cell is a plant cell.

37. The transformed host cell of claim 34, wherein the transformed host cell is a tobacco, wheat, potato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa cell.

38. The transformed host cell of claim 34, wherein:

the first nucleic acid sequence further encodes a chloroplast transit peptide;

the third nucleic acid sequence further encodes a chloroplast transit peptide.

39. A transformed plant comprising:

a first element comprising operatively linked in the 5′ to 3′ direction:

a first promoter that directs transcription of a first nucleic acid sequence;

a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;

a first 3′ transcription terminator; and

a first 3′ polyadenylation signal sequence;

a second element comprising operatively linked in the 5′ to 3′ direction:

a second promoter that directs transcription of a second nucleic acid sequence;

a second nucleic acid sequence encoding a β-ketoacyl reductase protein;

a second 3′ transcription terminator; and

a second 3′ polyadenylation signal sequence; and

a third element comprising operatively linked in the 5′ to 3′ direction:

a third promoter that directs transcription of a third nucleic acid sequence;

a third nucleic acid sequence encoding a β-ketothiolase protein;

a third 3′ transcription terminator; and

a third 3′ polyadenylation signal sequence;

40. The transformed plant of claim 39, wherein the transformed plant is a tobacco, wheat, potato, Arabidopsis, corn, soybean, canola, oil seed rape, sunflower, flax, peanut, sugarcane, switchgrass, or alfalfa plant.

41. The transformed plant of claim 39, wherein:

the first nucleic acid sequence further encodes a chloroplast transit peptide;

the third nucleic acid sequence further encodes a chloroplast transit peptide.

42. A method of preparing transformed host cells, the method comprising:

selecting a host cell;

transforming the selected host cell with a recombinant vector comprising:

a first element comprising operatively linked in the 5′ to 3′ direction:

a first promoter that directs transcription of the first nucleic acid sequence;

a first nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein;

a first 3′ transcription terminator; and

a first 3′ polyadenylation signal sequence;

a second element comprising operatively linked in the 5′ to 3′ direction:

a second promoter that directs transcription of the second nucleic acid sequence;

a second nucleic acid sequence encoding a β-ketoacyl reductase protein;

a second 3′ transcription terminator; and

a second 3′ polyadenylation signal sequence; and

a third element comprising operatively linked in the 5′ to 3′ direction:

a third promoter that directs transcription of the third nucleic acid sequence;

a third nucleic acid sequence encoding a β-ketothiolase protein;

a third 3′ transcription terminator; and

a third 3′ polyadenylation signal sequence; and

obtaining transformed host cells; wherein the transformed host cells produce polyhydroxyalkanoate polymer.

43. A method of preparing transformed host cells, the method comprising:

selecting a host cell;

transforming the selected host cell with a recombinant vector comprising

operatively linked in the 5′ to 3′ direction:

a promoter that directs transcription of a first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence;

a first nucleic acid sequence;

a second nucleic acid sequence;

a third nucleic acid sequence;

a 3′ transcription terminator; and

a 3′ polyadenylation signal sequence; and

obtaining transformed host cells; wherein:

the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence encode different proteins;

the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence

regenerating the transformed host plant cells to produce transformed plants; wherein:

the first nucleic acid sequence, second nucleic acid sequence, and third nucleic acid sequence are independently selected from the group consisting of a nucleic acid sequence encoding a polyhydroxyalkanoate synthase protein, a nucleic acid sequence encoding a β-ketoacyl reductase protein, and a nucleic acid sequence encoding a β-ketothiolase protein; and

the transformed plants produce polyhydroxyalkanoate polymer.

46. A method of producing polyhydroxyalkanoate comprising:

obtaining the transformed host cell of claim 29 or claim 34;

culturing the transformed host cell under conditions suitable for the production of polyhydroxyalkanoate; and

recovering polyhydroxyalkanoate from the transformed host cell.

47. The method of claim 46, wherein the polyhydroxyalkanoate is poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), or poly(3-hydroxybutyrate-co-4-hydroxybutyrate).

48. A method of producing polyhydroxyalkanoate comprising:

obtaining the transformed plant of claim 39;

growing the transformed plant under conditions suitable for the production of polyhydroxyalkanoate; and

recovering polyhydroxyalkanoate from the transformed plant.

49. The method of claim 48, wherein the polyhydroxyalkanoate is poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate).