Production of polyhydroxyalkanoate in plants

Abstract

The invention relates to the genetic manipulation of plants to produce polyhydroxyalkanoate, particularly in the peroxisomes. Methods for producing such polymers in plants and host cells are provided. Such methods find use in producing biodegradable thermoplastics in plants and other organisms. Nucleotide molecules, expression cassettes, and genetically manipulated host cell, plants, plant tissues, and seeds are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. application Ser. No. 10/089,281, which is the U.S. National Stage of International Application No. PCT/US00/26963, filed Sep. 29, 2000, which claims the benefit of U.S. Provisional Application Ser. No. 60/156,807, filed Sep. 29, 1999; all of which are hereby incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the genetic manipulation of plants for the production of biodegradable thermoplastics, particularly polyhydroxyalkanoate copolymers.

BACKGROUND OF THE INVENTION

[0003] Composed of polymers of a variety of organic compounds, plastics can be molded, extruded, cast into various shapes and films, and even drawn into fibers. It is such versatility that has led to incorporation of plastics into a seemingly endless number of products. Thus, plastic products have become an integral part of everyday life in industrialized society, and the demand for these products is expected to grow as the world population grows and developing countries move up the economic ladder. However, synthetic plastics are slow to degrade in landfills. If and when they do breakdown, the monomers and their derivatives resulting from degradation may actually be more hazardous to human health than the undegraded polymers (Selenskas et al. (1995) Amer. J. Indust. Med. 28:38R-398; Tosti et al. (1993) Toxicol. Indust. Health 9:493-502; Yin et al. (1996) J. Food Drug Anal. 4:313-318). These concerns have raised to a new level the urgency of exploring the use of the environmentally friendly, compostable polymers as substitutes for synthetic plastics. Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoic acids that are synthesized by a variety of bacteria as storage polymers under stressful conditions (Steinbuchel, A. (1991) Biomaterials: Novel materials from biological materials, D. Byrom, ed. (New York: Macmillan Publishers Ltd.), pp. 123-213). Since PHAs have thermoplastic properties, that is they become soft when heated and hard when cooled, and are fully biodegradable, they offer an attractive alternative to synthetic plastics (Brandl et al. (1995) Can. J. Microbiol. 41: 143-153; Byrom, D. (1993) Int. Biodeterior. Biodegrad. 31:199-208; Lee, S. Y. (1996) Biotechnol. Bioeng 49:1-14; Nawrath et al. (1993) Abst. Pap. Amer. Chem. Soc. 206:22-27; Poirier et al. (1995) Bio/technology Nat. Publ. Co. 13:142-150; Steinbuechel, A. (1992) Curr. Opin. Biotechnol. 3:291-297). Unlike man-made plastics, the production of PHA by living organisms is not dependent on finite natural resources like petroleum.

[0004] Currently, only one type of polyhydroxyalkanoate (PHA), Biopol, a copolymer made by fermentation, is commercially available (Poirier et al. (1995) Bio/technology Nat. Publ. Co. 13:142-150). However, at approximately $7 per pound, this polymer is much too expensive in comparison to the synthetic plastics that have similar properties but are cheaper with a price of approximately $0.5 per pound (Poirier et al. (1995) Bio/technology Nat. Publ. Co. 13:142-150). The higher cost of Biopol results primarily from its cost of production, the main contributing factor being the substrate (Poirier et al. (1995) Bio/technology Nat. Publ. Co. 13:142-150). If the PHAs can be produced in plants, the cost of production can be lowered substantially because these polymers would compete with seed oil as natural storage constituents of the cell. The current market price of plant seed oil is between 26 and 28 cents per pound (Anonymous (1998) Economic Research Service (Washington, D.C. 20036: U.S. Department of Agriculture). Only about 40% of the energy required to extend a fatty acid chain by two carbons is expended on extending a PHA chain by the same length. Starting with acetyl-CoA, a two carbon extension in oil biosynthesis requires two NADPH and one ATP. In comparison, only one NADPH is needed to accomplish the same for PHA biosynthesis (FIG. 1). Theoretically, more than two units of PHA should be formed for every unit of oil replaced.

[0005] Until recently, the only PHA that has been produced in plants was polyhydroxybutyrate (PHB), a homopolymer of 3 -hydroxybutyric acid (John et al. (1996) Proc. Natl. Acad. Sci. USA 93:12768-12773; Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA 91:12760-12764; Padgette et al. (1997) Plant Physiol. 114 (Suppl.) 3S; Poirier et al. (1992) Science 256.520-523)). Because this polymer is crystalline and brittle with a melting point too close to its degradation point, PHB is difficult to mold into desirable products (Lee, S. Y. (1996) Biotechnol. Bioeng. 491:1-14). Many bacteria make copolymers of 3-hydroxyalkanoic acids with a carbon chain length greater than or equal to five (Steinbuchel, A. (1991) Biomaterials: Novel materials from biological materials, D. Byrom, ed. (New York: Macmillan Publishers Ltd.), pp. 123-213). Such copolymers are polyesters composed of different 3-hydroxyalkanoic acid monomers. Depending on the composition, these copolymers can have properties ranging from firm to elastic (Anderson et al. (1990) Microbiol. Rev. 54:450-472; Lee, S. Y. (1996) Biotechnol. Bioeng. 49:1-14). Unlike PHB, the PHA copolymers are suitable for a variety of applications because they exhibit a wide range of physical properties.

[0006] Initial attempts at producing PHA in the cytosol proved toxic to the plant (Poirier et al. (1992) Science 256:520-523). This problem was overcome by targeting the PHA-producing enzymes to plastids (Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA 91:12760-12764). In either cellular compartment, however, only PHB was accumulated, not any of the copolymers. With both of these methods, the genes from Ralstonia eutropha (also known as Alcaligenes eutrophus) were used. The PHA synthase of this bacterium can utilize only short chain (C.sub.3-C.sub.5) monomers (Steinbuchel, A. (1991) Biomaterials: Novel materials from biological materials, D. Byrom, ed. (New York: Macmillan Publishers Ltd.), pp. 123-213).

[0007] Recently, the synthesis of PHA containing 3-hydroxyalkanoic acid monomers ranging from 6 to sixteen carbon in Arabidopsis thaliana was reported (Mittendorf et al. (1998) Proc. Natl. Acad. Sci. USA 95:13397-13402). To accumulate PHA, the Arabidopsis plants were transformed with a nucleotide sequence encoding PHA synthase from Pseudomonas aeuginosa that was modified for peroxisome targeting by the addition of a nucleotide sequence encoding the C-terminal 34 amino acids of a Brassica napus isocitrate lyase. In these plants, PHA was produced in glyoxysomes, leaf-type peroxisomes and vacuoles. However, PHA production was very low in the Arabidopsis plants, suggesting that either the introduced PHA synthase did not function properly in the intended organelle or more likely that the necessary substrates for the introduced PHA synthase were present at levels that were limiting for PHA synthesis. While this report demonstrated that PHA can be produced in peroxisomes of plants, the level of PHA produced in the plants was far below levels necessary for the commercial production of PHA in plants.

SUMMARY OF THE INVENTION

[0008] Methods are provided for producing PHA and intermediate molecules thereof in plants. The methods find use in the production of high-quality, biodegradable thermoplastics. The invention provides environmentally friendly alternatives to petroleum-based methods for producing plastics. The methods involve genetically manipulating a plant to produce enzymes for PHA synthesis in its peroxisomes. The methods comprise stably integrating in the genome of a plant nucleotide sequences encoding enzymes involved in the synthesis of PHA, preferably PHA copolymers.

[0009] Also provided are plants, plant tissues, plant cells, and seeds thereof, that are genetically manipulated to produce at least one enzyme involved in the synthesis of PHA in plant peroxisomes.

[0010] Nucleotide molecules and expression cassettes comprising nucleotide sequences encoding enzymes that can be employed in the synthesis of PHA in the peroxisomes of plants are provided. In particular, the invention provides nucleotide molecules encoding a maize MFP2-like polypeptide, and fragments and variants thereof. Additionally, the invention provides nucleotide molecules comprising nucleotide sequences which encode for either the hydratase or the dehydrogenase domain of the yeast multifunctional protein-2 (MFP2). Such nucleotide molecules encode novel enzymes which find use in PHA synthesis in host cells and plants, particularly in peroxisomes of plants. Isolated polypeptides encoded by the nucleotide molecules of the invention and host cells transformed with such nucleotide molecules are additionally provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 schematically depicts possible biosynthetic steps for producing PHA in plant peroxisomes utilizing enzymes from bacteria.

[0012] FIG. 2 schematically illustrates the full-length yeast multifunctional protein (yMFP), truncated versions of yMFP, and the maize MFP2-like polypeptide.

[0013] FIG. 3 schematically depicts the biosynthetic pathways for the synthesis PHB and PHA copolymers which are composed of 3-hydroxybutanoic acid monomers and other 3-hydroxyalkanoate monomers.

[0014] FIG. 4 schematically illustrates a functional assay for a 2-enoyl-CoA hydratase that catalyzes the synthesis of R-(-)-3-hydroxyacyl-CoA. This hydratase converts crotonyl-CoA to R-(-)-3-hydroxyacyl-CoA which is then utilized as the substrate by PHA synthase to form polymers. PHA synthase utiltizes R-(-)-3-hydroxyacyl-CoAs, but not S-3-hydroxyacyl-CoAs. Detection of polymer formation in the presence of crotonyl-CoA demonstrates that R-(-)-3-hydroxyacyl-CoA was produced.

[0015] FIG. 5 is a graphical depiction of the results of a functional assay for the activity of a 2-enoyl-CoA hydratase that catalyzes the synthesis R-(-)-3-hydroxyacyl-CoA.

DETAILED DESCRIPTION OF THE INVENTION

[0016] A number of terms used herein are defined and clarified in the following section.

[0017] By "PHA copolymer" is intended a polymer composed of at least two different 3-hydroxyalkanoic acid monomers.

[0018] By "PHA homopolymer" is intended a polymer that is composed of a single 3-hydroxyalkanoic acid monomer.

[0019] By "intermediate molecule" is intended a precursor in the biosynthetic pathway for PHA in a plant. Because PHA is not known to occur naturally in a plant, the biosynthetic pathway for PHA in plant additionally encompasses enzymes and products thereof that are involved in PHA synthesis which result from the genetic manipulation of the plant. Intermediate molecules of the present invention include, but are not limited to, fatty acids and .beta.-oxidation products derived therefrom, acetyl-CoA, acetoacetyl-CoA and other 3-ketoacyl-CoAs, 3-hydroxybutyryl-CoA, and other 3-hydroxyacyl-CoAs.

[0020] By "modified or unusual" fatty acids is intended fatty acids that have structural features such as, for example, an epoxy group, a triple bond, and methyl branching. Such "modified or unusual" fatty acids include, but are not limited to, vernolic acid, petroselinic acid, sterculic acid, chaulmoogric acid, erucic acid, ricinoleic acid, labellenic acid, crepenynic acid, and stearolic acid.

[0021] The present invention is drawn to methods and compositions for producing PHA in plants. Particularly, the present invention provides improved methods for producing PHA in plant peroxisomes. The methods involve increasing the level of PHA produced in a plant by increasing the synthesis of at least one intermediate molecule in PHA synthesis. Thus, the methods involve modifying the metabolic functions of the peroxisome to allow for increased production of PHA in a plant. Furthermore, the invention provides methods for producing PHA copolymers in plant peroxisomes.

[0022] Methods for producing PHB in the cytosol or plastids of plants and for producing PHA in plant peroxisomes are known in the art. However, such methods do not achieve the synthesis of high levels of PHA in plants. An object of the present invention is to provide improved methods for producing PHA, preferably PHA copolymers, in plants. The present invention involves genetically modifying plants in such a manner as to alter the metabolic functions of the peroxisome to increase the flux of carbon toward PHA synthesis. Such plants find use in preferred methods for producing high levels of PHA in plants, particularly in seeds, more particularly in oilseeds.

[0023] Methods for producing PHA in plants are provided. The methods involve genetically manipulating the genome of a plant to direct the synthesis of PHA to the peroxisomes, preferably peroxisomes in developing seeds. The invention encompasses plants and seeds thereof, that have been genetically manipulated to produce enzymes involved in PHA synthesis and expression cassettes containing coding sequences for such enzymes. The invention further encompasses genetically manipulated plant cells and plant tissues.

[0024] Peroxisomes, which are also known as microbodies, are small spherical organelles. In plants, there are generally two types of peroxisomes, leaf-type peroxisomes and glyoxysomes. Glyoxysomes are present in seeds containing oil, particularly during germination (Heldt (1997) Plant Biochemistry and Plant Molecular Biology, Oxford University Press, New York). In the present invention, "peroxisome" is intended to encompass all peroxisomes found in plant cells, including, but not limited to, leaf-type peroxisomes, microbodies, and glyoxysomes.

[0025] Methods are provided for producing PHA in a plant involving genetically manipulating the plant to produce in its peroxisomes at least two enzymes in the PHA biosynthetic pathway. The plants of the invention each comprise in their genomes at least two stably incorporated DNA constructs, each DNA construct comprising a coding sequence for an enzyme involved in PHA synthesis operably linked to a promoter that drives the expression of a gene in a plant. Plants of the invention are genetically manipulated to produce a PHA synthase (also known as a PHA polymerase) that catalyzes polymer synthesis. Preferably, such a PHA synthase catalyzes the synthesis of copolymers. More preferably such a PHA synthase catalyzes the synthesis of copolymers comprised of 3-hydroxybutanoic acid monomers and at least one additional monomer having a chain length of greater than four carbons. Most preferably such a PHA synthase catalyzes the synthesis of copolymers comprised of 3-hydroxybutanoic acid monomers and at least one additional monomer having a hydroxyacyl-chain length of from about 5 to about 18 carbons. Preferred PHA synthases include PHA synthases encoded by nucleotide sequences isolatable from Pseudomonas oleovorans (GenBank Accession No. M58445, SEQ ID NO: 8), Pseudomonas putida (GenBank Accession No. AF042276, SEQ ID NO: 9), Pseudomonas aeruginosa (EMBL Accession No. X66592, SEQ ID NO: 10), Aeromonas caviae (DDBJ Accession No. D88825, SEQ ID NO: 11), and Thiocapsa pfennigii (EMBL Accession No. A49465, SEQ ID NO: 12). The preferred PHA synthases additionally include the PHA synthases encoded by nucleotide sequences isolatable from Pseudomonas fluorescens (See U.S. Provisional Patent Application No. 60/156,770 filed Sep. 29, 1999; herein incorporated by reference.). In certain methods of the invention, the majority of PHA copolymers produced are comprised of monomers of chain-length C.sub.4 to C.sub.18.

[0026] The DNA constructs of the invention each comprise a coding sequence for an enzyme involved in PHA synthesis operably linked to a promoter that drives expression in a plant cell. Preferably, the promoters are selected from seed-preferred promoters, chemical-regulatable promoters, germination-preferred promoters, and leaf-preferred promoters. If necessary for directing the encoded proteins to the peroxisome, the DNA construct can include an operably linked peroxisome-targeting signal sequence.

[0027] It is recognized that for producing high levels of PHA copolymers in certain plants, particularly in their peroxisomes, it may be necessary to genetically manipulate plants to produce additional enzymes involved in PHA synthesis. Generally, the additional enzymes are directed to the peroxisome to increase the synthesis of at least one intermediate molecule. For example, such an intermediate molecule can be the substrate for a PHA synthase including, but not limited to, an R-(-)-3-hydroxyacyl-CoA. The methods of the invention comprise genetically modifying plants to produce, in addition to the PHA synthase described supra, one, two, three, four, or more additional enzymes involved in PHA synthesis. Preferably, each DNA construct comprising the coding sequence of one of these additional enzymes is operably linked to a promoter that drives expression in a plant and also to a nucleotide sequence encoding a peroxisome-targeting signal sequence. Depending on the plant, the addition of one or more of these enzymes may be necessary to achieve high-level PHA synthesis in the plant. The additional enzymes include, but are not limited to, an enzyme that catalyzes the synthesis of R-(-)-3-hydroxyacyl-CoA, a 3-ketoacyl-CoA reductase, and an acetyl-CoA:acetyl transferase.

[0028] Additionally, the plant of the invention can comprise in its genome a DNA construct comprising a coding sequence for second PHA synthase. Preferably, the second PHA synthase is capable of synthesizing PHB. Preferred second PHA synthases include those encoded by nucleotide sequences isolatable from Ralstonia eutropha (GenBank Accession No. J05003, SEQ ID NO: 13), Acinetobacter sp. (GenBank Accession No. U04848, SEQ ID NO: 14), Alcaligenes latus (GenBank Accession No. AF078795, SEQ ID NO: 15), Azorhizobium caulinodans (EMBL Accession No. AJ006237, SEQ ID NO: 16), Comamonas acidovorans (DDBJ Accession No. AB009273, SEQ ID NO: 17), Methylobacterium extorquens (GenBank Accession No. L07893, SEQ ID NO: 18), Paracoccus denitrificans (DDBJ Accession No. D43764, SEQ ID NO: 19), and Zoogloea ramigera (GenBank U66242, SEQ ID NO: 20)

[0029] The methods of the invention additionally comprise growing the plant under conditions favorable for PHA production, harvesting the plant, or one or more parts thereof, and isolating the PHA from the plant or part thereof. Such parts include, but are not limited to, seeds, leaves, stems, roots, fruits, and tubers. The PHA can be isolated from the plant or part thereof by methods known in the art. See, U.S. Pat. Nos. 5,942,597; 5,918,747; 5,899,339; 5,849,854; and 5,821,299; herein incorporated by reference. See also, EP 859858A1, WO 97/07229, WO 97/07230, and WO 97/15681; herein incorporated by reference.

[0030] The invention provides methods for producing increased levels of PHA in the peroxisomes of plants that involve increasing the synthesis of one or more intermediate molecules in the peroxisome. The methods involve diverting the flux of carbon in the peroxisome to favor PHA synthesis over endogenous metabolic processes such as, for example, .beta.-oxidation. In a first aspect of the invention, plants are genetically manipulated to increase the synthesis of R-(-)-3-hydroxyacyl-CoAs. In a second aspect of the invention, plants are genetically manipulated to increase the synthesis of a specific R-(-)-3-hydroxyacyl-CoA, R-(-)-3-hydroxybutyryl-CoA. In a third aspect of the invention, the first and second aspects are combined to provide plants that are genetically manipulated to increase the synthesis of both R-(-)-3-hydroxyacyl-CoAs and R-(-)-3-hydroxybutyryl-CoA.

[0031] Further, it is recognized that each of the aspects of the invention can be used to produce PHA with substantially different monomer compositions. In particular, the level of 3-hydroxybutanoic acid in the PHA produced in a plant will vary with each aspect. For each particular type of plant, PHA produced by plants of the second or third aspect of the invention is expected to have a higher 3-hydroxybutanoic acid monomer content than PHA produced by plants of the first aspect. Similarly, PHA produced by plants of the second aspect is expected to have a higher 3-hydroxybutanoic acid monomer content than PHA produced by plants of the third aspect.

[0032] In a first embodiment of the invention, methods are provided for producing PHA involving genetically manipulating a plant for increased synthesis of R-(-)-3-hydroxyacyl-CoA, a key intermediate molecule in PHA synthesis in the peroxisome. The methods comprise stably integrating into the genome of a plant a first DNA construct comprising a coding sequence for a PHA synthase, and a second DNA construct comprising a coding sequence for an enzyme that catalyzes the formation R-(-)-3-hydroxyacyl-CoA, a substrate of PHA synthase. In .beta.-oxidation in plant peroxisomes, acyl-CoA oxidase catalyzes the conversion of fatty acyl-CoA into 2-enoyl-CoA which is subsequently converted to S-(+)-3-hydroxyacyl-CoA via the 2-enoyl-CoA hydratase of a multifunctional protein. While some R-(-)-3-hydroxyacyl-CoA may be present in peroxisomes, the level is believed to be very low and insufficient to allow for the synthesis of an economically acceptable level of PHA in a plant. Furthermore, all known PHA synthases require that 3-hydroxyacyl-CoA monomers be in R-(-)-form for PHA synthesis. To overcome the substrate limitation for PHA synthesis, the present invention discloses methods for PHA synthesis which involve providing a plant with an enzyme in its peroxisomes that catalyzes the formation of R(-)-3-hydroxyacyl-CoA. By genetically manipulating a plant to increase the synthesis of R-(-)-3-hydroxyacyl-CoA, the present invention overcomes a major impediment to achieving high-level production in plants of PHA, particularly copolymers. Such an enzyme can be an enoyl-CoA hydratase that catalyzes the synthesis of R-(-)-3-hydroxyacyl-CoA, particularly an 2-enoyl-CoA hydratase from Aeromonas caviae (DDBJ Accession No. E15860, SEQ ID NO: 21).

[0033] Alternatively, two proteins from yeast and one from maize can each be utilized as the enzyme. One such protein is the yeast multifunctional protein (encoded by GenBank Accession No. M86456, SEQ ID NO: 3) which possesses an 2-enoyl-CoA hydratase activity and a 3-hydroxyacyl dehydrogenase (reductase) activity. Similarly, the invention provides an isolated MFP2-like polypeptide (previously designated as a multifunctional protein-2 or MFP-2) from maize (SEQ ID NO: 2) which also possesses an 2-enoyl-CoA hydratase activity. The invention further provides isolated nucleotide molecules encoding such a maize MFP2-like polypeptide (SEQ ID NO: 1). The hydratase of the yeast multifunctional protein and maize MFP2-like polypeptide is known to yield R-(-)-3-hydroxyacyl-CoA products. If necessary, the dehydrogenase activity of the yeast multifunctional protein can be eliminated or neutralized by methods known to those of ordinary skill in the art such as, for example, site-directed mutagenesis, and truncation of the coding sequence to only the portion necessary to encode the desired hydratase activity. The invention provides isolated nucleotide molecules comprising nucleotide sequences which encode either the hydratase or reductase of the yeast multifunctional protein (SEQ ID NOs: 4 and 6). Additionally provided are isolated polypeptides encoded by such sequences (SEQ ID NOs: 5 and 7).

[0034] Other multifunctional proteins known in the art can be utilized in the methods of the present invention. Any multifunctional protein possessing a domain comprising a 2-enoyl-CoA hydratase that is capable of catalyzing the synthesis of R-(-)-3-hydroxyacyl-CoA can be employed in the methods of the invention.

[0035] The other yeast protein that can be utilized as the enzyme that catalyzes the formation of R-(-)-3-hydroxyacyl-CoA is an enzyme identified as a 3-hydroxybutyryl-CoA dehydrogenase (Leaf et al. (1996) Microbiology 142:1169-1180). The gene encoding this enzyme can be cloned from Saccharomyces cervisae, sequenced and employed in the methods of the present invention. It is recognized that the nucleotide sequence encoding this enzyme can be modified to alter the amino acid sequence of the enzyme in such a manner as to favorably affect the production of R-(-)-3-hydroxyacyl-CoA in a plant. Such modifications can affect characteristics of the enzyme such as, for example, substrate specificity, product specificity, product inhibition, substrate binding affinity, product binding affinity, and the like. A method such as, for example, DNA shuffling can be employed to modify this enzyme in the desired manner. Any method known in the art for altering the characteristics of an enzyme to favorably affect the mass action ratio toward the desired product is encompassed by the methods of the present invention. Such methods typically involve modifying at least a portion of the nucleotide sequence encoding the enzyme and include, but are not limited to, DNA shuffling, site-directed mutagenesis, and random mutagenesis.

[0036] In a second embodiment of the invention, methods are provided for producing PHA involving genetically manipulating a plant for increased synthesis of R-(-)-3-hydroxybutyryl-CoA, a substrate of PHA synthase, in peroxisomes. The methods of the invention provide a plant that is genetically manipulated for increased synthesis of a substrate for a PHA synthase and thus provide a plant that is genetically manipulated for high-level PHA synthesis in its peroxisomes. The methods involve stably integrating into the genome of a plant a first DNA construct comprising a coding sequence for a PHA synthase, and a second DNA construct comprising a coding sequence for 3-ketoacyl-CoA reductase and a third DNA construct comprising a coding sequence for an acetyl-CoA:acetyl transferase. The first, second, and third DNA constructs each additionally comprise an operably linked promoter that drives expression in a plant cell, and if necessary, an operably linked peroxisome-targeting signal sequence. Acetyl-CoA:acetyl transferase, also referred to as ketothiolase, catalyzes the synthesis of acetoacetyl-CoA from two molecules of acetyl-CoA. Acetoacetyl-CoA can then be converted into R-(-)-3-hydroxybutyryl-CoA via a reaction catalyzed by a 3-ketoacyl-CoA reductase, particularly an acetoacetyl-CoA reductase.

[0037] Preferred 3-ketoacyl-CoA reductases of the invention are those that utilize NADH and include, but are not limited to, at least a portion of the multifunctional proteins from yeast (encoded by GenBank Accession No. M86456, SEQ ID NO: 3) and rat (encoded by GenBank Accession No. U37486, SEQ ID NO: 22), wherein such a portion comprises a 3-ketoacyl-CoA reductase domain. Any multifunctional protein having a 3-ketoacyl-CoA reductase (dehydrogenase) domain can be employed in the methods of the invention. However, in the methods of the invention, NADPH-dependent 3-ketoacyl-CoA reductases can also be employed including, but not limited to, the 3-ketoacyl-CoA reductases encoded by GenBank Accession No. J04987 (SEQ ID NO: 23).

[0038] Preferred acetyl-CoA:acetyl transferases of the invention include a radish acetyl-CoA:acetyl transferase encoded by the nucleotide sequence having EMBL Accession No. X78116 (SEQ ID NO: 24).

[0039] If necessary to increase the level of NADPH in the peroxisome, the methods of the second embodiment can additionally involve, stably integrating into the genome of a plant a fourth DNA construct comprising a nucleotide sequence encoding an NADH kinase or an NAD.sup.+ kinase and an operably linked promoter that drives expression in a plant cell. Such NADH and NAD.sup.+ kinases catalyze the synthesis of NADPH and NADP.sup.+, respectively. Nucleotide sequences encoding such kinases include, but are not limited to, DDJB Accession No. E13102 (SEQ ID NO: 25) and EMBL Accession Nos. Z73544 (SEQ ID NO: 26) and X84260 (SEQ ID NO: 27). The fourth construct can additionally comprise an operably linked peroxisome-targeting signal sequence. By targeting such NADH and NAD.sup.+ kinases to the peroxisome, the level of NADPH and NADP.sup.+ can be increased in the plant peroxisome for use by enzymes, such as, for example, an NADPH-dependent 3-ketoacyl-CoA reductase.

[0040] In a third embodiment of the invention, methods are provided for producing PHA in a plant involving genetically manipulating a plant for increased synthesis of R-(-)-3-hydroxybutyryl-CoA and other R-(-)-3-hydroxyacyl-CoA molecules. Such methods provide a plant that is genetically manipulated to overcome substrate limitations for PHA copolymer synthesis in its peroxisomes. The methods involve stably integrating into the genome of a plant a first, a second, a third, and a fourth DNA construct comprising a coding sequence for an enzyme involved in PHA synthesis in a plant. The first DNA construct comprises a coding sequence for a PHA synthase that is capable of catalyzing the synthesis of PHA copolymers. The second DNA construct comprises a coding sequence for an enzyme that catalyzes the synthesis of R-(-)-3-hydroxyacyl-CoA. The third DNA construct comprises a coding sequence for a 3-ketoacyl-CoA reductase, and the fourth DNA construct comprises a coding sequence for an acetyl-CoA:acetyl transferase. If desired, a fifth DNA construct can also be stably integrated into the genome of the plant. The fifth DNA construct comprises a nucleotide sequence encoding a NADH kinase or an NAD.sup.+ kinase.

[0041] Preferred enzymes of the third embodiment include the enzymes of the first and second embodiments, described supra. The DNA constructs each additionally comprise an operably linked promoter and, if necessary, an operably linked peroxisome-targeting signal to direct the encoded protein to the peroxisome. By targeting such enzymes to the peroxisome, the plant is capable of increased synthesis of intermediate molecules, particularly intermediate molecules that are substrates for a PHA synthase that catalyzes the formation of copolymers.

[0042] Methods are provided for increasing the synthesis of PHA in a plant. Such methods find use with methods known in the art for producing PHA in plants, particularly in peroxisomes. The methods of the invention involve increasing the synthesis of an intermediate molecule in PHA synthesis. Preferably, such an intermediate molecule is limiting for PHA synthesis in the peroxisome and that increasing the synthesis of such a molecule in the peroxisome increases the level of PHA produced in a plant. It is recognized that increasing the synthesis of an intermediate molecule in a plant peroxisome might not lead to an increased level of the intermediate molecule in the plant because the intermediate molecule can be further metabolized into, for example, PHA.

[0043] The methods for increasing the synthesis of PHA in a plant involve stably incorporating into the genome of a plant at least one DNA construct comprising a coding sequence for an enzyme involved in the synthesis of an intermediate molecule, operably linked to a promoter that drives expression in a plant. If necessary for peroxisome-targeting of the encoded enzyme, the DNA construct additionally comprises a peroxisome-targeting signal operably linked to the coding sequence.

[0044] The methods of the invention can be used to increase the synthesis of any intermediate molecule in PHA synthesis. Preferred intermediate molecules include those that can be limiting for PHA synthesis, particularly in the peroxisome, such as, for example, R-(-)-3-hydroxybutyryl-CoA, other R-(-)-3-hydroxyacyl-CoAs, acetoacetyl-CoA, and other 3-ketoacyl-CoAs.

[0045] A plant can be genetically manipulated to produce any one or more of the enzymes involved in the synthesis of the intermediate molecule in the plant including, but not limited to, the enzymes for PHA synthesis of the present invention described supra. Preferred enzymes for increasing the synthesis of an intermediate molecule include enzymes, described supra, that catalyze the formation of R-(-)-3-hydroxyacyl-CoA, 3-ketoacyl-CoA reductases that utilize NADH and acetyl-CoA:acetyl transferases.

[0046] In a fourth embodiment of the invention, methods are provided for increasing in a plant the synthesis of a R-(-)-3-hydroxyacyl-CoA, key intermediate molecule in PHA synthesis. The level in the peroxisome of R-(-)-3-hydroxyacyl-CoA, a substrate of PHA synthase, is known to be very low and is believed to limit the level of PHA produced in the peroxisome. Thus, increasing the synthesis of R-(-)-3-hydroxyacyl-CoA can increase the synthesis of PHA in a plant. The methods comprise genetically manipulating plants to produce an enzyme that catalyzes the synthesis of R-(-)-3-hydroxyacyl-CoA, preferably an enzyme selected from a 2-enoyl-CoA hydratase from Aeromonas caviae (encoded by DDBJ Accession No. E15860, SEQ ID NO: 21), a maize MFP2-like polypeptide (SEQ ID NO: 2), a modified yeast multifunctional protein (SEQ ID NO: 5) possessing a 2-enoyl-CoA hydratase activity and a 3-hydroxyacyl-CoA dehydrogenase activity wherein the latter activity is neutralized, or a modified yeast 3-hydroxyacyl-CoA dehydrogenase, wherein the enzyme utilizes NADH and its mass action ratio has been shifted in favor of R-(-)-3-hydroxyacyl-CoA over 3-ketoacyl-CoA.

[0047] Methods are provided for producing novel enzymes for the synthesis of PHA, particularly in peroxisomes, more particularly in plant peroxisomes. The methods find use in providing novel peroxisome-localized enzymes for PHA synthesis in plant peroxisomes. Additionally, the methods find further use in providing DNA constructs that can be used to transform a plant for the production of a PHA synthesis enzyme in its peroxisomes. The methods involve modifying the coding sequence of a multifunctional protein, particularly an MFP2, more particularly an MFP2 from yeast (encoded by GenBank Accession No. M86456, SEQ ID NO: 3) or rat (encoded by GenBank Accession No. U37486, SEQ ID NO: 22). The coding sequence is modified to eliminate or substantially reduce of one of the two separate enzymatic activities of the protein encoded thereby. The coding sequence can be modified by methods known in the art including, but not limited, to site-directed mutagenesis and deletion of a portion of the coding sequence. If necessary, an initiation codon, a stop codon or both can be added to facilitate translation in a host cell. The methods can further involve preparing a DNA construct by operably linking a nucleotide sequence encoding a peroxisome-targeting signal to the modified coding sequence. If desired for expression in a plant or host cell, the DNA construct can additionally comprise an operably linked promoter that drives expression in the plant or cell. The novel enzymes of the invention can be produced by transforming a plant or host cell with such a DNA construct.

[0048] In a fifth embodiment of the invention, methods are provided for producing a peroxisome-targeted 2-enoyl-CoA hydratase employing the coding sequence of a yeast MFP2 (GenBank Accession No. M86456, SEQ ID NO: 3). The methods involve modifying the coding sequence to eliminate the dehydrogenase (also referred to as reductase) activity of the encoded protein. This can be accomplished by, for example, deleting from the 5' end of the coding sequence a portion that encoded at least part of the dehydrogenase domain (FIG. 2, SEQ ID NO: 4). To facilitate translation of the encoded protein, an initiation codon can be added to the truncated nucleotide sequence (SEQ ID NO: 4). A DNA construct can be prepared by operably linking a nucleotide sequence encoding a plant peroxisome-targeting signal to such a truncated coding sequence. Such a DNA construct can additionally comprise an operably linked promoter that drives expression in a plant.

[0049] In a sixth embodiment of the invention, methods are provided for producing a NADH-dependent, peroxisome-targeted 3-ketoacyl-CoA reductase employing the coding sequence of a yeast multifunctional protein (GenBank Accession No. M86456, SEQ ID NO: 3). The methods involve modifying the coding sequence of a multifunctional protein to eliminate the hydratase activity of the encoded protein. Because the reductase of the yeast multifunctional protein is known to be NADH dependent, a peroxisome-targeted 3-ketoacyl-CoA reductase is expected to be NADH dependent. Such an NADH-dependent reductase finds use in the high-level synthesis of PHA in plant peroxisomes where NADPH, but not NADH, is known to be limiting. This can be accomplished by, for example, deleting from the 3' end of the coding sequence a portion that encoded at least a part of the hydratase domain (FIG. 2, SEQ ID NO: 6). To facilitate translation of the desired polypeptide, an appropriate stop codon can be operably linked to the 3' end of the truncated coding sequence (SEQ ID NO: 6). In addition, a DNA construct can be prepared by operably linking a nucleotide sequence encoding a plant peroxisome-targeting signal to such a truncated coding sequence. Such a DNA construct can additionally comprise an operably linked promoter that drives expression in a plant.

[0050] Methods are provided for increasing the level in a plant of at least one intermediate molecule in PHA synthesis in the plant. Such intermediate molecules include molecules naturally synthesized by the plant as well as those that are synthesized by the plant after being genetically manipulated to comprise at least one enzyme involved in PHA synthesis that does not occur naturally in the plant. The methods involve the buildup of intermediate molecules as well as the use of additional enzymes for the production of specialty chemicals. Thus, plants containing intermediate molecules or PHA can be obtained. It is recognized that methods of the present invention can be used in combination with methods for producing PHA homopolymers, copolymers or both.

[0051] To increase the level of at least one intermediate molecule in a plant, the plant can be genetically manipulated to produce any one or more of the enzymes involved in the synthesis of the intermediate molecule in the plant including, but not limited to, the enzymes for PHA synthesis of the present invention described supra. Preferred enzymes for increasing the synthesis of an intermediate molecule include enzymes, described supra, that catalyze the formation of R-(-)-3-hydroxyacyl-CoA, 3-ketoacyl-CoA reductases that utilize NADH and acetyl-CoA:acetyl transferases.

[0052] Further, it is recognized that it may be necessary to lower or eliminate the activity of an endogenous enzyme in a plant that in some way limits the synthesis of the desired intermediate molecule. Such an endogenous enzyme may, for example, catabolize or modify the intermediate molecule in an undesirable way. Methods for lowering or eliminating the activity of an enzyme in a plant include, but are not limited to, sense and antisense suppression methods. For example, the activity of the 2-enoyl-CoA hydratase of an endogenous multifunctional protein that catalyzes the formation of S-(+)-3-hydroxyacyl-CoA can be reduced or eliminated in the peroxisome to favor, instead, the synthesis of R-(-)-3-hydroxyacyl-CoA therein.

[0053] While the methods of the invention can be used with any plant, preferred plants are oilseed plants genetically manipulated to produce PHA copolymers in their peroxisomes, particularly in seeds. More preferably, the oilseed plants have been genetically manipulated to have seeds with increased levels of short-chain fatty acids, modified or unusual fatty acids, cytosolic acyl-CoA oxidase activity, or combinations thereof. Such oilseed plants have increased rates of .beta.-oxidation in their seeds and find use in methods of producing high levels of PHA copolymers, particularly in seeds.

[0054] While the compositions and methods disclosed herein are drawn to the production of PHA and PHA intermediates in plants, the present invention is not limited to methods involving PHA production in plant and cells thereof. Those skilled in the art will recognize that the compositions and methods can be employed with any host cell for the production of PHA and intermediates thereof. Host cells include, but are not limited to, plant cells, animal cells, bacterial cells and fungal cells, particularly yeast cells.

[0055] Compositions comprising nucleic acid molecules which comprise coding sequences for enzymes involved in the synthesis of PHA in the peroxisomes of plants are provided. Compositions of the invention include nucleotide molecules encoding a maize MFP2-like polypeptide and fragments and variants thereof. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence set forth in SEQ ID NO: 2. Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, for example that set forth in SEQ ID NO: 1, and fragments and variants thereof.

[0056] The invention encompasses isolated or substantially purified nucleic acid or protein compositions. An "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0057] Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein of the invention. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes generally do not retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.

[0058] A fragment of a nucleotide sequence of the invention that encodes a biologically active portion of a multifunctional protein will encode at least 15, 25, 30, 50, 100, 150, 200, 250 or 300 contiguous amino acids, or up to the total number of amino acids present in a full-length multifunctional protein of the invention (for example, 314 amino acids for SEQ ID NO: 2). Fragments of a multifunctional protein nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of a multifunctional protein.

[0059] Thus, a fragment of a multifunctional protein nucleotide sequence may encode a biologically active portion of a multifunctional protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a multifunctional protein can be prepared by isolating a portion of one of the multifunctional protein nucleotide sequences of the invention, expressing the encoded portion of the multifunctional protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the multifunctional protein. Nucleic acid molecules that are fragments of a multifunctional protein nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200 or 1,300 nucleotides, or up to the number of nucleotides present in a full-length multifunctional protein nucleotide sequence disclosed herein (for example, 1362 nucleotides for SEQ ID NO: 1).

[0060] By "variants" is intended substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the multifunctional proteins or other enzymes involved in PHA synthesis of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a multifunctional protein or other enzyme of the invention. Generally, variants of a particular nucleotide sequence of the invention will have at least about 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.

[0061] The nucleotide sequences of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequence set forth herein. Sequences isolated based on their sequence identity to the entire maize MFP2-like polypeptide nucleotide sequence set forth herein or to fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. By "orthologs" is intended genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.

[0062] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning. A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

[0063] In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as .sup.32P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the maize MFP2-like polypeptide nucleotide sequence of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0064] For example, the entire maize MFP2-like polypeptide nucleotide sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding MFP2 sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among MFP2 sequences and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Such probes may be used to amplify corresponding MFP2 sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in a organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0065] Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

[0066] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

[0067] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (%GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by about 1 .degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with .gtoreq.90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T.sub.m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0068] Thus, isolated sequences that encode for an MFP2 and which hybridize under stringent conditions to the MFP2 sequence disclosed herein, or to fragments thereof, are encompassed by the present invention. Such sequences will be at least about 50% to 60% homologous, about 60%, to 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous with the disclosed sequences. That is, the sequence identity of sequences may range, sharing at least about 50% to 60%, about 65% or 70%, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0069] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity". [0070] (a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. [0071] (b) As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

[0072] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-similarity-method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0073] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.

[0074] Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP version 10 using the following parameters: % identity using GAP Weight of 50 and Length Weight of 3; % similarity using Gap Weight of 12 and Length Weight of 4, or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0075] GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

[0076] GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915). [0077] (c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.). [0078] (d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. [0079] (e)(i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.

[0080] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1.degree. C. to about 20.degree. C., depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. [0081] (e)(ii) The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides that are "substantially similar" share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.

[0082] The use of the term "DNA constructs" herein is not intended to limit the present invention to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. Thus, the DNA constructs of the present invention encompass all nucleotide constructs that can be employed in the methods of the present invention including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0083] In the practice of embodiments of the invention, heterologous DNA can be employed. By "heterologous DNA" is intended DNA that is foreign to the genome of an organism. Such foreign DNA encompasses any DNA present in the genome of an organism that originated in the present organism or one of its progenitors by artificial methods such as, for example, transformation. Such foreign DNA also encompasses DNA native to an organism introduced into the genome of the organism via non-natural methods such as, for example, transformation. Related terms include "heterologous protein" and "heterologous enzyme" which are encoded by "heterologous DNA." It is recognized that such a heterologous enzyme or protein can possess an amino acid sequence that is identical to that of a native enzyme or protein of an organism. Further, a "heterologous coding sequence" is coding sequence composed of heterologous DNA.

[0084] It is recognized that enzymes similar to those described herein, referred to as "variant enzymes," may be utilized. By "variant enzyme" or "variant protein" is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess at least one desired biological activity of the native protein, that is, for example, 2-enoyl-CoA hydratase or 3-ketoacyl-CoA reductase activity as described herein for MFP2. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native enzyme or protein of the invention will have at least about 40%, 50%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0085] The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptide can be prepared by mutations in the nucleotide sequence encoding the native protein of interest which is also referred to as the coding sequence of the native protein. Thus, proteins and their respective coding sequences include the native forms as well as variants thereof.

[0086] A variety of methods can be used to produce variant enzymes such as, for example, mutagenesis of the coding sequences of the native enzyme. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred.

[0087] In constructing variants of the enzyme of interest, modifications to the nucleotide sequences encoding the variants will be made such that variants continue to possess the desired activity. Obviously, any mutations made in the DNA encoding the variant protein must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See EP Patent Application Publication No. 75,444.

[0088] In some cases it may be necessary to utilize a protein that possesses more than one enzymatic function. If only one or subset of enzymatic activities of such a protein is desired, it will be necessary to "neutralize," that is eliminate or substantially minimize, the undesirable enzymatic activity or activities. Those skilled in the art of modifying proteins and enzymes know that a variety of methods can be used singly or in combination to neutralize an enzymatic activity. Generally such methods involve modifying the coding sequences of the protein such that the desired activity or activities are retained and the undesirable activity or activities are eliminated. Such modifications to the coding sequence include deletions, substitutions, and insertions.

[0089] The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by enzyme activity assays, such as, for example, a PHA synthase assay, an enoyl-CoA hydratase assay, a ketoacyl-CoA reductase assay and an acetyl-CoA:acetyl transferase assay. See, for example, Schubert et al. (1988) J. Bacteriol. 170:5837-5847 (PHA synthase), Valentin and Steinbuechel (1994) Appl. Microbiol. Biotechnol. 40:699-709 (PHA synthase), Moskowitz and Merrick (1969) Biochemistry 8:2748-2755 (enoyl-CoA hydratase), Lynen and Wieland (1955) Meth. Enzymol. 1:566-573 (ketoacyl-CoA reductase), Nishimura et al. (1978) Arch Microbiol. 116:21-27 (acetyl-CoA:acetyl transferase) and Iwahashi et al. (1989) J. Biochem. 105:588-593 (NAD/NADH kinase); all of which are herein incorporated by reference.

[0090] Nucleotide sequence encoding the enzymes of the invention may be derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different coding sequences of an enzyme of the invention can be manipulated to create a new enzyme possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, nucleotide sequence motifs encoding a domain of interest in an enzyme of the invention may be shuffled to obtain a new gene coding for an enzyme with an improved or modified property of interest, such as an increased K.sub.m or modification that results in changes in substrate or product specificities of the enzyme. Such changes in substrate and product specificities include, but are not limited, to changes related to stereochemistry of the substrate utilized and/or the product formed. For example, an enzyme may only catalyze the formation of a (+)-epimer of a particular product. However, after shuffling one or more nucleotide sequences encoding such an enzyme, a variant enzyme is produced that produces only the (-)-epimer of the same product. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458, herein incorporated by reference.

[0091] In embodiments of the invention, it is necessary to direct an enzyme for PHA synthesis to the peroxisomes of a plant. Methods for directing an enzyme to the peroxisome are well known in the art. Typically, such methods involve operably linking a nucleotide sequence encoding a peroxisome-targeting signal to the coding sequence of the enzyme or modifying the coding sequence of the enzyme to additionally encode the peroxisome-targeting signal without substantially affecting the intended function of the encoded enzyme. See, for example, Olsen et al. (1993) Plant Cell 5:941-952, Mullen et al. ( 1997) Plant Physiol. 115:881-889, Gould et al. (1990) EMBO J. 9:85-90, Flynn et al. (1998) Plant J. 16:709-720; Preisig-Muller and Kindl (1993) Plant Mol. Biol. 22:59-66 and Kato et al. (1996) Plant Cell 8:1601-161 1; herein incorporated by reference.

[0092] It is recognized that an enzyme of the invention may be directed to the peroxisome by operably linking a peroxisome-targeting signal to the C-terminus or the N-terminus of the enzyme. It is further recognized that an enzyme which is synthesized with a peroxisome-targeting signal may be processed proteolytically in vivo resulting in the removal of the peroxisome-targeting signal from the amino acid sequence of the mature, peroxisome-localized enzyme.

[0093] It is recognized that it may be necessary to reduce or eliminate the activity of one or more enzymes in a plant that interfere with PHA production by the methods of the present invention. The activity of such an interfering enzyme may be reduced or eliminated by reducing or eliminating the synthesis of the interfering enzyme. Methods for reducing or eliminating the synthesis of a particular protein or enzyme in a plant, such as, for example, sense and antisense suppression methods, are known in the art.

[0094] Antisense suppression methods involve the use of DNA construct that is portion complementary to at least a portion of a transcript encoding the protein of interest. The antisense DNA construct is designed for the production of antisense transcripts when transcribed in a plant. Such antisense transcripts are capable of hybridizing with the corresponding native or sense transcript of the protein of interest. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding sense transcript. In this manner, antisense constructions having 70%, preferably 80%, more preferably 85% sequence identity to the corresponding sense transcript may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used.

[0095] Sense suppression methods, also known as cosuppression methods, involve the use of DNA construct that is designed to produce of a transcript that is in the same orientation, the sense orientation, as the transcript of the protein of interest. Methods for suppressing the production of a protein in a plant using nucleotide sequences in the sense orientation to are known in the art. The methods generally involve transforming plants with a DNA construct comprising a promoter that drives expression in a plant operably linked to at least a portion of a nucleotide sequence that corresponds to the transcript of the endogenous gene. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, preferably greater than about 65% sequence identity, more preferably greater than about 85% sequence identity, most preferably greater than about 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323; herein incorporated by reference.

[0096] In the methods of the present invention, expression cassettes can be utilized. The cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest. Generally, the expression cassette is provided with a plurality of restriction sites for insertion of the sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0097] By "operably linked" is intended the joining of two or more contiguous nucleotide sequences in such a manner that the desired functions or functions are achieved. "Operably linked" includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. In the case of protein coding sequences, "operably linked" includes joining two protein coding sequences in such a manner that both sequences are in the same reading frame for translation. For example, a nucleotide sequence encoding a peroxisome-targeting signal may be joined to the 3' end of a coding sequence of a protein of the invention in such manner that both sequences are in the same reading frame for translation to yield a the protein of the invention with a C-terminal addition of the peroxisome-targeting signal.

[0098] The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.

[0099] The expression cassette will include in the 5'-to-3' direction of transcription, a transcriptional and translational initiation region, a nucleotide sequence of interest, and a transcriptional and translational termination region functional in plants. The transcriptional initiation region, the promoter, may be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By "foreign" is intended that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced.

[0100] While it may be preferable to express the sequences using heterologous promoters, the native promoter sequences may be used. Such constructs would change expression levels of MFP2 in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.

[0101] The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell. 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

[0102] The coding sequences of the enzymes for PHA biosynthesis used in the practice of the invention can be optimized for enhanced expression in plants of interest. That is, the coding sequences can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1 -11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Thus, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the nucleotide sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.

[0103] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0104] The expression cassettes may additionally contain 5'-leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5'-noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989) Proc. Natl. Acad Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G., and P. Sarnow (1991) Nature 353:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L., (1987) Nature 325:622-625; tobacco mosaic virus leader (TmV), (Gallie, D. R. et al. (1989) Molecular Biology of RNA, pages 237-256; and maize chlorotic mottle virus leader (MCMV) (Lommel, S. A. et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiology, 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.

[0105] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitutions, e.g., transitions and transversions, may be involved. Such a fragment of DNA that has been manipulated by any method known to those skill in the art is referred to herein as a "DNA construct." The term "DNA construct " also encompasses expression cassettes, chimeric genes, synthetic genes, genes with modified coding sequences, and the like.

[0106] A number of promoters can be used in the practice of the invention. The promoters may be selected based on the desired timing, localization and level of expression genes encoding enzymes in a plant. Constitutive, seed-preferred, germination-preferred, tissue-preferred and chemical-regulatable promoters can be used in the practice of the invention. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

[0107] The methods of the invention are useful for producing PHA copolymers in seeds. Toward this end, the coding sequences for the enzymes of the invention may be utilized in expression cassettes or DNA constructs with seed-preferred promoters, seed-development promoters (those promoters active during seed development), as well as seed-germination promoters (those promoters active during seed germination). Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and celA (cellulose synthase) (see the copending application entitled "Seed-Preferred Promoters," U.S. application Ser. No. 09/377,648, filed Aug. 19, 1999, herein incorporated by reference). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, particular promoters include those from the following genes: phaseolin, napin, .beta.-conglycinin, soybean lectin, and the like. For monocots, particular promoters include those from the following genes: maize 15Kd zein, 22KD zein, 27kD zein, waxy, shrunken 1, shrunken 2, and globulin 1.

[0108] For tissue-preferred expression, the coding sequences of the invention can be operably linked to tissue-preferred promoters. For example, leaf-preferred promoters may be utilized if expression in leaves is desired. Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0109] Other tissue-preferred promoters include, for example, Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Lam (1994) Results Probl Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.

[0110] In the practice of the invention, it may be desirable to use chemical-regulatable promoters to control the expression of gene in a plant. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulatable promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

[0111] It is further recognized that the components of the expression cassette may be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. See, for example Perlak et al.(1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; Murray et al. (1989) Nucleic Acid Research 17:477-498; and WO 91/16432.

[0112] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

[0113] The modified plant may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell. Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.

[0114] In the methods of the present invention, plants genetically manipulated to produce PHA are utilized. By "genetically manipulated" is intended modifying the genome of an organism, preferably a plant, including cells and tissue thereof, by any means known to those skilled in the art. Modifications to a genome include both losses and additions of genetic material as well as any sorts of rearrangements in the organization of the genome. Such modifications can be accomplished by, for example, transforming a plant's genome with a DNA construct containing nucleotide sequences which are native to the recipient plant, non-native or a combination of both, conducting a directed sexual mating or cross pollination within a single species or between related species, fusing or transferring nuclei, inducing mutagenesis and the like.

[0115] In the practice of certain specific embodiments of the present invention, a plant is genetically manipulated to produce more than one heterologous enzyme involved in PHA synthesis. Those of ordinary skill in the art realize that this can be accomplished in any one of a number of ways. For example, each of the respective coding sequences for such enzymes can be operably linked to a promoter and then joined together in a single continuous fragment of DNA comprising a multigenic expression cassette. Such a multigenic expression cassette can be used to transform a plant to produce the desired outcome. Alternatively, separate plants can be transformed with expression cassettes containing one or a subset of the desired set of coding sequences. Transformed plants that express the desired activity can be selected by standard methods available in the art such as, for example, assaying enzyme activities, immunoblotting using antibodies which bind to the enzymes of interest, assaying for the products of a reporter or marker gene, and the like. Then, all of the desired coding sequences can be brought together into a single plant through one or more rounds of cross pollination utilizing the previously selected transformed plants as parents.

[0116] Methods for cross pollinating plants are well known to those skilled in the art, and are generally accomplished by allowing the pollen of one plant, the pollen donor, to pollinate a flower of a second plant, the pollen recipient, and then allowing the fertilized eggs in the pollinated flower to mature into seeds. Progeny containing the entire complement of heterologous coding sequences of the two parental plants can be selected from all of the progeny by standard methods available in the art as described supra for selecting transformed plants. If necessary, the selected progeny can be used as either the pollen donor or pollen recipient in a subsequent cross pollination.

[0117] The invention can be practiced with any plant species including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Caricapapaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[0118] Preferably, plants of the present invention are crop plants (for example, maize, alfalfa, sunflower, Brassica sp., soybean, cotton, safflower, peanut, sorghum, wheat, rice, potatoes, millet, tobacco, etc.), more preferably maize and oilseed plants, yet more preferably maize plants. Such oilseed plants include, but are not limited to, Brassica sp., sunflower, safflower, soybean, peanut, cotton, flax, coconut and oil palm.

[0119] The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1

Engineering Plants to Produce PHA Copolymers

[0120] Bacteria can produce PHA copolymers because these substrates are apparently derived from the .beta.-oxidation cycle; as bacterial cells are uncompartmented, both .beta.-oxidation and PHA synthesis take place in the cytosol. In plants, however, .beta.-oxidation is confined primarily to peroxisomes and thus offers a suitable site for copolymer production. Using methods known to those of ordinary skill in the art, signal sequences for targeting proteins to peroxisomes can be added to PHA-producing enzymes, allowing the localization of these enzymes in the peroxisomes. Such signal sequences for targeting proteins to plant peroxisomes are well known (Mullen et al. (1997) Plant Journal 12:313-322; Trelease et al. (1 996) Protoplasma 195:156-167).

[0121] An intermediate in .beta.-oxidation is S-(+)-3-hydroxyacyl-CoA. However, its configuration is unsuitable for PHA synthases, which require R-(-)-3-hydroxyacyl-CoA as a substrate (Gemgross et al. (1995) Proc. Natl. Acad. Sci. USA 92:6279-6283; Steinbuchel, A. (1991) Biomaterials: Novel materials from biological materials, D. Byrom, ed. (New York: Macmillan Publishers Ltd.), pp. 123-213). Moreover, the concentration of R-(-)-3-hydroxyacyl-CoA in the peroxisomes must be very low, as the enzyme catalyzing the proximal and distal reactions of this intermediate is multifunctional in nature (Engeland et al. (1991) Eur. J. Biochem. 200:171-178; Guhnemann Schafer et al. (1995) Biochim. Biophys. Acta. 1256:181-186). As acyl-CoA oxidase appears to be an independent enzyme (i.e., not a component of a multi-enzyme complex) in .beta.-oxidation, its product, 2-enoyl-CoA, might be readily available for hydration into the R-(-)-epimer, provided that a hydratase capable of catalyzing this reaction is available (FIG. 1).

[0122] A multifunctional protein from yeast reportedly has only the activities of enoyl-CoA hydratase and R-(-)-3-hydroxyacyl-CoA dehydrogenase (Hiltunen et al. (1992) J. Biol. Chem. 267:6646-6653). Interestingly, this enoyl-CoA hydratase forms R-(-)-3-hydroxyacyl-CoA in contrast to its plant counterpart (Guhnemann et al. (1994) Eur. J. Biochem. 226:909-915; Kindl, H. (1993) Biochimie 75:22R-230). Engineering the yeast multifunctional protein (encoded by GenBank Accession No. M86456, SEQ ID NO: 3) to carry only the hydratase activity and then targeting it to the plant peroxisomes along with a PHA synthase should lead to PHA copolymer formation.

[0123] Leaf et al. ((1996) Microbiology 142:1169-1180) were able to produce PHB granules in the cytosol of yeast transformed with only PHB synthase. Although they were unable to pinpoint the compartment in which it was localized, they identified a 3-hydroxybutyryl-CoA dehydrogenase that catalyzed the reversible reaction between acetoacetyl-CoA and R-(-)-3-hydroxybutyryl-CoA. No further information (i.e. gene or protein sequence) is available on this enzyme yet. Once the gene for 3-hydroxyacyl-CoA dehydrogenase enzyme becomes available, it can be used in producing substrate for PHA copolymer formation in peroxisomes. It is recognized that the coding sequence of this enzyme can be manipulated to alter characteristics of the encoded enzyme to favor the synthesis R-(-)-3-hydroxyacyl-CoA over 3-ketoacyl-CoA in plant peroxisomes by techniques well known to those skilled in the art. Such techniques include, for example, e.g., DNA shuffling (Crameri et al. (1998) Nature 391:288-291; Stemmer, W. P. C. (1994) Proc. Natl. Acad Sci. USA 91:10747-10751; Stemmer, W. P. C. (1994) Nature 370:389-391).

[0124] Mammalian mitochondrial enoyl-CoA hydratase is an independent enzyme (Minami-Ishi et al. (1989) Eur. J. Biochem. 185:73-78) which catalyzes the formation of S-(+)-3-hydroxyacyl-CoA (encoded by GenBank Accession No. U37486, SEQ ID NO: 22). The DNA encoding this enzyme can be subjected to shuffling to alter the activity of this enzyme to form the R-(-) instead of the S-(+)-epimer. Then, the shuffled DNA can be modified further to include nucleotide sequences which encodes a signal sequence for targeting to the peroxisomes.

[0125] Recently, a 2-enoyl-CoA hydratase from Aeromonas caviae was reported to hydrate 2-enoyl-CoA to R-3-hydroxyacyl-CoA (DDBJ Accession No. E 15860, SEQ ID NO: 21) (Fukui et al. (1998) J. Bacteriol. 180:667-673). This enzyme can be modified to include a signal sequence for targeting to plant peroxisomes. Together with a peroxisome-localized PHA synthase, such an enoyl-CoA hydratase can catalyze the biosynthesis of copolymers.

Example 2

Production of Specific Types of PHA in Plants

[0126] It is desirable to produce a pure copolymer of a defined monomer composition. A relatively pure copolymer would have predictable properties in comparison to a mixture of copolymers as the composition of the latter can vary according to the environment. The ability of Pseudomonas sp. to make copolymers of PHAs from various substrates is well known to those skilled in the art. However, the PHA synthases from these species have a broad substrate range (Caballero et al. (1995) Int. J. Biol. Macromol. 17:86-92; Huisman et al. (1989) Appl. Environ. Microbiol. 55:1949-1954; Lee et al. (1995) Appl. Environ. Microbiol. 42:901-909; Ramsay et al. (1990) Appl. Environ. Microbiol. 56:2093-2098; Steinbuechel et al. (1992) Appl. Environ. Microbiol. 37:691-697;Timm et al. (1992) Eur. J. Biochem. 209:1R-30). When a genomic fragment containing the PHA synthase gene from Thiocapsa pfennigii (see, WO 96/08566) was introduced into Pseudomonas putida or A. eutrophus strains deficient in PHA synthase, majority of the copolymer made was polyhydroxybutyrate-co-hydroxyhexanoate (HB-co-HHX) (Liebergesell et al. (1993) Appl. Environ. Microbiol. 40:292-300; Valentin et al. (1994) Appl. Environ. Microbiol 40:710-716). These are the first reports of an enzyme overcoming the barrier between short- and medium-chain monomers with respect to substrate specificity for copolymer synthesis.

[0127] Recently, a PHA synthase has been identified from A. caviae (DDBJ Accession No. D88825, SEQ ID NO: 11) that also makes primarily poly(hydroxybutyrate-co-hydroxyhexanoate) copolymer when the bacteria are grown in cultures containing octanoate or (Fukui et al. (1997) J. Bacteriol. 179:4821-4830). This enzyme, when transformed into an A. eutrophus strain that is deficient in PHB synthase, confers upon it the ability to make poly(hydroxybutyrate-co-hydroxyhexanoate), indicating that this enzyme is specific for these two substrates (Fukui et al. (1997) J. Bacteriol. 179:4821-4830). Copolymers consisting mainly of poly(hydroxybutyrate-co-hydroxyhexanoate) can be produced if either T. pfennigii or A. caviae synthase is targeted to the plant peroxisomes along with an enoyl-CoA hydratase, 3-ketoacyl-CoA reductase, or 3-hydroxyacyl-CoA dehydrogenase that is capable of producing the R-(-) epimer (FIG. 3).

[0128] By introducing the other two enzymes (ketothiolase and reductase) of the PHA biosynthetic pathway into the peroxisomes, a large portion of acetyl-CoA should be partitioned to the synthesis of PHA (FIGS. 1 and 3). As ketothiolase is the most limiting enzyme in .beta.-oxidation and is not associated with other enzymes (Kindl, H. (1987) Lipids:Structure and Function, P. K. Stumpf, ed. (Orlando, Fla.: Academic Press, Inc.), pp. 31-52), 3-ketobutyryl-CoA, the penultimate product of the last cycle of .beta.-oxidation, should be readily available to the introduced reductase for conversion into 3-hydroxybutyryl-CoA. Assuming the dominant fatty acid being degraded through .beta.-oxidation is C.sub.18, 3-ketobutyryl-CoA would constitute >20% of the carbon flux through .beta.-oxidation. Even if the reductase can use 25% of this intermediate, that would entail a diversion of 5% carbon from fatty acids passing through .beta.-oxidation toward PHA formation. Introducing ketothiolase would further augment the level of 3-ketobutyryl-CoA in peroxisomes.

[0129] Expression of PHB biosynthetic machinery in the peroxisomes along with that in the plastids as well as cytosol can lead to more PHB deposition in seeds. Previously, it has been reported that the expression of PHB biosynthetic enzymes in the cytosol of plants resulted in plants being of reduced vigor or "sick" (Poirier et al. (1992) Science 256:520-523). In this study, only reductase and synthase were expressed, however, allowing the cytosolic ketothiolase to supply acetoacetyl-CoA for PHB synthesis. Acetoacetyl-CoA, however, is a substrate for the synthesis of other cellular components, such as secondary metabolites and phytohormones. Enough acetoacetyl-CoA may have been diverted toward the formation of PHB that the homeostatic limits for normal cell metabolism were diminished. Alternatively, PHB granules might have physically caused disturbance in the leaf cytosol, affecting metabolism in general. The physiology of leaves may limit their usefulness as a site of PHB synthesis. A mature leaf is a source of photosynthate and as such produces and supplies photosynthate to sinks within the plant. On the other hand, a developing seed is a strong sink. Due to the myriad physiological differences between a source leaf and a strong sink like a developing seed, expression of genes encoding enzymes involved in PHB synthesis in the cytosol of a developing seed may not be as toxic as in that of a leaf. Expression of these genes in the peroxisomes of seeds can be driven by seed-preferred, chemical-regulatable or germination-preferred promoters. If these genes need to be expressed during both seed fill and germination or during only a limited portion of the seed fill period, a chemical-regulatable promoter may be desirable.

[0130] It is further recognized that in the practice of the invention, it can be advantageous to make use of transgenic or naturally occurring lines of oilseed plants that are known to have higher rates of .beta.-oxidation in their seeds to achieve optimal PHA production in a plant.

Example 3

Engineering a Peroxisomal 2-Enoyl-CoA Hydratase from a Yeast Multifunctional Protein

[0131] To produce PHA in plant peroxisomes, it is essential to effectively divert 2-enoyl-CoA from .beta.-oxidation and to the synthesis of R-(-)-3-hydroxyacyl-CoA, the substrate of PHA synthase. In contrast to the multifunctional protein in other organisms, yeast multifunctional protein (encoded by GenBank Accession No. M86456, SEQ ID NO: 3) converts trans-2-enoyl-CoA to R-(-)-3-hydroxyacyl-CoA. The hydratase domain of the yeast multifunctional protein utilizes a broader chain-length range of substrates than does the hydratase isolated from Aeromonas caviae (Fukui et al. (1998) J. Bacteriol. 180:667-673). Such a hydratase with such a broad substrate range finds use in the production of a wide variety of copolymers in plants.

[0132] Thus, the R-specific enoyl-CoA hydratase of the yeast multifunctional protein can used to produce R-(-)-3-hydroxyacyl-CoA for PHA synthesis in plant peroxisomes. Since the R-(-)-3-hydroxyacyl-CoA dehydrogenase of the yeast multifunctional protein requires NADH and the NADH-binding sites are located in the N-terminal portion of the polypeptide, the hydratase is likely located in the C-terminal portion of the yeast multifunctional protein. Filppula et al. ((1995) J. Biol. Chem. 270:27453-27457) constructed a C-terminally truncated form of the yeast multifunctional protein and showed that the mutant enzyme contained only R-(-)-3-hydroxyacyl-CoA dehydrogenase activity and thus demonstrated that the deleted C-terminal portion of the full-length yeast multifunctional protein is essential for hydratase activity. Qin et al. (1997) Biochem. J. 321: 21-28 described an N-truncated rat multifunctional protein with 318 amino acid residue deletion. This mutant enzyme remained full hydratase activity while its dehydrogenase activity was completely lost. These observations are in agreement with the prediction that an N-terminally truncated form of yeast multifunctional protein possess hydratase activity and lack dehydrogenase activity.

[0133] To engineer a 2-enoyl-CoA hydratase that catalyzes the synthesis of R-(-)-hydroxyacyl-CoA, the nucleotide sequence encoding the yeast multifunctional protein (GenBank Accession No. M86456, SEQ ID NO: 3) can be modified by site-directed mutagenesis and/or N-terminal truncation to remove the dehydrogenase activity. After analyzing the primary structure of the yeast multifunctional protein, two putative NADH-binding domains (residues 152-180 and residues 456-484) were identified. These two putative NADH-binding domains each possess the conserved Y and K that are a signature NADH-binding-sites. Y165 and K169 lie in the first domain while Y478 and K482 are found in the second domain, although it is unknown whether the first domain, the second one or both of them serves for NAD/NADH-binding. To eliminate dehydrogenase activity, the NADH binding sites can be disrupted to generate mutant yeast multifunctional proteins such as, for example, a first mutant enzyme having the two amino acid substitutions, Y 165F and K 169A and a second mutant enzyme having the two amino acid substitutions Y478F and K482A and a third mutant enzyme having the four amino acid substitutions, Y165F, K169A, Y478F, and K482A. Alternatively, two N-terminally truncated versions of the yeast multifunctional protein can be constructed by eliminating one and both NADH-binding sites (FIG. 2). Methods for assaying such enzyme activities are known in the art. See, for example, Moskowitz and Merrick (1969) Biochemistry 8:2748-2755 (enoyl-CoA hydratase), and Lynen and Wieland (1955) Meth. Enzymol. 1:566-573 (ketoacyl-CoA reductase) and Example 6 infra.

[0134] An N-terminally truncated version of the yeast multifunctional protein is set forth in SEQ ID NO: 5. A nucleotide sequence for the truncated yeast multifunctional protein is set forth in SEQ ID NO: 4. A similar approach can be used to modify any multifunctional protein known in the art.

[0135] The resulting 2-enoyl-CoA hydratase enzyme can then be modified for targeting to the peroxisome by operably linking a peroxisome-targeting signal sequence to the coding sequence for the mutant enzyme.

Example 4

Engineering a Peroxisomal 3-ketoacyl-CoA Reductase from a Yeast Multifunctional Protein

[0136] R-(-)-3-hydroxybutyryl-coenzyme A dehydrogenase (also known as acetoacetyl-CoA reductase) is encoded by phaB in PHA biosynthesis in a number of microorganisms. Such R-(-)-3-hydroxybutyryl-coenzyme A dehydrogenases all utilize NADPH as the electron donor. In one report an acetoacetyl-CoA reductase was shown to be NADH-dependent, but it produced S-(+)-3-hydroxybutyryl-CoA as its product (Liebergesell and Steinbuchel (1992) Eur. J. Biochem. 209:135-150). Another report showed that an NADH-dependent acetoacetyl-CoA reductase was isolated from Parracoccus denitrificans (Yabutani (1995) FEMS Microbiol. Lett. 133:85-90). Subsequent characterization confirmed that it utilized NADPH, but not NADH as an electron donor (Madison and Huisman (1999) Microbiol. Mol. Biol. Rev. 63:21-53).

[0137] In plant peroxisomes, it is postulated that NADPH pool is limited while NADH predominates. It is doubtful that the phaB gene product, an NADPH-dependent reductase, will function in peroxisomes. Therefore, a new enzyme that utilizes NADH as the electron donor is desired. The desired enzyme must also be able to convert 3-acetoacetyl-CoA and to R-(-)-3-hydroxybutyryl-CoA. Preferably, the desired enzyme is also capably of converting any 3-ketoacyl-CoA to an R-(-)-3-hydroxyacyl-CoA.

[0138] Using protein engineering methods, the coding sequence of the yeast multifunctional protein can be modified to produce the desired enzyme. The dehydrogenase moiety of the yeast multifunctional protein utilizes R-(-)-3-hydroxyacyl-CoA and requires NADH as its electron acceptor. Since the dehydrogenase of the yeast multifunctional protein enzyme utilizes substrates of fatty acyl-CoAs with C.sub.4 or longer chain length, the desired enzyme is expected to catalyze the NADH-dependent reduction of acetoacetyl-CoA to R-(-)-3-hydroxybutyryl-CoA. Based on sequence analysis of primary structure of the protein, two NADH-binding sites residing at the N-terminal portion were identified. For example, to produce the desired 3-ketoacyl-CoA reductase, the nucleotide sequence encoding the of yeast multifunctional protein can be truncated to produce a nucleotide sequence that encodes a C-terminally truncated version of the yeast multifunctional protein which lacks the final 271 amino acid of amino acid sequence of the yeast multifunctional protein. The C-terminally truncated version of the yeast multifunctional protein is set forth in SEQ ID NO: 7. A nucleotide sequence encoding the truncated yeast multifunctional protein is set forth in SEQ ID NO: 6.

[0139] The desired 3-ketoacyl-CoA reductase can then be modified, if necessary, for targeting to the peroxisome by operably linking a peroxisome-targeting signal sequence to the coding sequence for the desired enzyme.

Example 5

A Novel Maize Protein with Homology to Yeast MFP2

[0140] To search for homologous sequences in maize, the nucleotide sequence encoding the yeast MFP2 (GenBank Accession No. M86456, SEQ ID NO: 3) was used to search a Pioneer Hi-Bred maize EST database. An EST clone with substantial homology to the yeast sequence was identified. The 1362 bp maize EST clone was sequenced and found to be full-length (SEQ ID NO: 1) with an open reading frame encoding a polypeptide of 314 amino acids (SEQ ID NO: 2). Interestingly, in comparison, the yeast MFP2 is comprised of an amino acid sequence which is 900 amino acids in length. Further sequence analysis revealed that the maize amino acid sequence corresponds to the C-terminal portion of the yeast MFP2 that is believed to be a 2-enoyl-CoA hydratase domain (FIG. 2) Unlike the yeast MFP2, the smaller maize MFP2-like polypeptide lacks the dehydrogenase domain of the yeast and mammalian MFP2s and does not appear to contain a peroxisome-targeting sequence.

[0141] Thus, on the basis of homology to the yeast MFP2, the maize MFP2-like polypeptide is predicted to encode a 2-enoyl-CoA hydratase. Such a hydratase can be targeted to the peroxisome for use in PHA production therein by operably linking a peroxisome-targeting signal sequence to the coding sequence for the maize polypeptide.

[0142] The present invention discloses a novel protein from maize with homology to MFP2s. While MFP2s are found in several fungi and in several mammals including, but not limited to, mouse, guinea pig, human and pig, no plant sequence has yet been publically disclosed. It has been generally believed that MFPs are unique to fungi and animals. The discovery of the maize MFP2-like polypeptide, which shares substantial homology with the yeast MFP2, indicates, however that, at least, the hydratase domain of MFP2 is also found in plants.

[0143] While the maize MFP2-like polypeptide shares significant homology to MFP2s from fungi and mammals, the maize protein possesses several notable differences. In addition to the lack of a dehydrogenase domain, the maize MFP2-like polypeptide does not contains any known peroxisomal-targeting sequences in its N-terminal or C-terminal portions. This is different from yeast and mammalian MFP2s which are considered to be localized in peroxisomes and play roles in fatty acid .beta.-oxidation. Thus, the maize MFP2-like polypeptide is likely to play a physiological role in plants that is distinct from that of known MFP2s.

Example 6

Assaying the Activities of PHA Synthesis Enzymes

[0144] To test the activity of the maize MFP2-like polypeptide and the truncated yeast MFP2 (hydratase domain) prepared as described supra, their respective nucleotide sequences (SEQ ID NOs: 1 and 4) and that of the full-length yeast MFP2 (SEQ ID NO: 3) were cloned into appropriate bacterial expression vectors and used to transform E. coli using standard techniques known in the art for expressing recombinant proteins and transforming bacteria. Total protein extracts were isolated from IPTG-induced E. coli (strain BL2 1) harboring a plasmid comprising the nucleotide sequence of SEQ ID NO: 3 (pYMFP), the nucleotide sequence of SEQ ID NO: 4 (pYHL) or the nucleotide sequence of SEQ ID NO: 1 (pMHL). The activities of the truncated proteins were tested using the functional assay schematically illustrated in FIG. 4. The reaction mixture consisted of 20 .mu.l of PHA synthase preparation (19 mg/ml), 50 .mu.l of E. coli extract, 50 mM Tris-HCl, pH 7.5 and .mu.M of crotonyl-CoA, acetoacetyl-CoA (AcAcCoA), or 3-hydroxybutryl-CoA (3HB-CoA) in total volume of 300 .mu.l. The reaction was started by the addition of the substrate and incubated at 37.degree. C. for two hours.

[0145] The results are presented in Table 1 and FIG. 5. With crotonyl-CoA as a substrate, polymer formation was detected in cultures of E. coli that were transformed with a nucleotide sequences encoding either the full-length yeast MFP2 (pYMFP), the truncated yeast MFP2 (yYHL), or the maize MFP2-like polypeptide (yMHL). The vector-only control (Table 1, reaction 1) indicated that polymer was not formed in the presence of crotonyl-CoA without one of the three nucleotide sequences (SEQ ID NOs: 1, 3, and 4). Thus, proteins encoded by each of the nucleotide sequences can catalyze the conversion of a crotonyl-CoA, which is a 2-enoyl-CoA, into R-(-)-3-hydroxyacyl-CoA, which can then be used for PHA synthesis as a substatrate for PHA synthase.

[0146] Comparison of the relative hydratase activities, as measured by PHA formation, for the proteins encoded by SEQ ID NOs: 1, 3 and 4 is provided in FIG. 5. The highest relative hydratase activity was detected in the cultures expressing the maize MFP2-like polypeptide (maize hydratase), followed by the truncated yeast MFP2 (yeast hydratase) and the full-length yeast MFP2 (yeast MFP). The results indicate that the removal an N-terminal portion of an MFP2 can lead to increased hydratase activity, as determined by increased PHA production, when compared to the hydratase activity of the full-length MFP2.

[0147] In general, the results presented in Table 1 and FIG. 5 demonstrate that both the maize MFP2-like polypeptide and the truncated yeast MFP2 possess a hydratase activity that can be used for PHA synthesis in a host cell. Given that 2-enoyl-CoA substrates are found in the peroxisome, the maize MFP2-like polypeptide and the truncated yeast MFP2 of the invention can be targeted to peroxisome, along with other necessary enzymes, for PHA synthesis therein. The results further demonstrate that a truncated MFP2 can provide an improvement in PHA production in an living organism, when compared to the full-length MFP. TABLE-US-00001 TABLE 1 Functional Assay for the Presence of an 2-enoyl-CoA Hydratase Activity Reaction Substrate Hydratase Polymer Formed 1 crotonyl-CoA vector only No 2 crotonyl-CoA pMHL Yes 3 crotonyl-CoA pYHL Yes 4 crotonyl-CoA pYMFP Yes 5 3HB-CoA 50 .mu.l buffer Yes 6 AcAcCoA 50 .mu.l buffer No 7 AcAcCoA pMHL No

Example 7

Transformation and Regeneration of Transgenic Maize Plants by Particle Bombardment

[0148] Immature maize embryos from greenhouse donor plants are bombarded with a plasmid comprising a nucleotide sequence of the invention encoding an enzyme involved in PHA synthesis operably linked to a seed-preferred promoter and the selectable marker gene PAT (Wohlleben et al. (1998) Gene 70:25-37), which confers resistance to the herbicide Bialaphos. Alternatively, the selectable marker gene is provided on a separate plasmid. Transformation is performed as follows. Media recipes follow below.

Preparation of Target Tissue

[0149] The ears are husked and surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5-cm target zone in preparation for bombardment.

Preparation of DNA

[0150] A plasmid vector comprising the nucleotide sequence of the invention encoding an enzyme involved in PHA synthesis linked to a seed-preferred promoter is made. This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 .mu.m (average diameter) tungsten pellets using a CaCl.sub.2 precipitation procedure as follows: [0151] 100 .mu.l prepared tungsten particles in water [0152] 10 .mu.l (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g total DNA) [0153] 100 .mu.l 2.5 M CaCl.sub.2 [0154] 10 .mu.l 0.1 M spermidine

[0155] Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 .mu.l 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 .mu.l spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.

Particle Gun Treatment

[0156] The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

[0157] Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5'' pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for PHA content and/or the activity or level of the enzyme of the invention.

Bombardment and Culture Media

[0158] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times. SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H.sub.2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after bringing to volume with D-I H.sub.2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times. SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H.sub.2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H.sub.2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos(both added after sterilizing the medium and cooling to room temperature).

[0159] Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H.sub.2O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H.sub.20 after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with D-I H.sub.2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 111 17-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H.sub.2O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H.sub.2O after adjusting pH to 5.6); and 6 g/l bacto-agar (added after bringing to volume with polished D-I H.sub.2O), sterilized and cooled to 60.degree. C.

Example 8

Production of Transgenic Maize Plants via Agrobacterium-Mediated Transformation

[0160] For Agrobacterium-mediated transformation of maize with a nucleotide sequence of the invention encoding an enzyme involved in PHA synthesis, preferably the method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the nucleotide sequence of the invention to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are preferably immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). Preferably the immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional "resting" step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). Preferably the immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). Preferably, the immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and preferably calli grown on selective medium are cultured on solid medium to regenerate the plants.

Example 9

Production of Transformed Soybean Plants

[0161] Soybean embryos are bombarded with a plasmid comprising a nucleotide sequence of the invention encoding an enzyme involved in PHA synthesis operably linked to a seed-preferred as follows. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface-sterilized, immature seeds of the soybean cultivar A2872, are cultured in the light or dark at 26.degree. C. on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.

[0162] Soybean embryogenic suspension cultures can maintained in 35 ml liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.

[0163] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0164] A selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188), and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette comprising the nucleotide sequence of the invention operably linked to the seed-preferred promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

[0165] To 50 .mu.l of a 60 mg/ml 1 .mu.m gold particle suspension is added (in order): 5 .mu.l DNA (1 .mu.g/.mu.l), 20 .mu.l spermidine (0.1 M), and 50 .mu.l CaCl.sub.2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 .mu.l 70% ethanol and resuspended in 40 .mu.l of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk.

[0166] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60.times.15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

[0167] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 10

Genetic Transformation of Sunflower Plants

[0168] Sunflower meristem tissues are transformed with an expression cassette comprising a nucleotide sequence of the invention encoding an enzyme involved in PHA synthesis operably linked to a seed-preferred promoter as follows (see also European Patent Number EP 0 486233, herein incorporated by reference, and Malone-Schoneberg et al. (1994) Plant Science 103:199-207). Mature sunflower seed (Helianthus annuus L.) are dehulled using a single wheat-head thresher. Seeds are surface sterilized for 30 minutes in a 20% Clorox bleach solution with the addition of two drops of Tween 20 per 50 ml of solution. The seeds are rinsed twice with sterile distilled water.

[0169] Split embryonic axis explants are prepared by a modification of procedures described by Schrarnmeijer et al. (Schrammeijer et al.(1990) Plant Cell Rep. 9: 55-60). Seeds are imbibed in distilled water for 60 minutes following the surface sterilization procedure. The cotyledons of each seed are then broken off, producing a clean fracture at the plane of the embryonic axis. Following excision of the root tip, the explants are bisected longitudinally between the primordial leaves. The two halves are placed, cut surface up, on GBA medium consisting of Murashige and Skoog mineral elements (Murashige et al. (1962) Physiol. Plant. 15: 473-497), Shepard's vitamin additions (Shepard (1980) in Emergent Techniquesfor the Genetic Improvement of Crops (University of Minnesota Press, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/l sucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-acetic acid (IAA), 0.1 mg/l gibberellic acid (GA3), pH 5.6, and 8 g/l Phytagar.

[0170] The explants are subjected to microprojectile bombardment prior to Agrobacterium treatment (Bidney et al. (1992) Plant Mol. Biol. 18: 301-313). Thirty to forty explants are placed in a circle at the center of a 60.times.20 mm plate for this treatment. Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each plate is bombarded twice through a 150 mm nytex screen placed 2 cm above the samples in a PDS 1000.RTM. particle acceleration device.

[0171] Disarmed Agrobacterium tumefaciens strain EHA 105 is used in all transformation experiments. A binary plasmid vector comprising the expression cassette that contains the nucleotide sequence of the invention encoding an enzyme involved in PHA synthesis operably linked to the seed-preferred promoter is introduced into Agrobacterium strain EHA 105 via freeze-thawing as described by Holsters et al. (1978) Mol. Gen. Genet. 163:181-187. This plasmid further comprises a kanamycin selectable marker gene (i.e, nptII). Bacteria for plant transformation experiments are grown overnight (28.degree. C. and 100 RPM continuous agitation) in liquid YEP medium (10 gm/l yeast extract, 10 gm/l Bactopeptone, and 5 gm/l NaCl, pH 7.0) with the appropriate antibiotics required for bacterial strain and binary plasmid maintenance. The suspension is used when it reaches an OD.sub.600 of about 0.4 to 0.8. The Agrobacterium cells are pelleted and resuspended at a final OD.sub.600 of 0.5 in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH.sub.4Cl, and 0.3 gm/l MgSO.sub.4.

[0172] Freshly bombarded explants are placed in an Agrobacterium suspension, mixed, and left undisturbed for 30 minutes. The explants are then transferred to GBA medium and co-cultivated, cut surface down, at 26.degree. C. and 18-hour days. After three days of co-cultivation, the explants are transferred to 374B (GBA medium lacking growth regulators and a reduced sucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/l kanamycin sulfate. The explants are cultured for two to five weeks on selection and then transferred to fresh 374B medium lacking kanamycin for one to two weeks of continued development. Explants with differentiating, antibiotic-resistant areas of growth that have not produced shoots suitable for excision are transferred to GBA medium containing 250 mg/l cefotaxime for a second 3-day phytohormone treatment. Leaf samples from green, kanamycin-resistant shoots are assayed for the presence of NPTII by ELISA and for the presence of transgene expression by assaying, for example, for PHA production as described supra.

[0173] NPTII-positive shoots are grafted to Pioneer.RTM. hybrid 6440 in vitro-grown sunflower seedling rootstock. Surface sterilized seeds are germinated in 48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH 5.6) and grown under conditions described for explant culture. The upper portion of the seedling is removed, a 1 cm vertical slice is made in the hypocotyl, and the transformed shoot inserted into the cut. The entire area is wrapped with parafilm to secure the shoot. Grafted plants can be transferred to soil following one week of in vitro culture. Grafts in soil are maintained under high humidity conditions followed by a slow acclimatization to the greenhouse environment. Transformed sectors of To plants (parental generation) maturing in the greenhouse are identified by NPTII ELISA and/or by analysis in leaf extracts of enzyme activity of the enzyme encoded by the nucleotide sequence of the invention of leaf extracts, while transgenic seeds harvested from NPTII-positive T.sub.0 plants are identified by similar enzyme activity analyses of small portions of dry seed cotyledons.

[0174] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0175] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Sequence CWU 1

1

27 1 1362 DNA Zea mays CDS (233)..(1174) 1 gaattcccgg gtcgacccac gcgtccggtc ggcggggctg ccgcctgccg catcctcccc 60 tcgcccaccg tcgacgactt gatcccctcc caccgttgag gccgcctcct cacggcagcg 120 agccagcgac tcctctcttc gtctcctaca agtagccaga caccactccg actttgccgg 180 caacccgtcg acagcgacga ggcgcagcat aaggcatacg ggcacggcgg cc atg gcg 238 Met Ala 1 acc agc tcc aaa ccc gcc gcg ccc gtg gac ccc atg gtc gtg ctc gcc 286 Thr Ser Ser Lys Pro Ala Ala Pro Val Asp Pro Met Val Val Leu Ala 5 10 15 cac gag ttc ccc gag gtg tca ttc gac tac gac gag agg gat gta gcg 334 His Glu Phe Pro Glu Val Ser Phe Asp Tyr Asp Glu Arg Asp Val Ala 20 25 30 ttg tac gcg ctc ggg gtt ggt gcc tgc ggc gat gac gcc gtc gac gag 382 Leu Tyr Ala Leu Gly Val Gly Ala Cys Gly Asp Asp Ala Val Asp Glu 35 40 45 50 aag gag ctt cac ttc gtg tac cac agg gat ggg cag cca cac att aag 430 Lys Glu Leu His Phe Val Tyr His Arg Asp Gly Gln Pro His Ile Lys 55 60 65 acc ctt cct act ttt gtt tct tta ttt ccc aac aag aac agc aat ggg 478 Thr Leu Pro Thr Phe Val Ser Leu Phe Pro Asn Lys Asn Ser Asn Gly 70 75 80 ctt gga ttt gtt gat gtg cct ggc ctt aac ttt gat gca agc ctt cta 526 Leu Gly Phe Val Asp Val Pro Gly Leu Asn Phe Asp Ala Ser Leu Leu 85 90 95 ctg cat ggt caa caa tac ata gag atc tat agg cca atc cct tcg tat 574 Leu His Gly Gln Gln Tyr Ile Glu Ile Tyr Arg Pro Ile Pro Ser Tyr 100 105 110 gtc agt gtt gta aac agg gtt aaa gta gtt ggt ttg cac gac aag ggg 622 Val Ser Val Val Asn Arg Val Lys Val Val Gly Leu His Asp Lys Gly 115 120 125 130 aaa gca act att ctt gag ctc gaa act acc aca agc ctc aaa gag tca 670 Lys Ala Thr Ile Leu Glu Leu Glu Thr Thr Thr Ser Leu Lys Glu Ser 135 140 145 ggg gaa att tta tgc atg aac agg agt act atc tac ttg cgt ggt gct 718 Gly Glu Ile Leu Cys Met Asn Arg Ser Thr Ile Tyr Leu Arg Gly Ala 150 155 160 gga ggg ttt tca gac tct tca cgg cca tac tca tat gct acc tat cct 766 Gly Gly Phe Ser Asp Ser Ser Arg Pro Tyr Ser Tyr Ala Thr Tyr Pro 165 170 175 gct aat caa gtt tct cgc att tct att cca aat tcg gca cct tct gca 814 Ala Asn Gln Val Ser Arg Ile Ser Ile Pro Asn Ser Ala Pro Ser Ala 180 185 190 gta tgc gac gac cag aca aag caa tcc cag gca ttg tta tac agg cta 862 Val Cys Asp Asp Gln Thr Lys Gln Ser Gln Ala Leu Leu Tyr Arg Leu 195 200 205 210 tct ggg gat tac aat cct ttg cat tca gac cca gat att gca cag ctt 910 Ser Gly Asp Tyr Asn Pro Leu His Ser Asp Pro Asp Ile Ala Gln Leu 215 220 225 gct ggg ttc acc cgt cca atc ctg cac ggc ctc tgc acc cta gga ttc 958 Ala Gly Phe Thr Arg Pro Ile Leu His Gly Leu Cys Thr Leu Gly Phe 230 235 240 gct gct cgc gcc gtc ata aaa tct ttc tgc aac ggc gaa ccg act gcg 1006 Ala Ala Arg Ala Val Ile Lys Ser Phe Cys Asn Gly Glu Pro Thr Ala 245 250 255 gtg aag agc atc ttc ggc cgt ttc ctt ctg cac gtc tac ccc ggg gaa 1054 Val Lys Ser Ile Phe Gly Arg Phe Leu Leu His Val Tyr Pro Gly Glu 260 265 270 acg ttg tcc act gag atg tgg ctt gac ggc cag aag gtg cac tac caa 1102 Thr Leu Ser Thr Glu Met Trp Leu Asp Gly Gln Lys Val His Tyr Gln 275 280 285 290 acg aag gcc aag gag cgg aac cga gct gtc ctc tct gga tat gtg ttg 1150 Thr Lys Ala Lys Glu Arg Asn Arg Ala Val Leu Ser Gly Tyr Val Leu 295 300 305 ctc cag cac atc ccc tcg tca ttg taagtaaaag ttttgtttct taaaattggt 1204 Leu Gln His Ile Pro Ser Ser Leu 310 gcccgctgaa agcatttggc ttggctgtga taaattgagc acgaggtggg ctgccactgt 1264 aatgatagcc atgtatgggt ctgcacataa cacattcgta ctgtattgat aagaagcacg 1324 taccacttaa atacgcagat ccgaacgtct tgattttt 1362 2 314 PRT Zea mays 2 Met Ala Thr Ser Ser Lys Pro Ala Ala Pro Val Asp Pro Met Val Val 1 5 10 15 Leu Ala His Glu Phe Pro Glu Val Ser Phe Asp Tyr Asp Glu Arg Asp 20 25 30 Val Ala Leu Tyr Ala Leu Gly Val Gly Ala Cys Gly Asp Asp Ala Val 35 40 45 Asp Glu Lys Glu Leu His Phe Val Tyr His Arg Asp Gly Gln Pro His 50 55 60 Ile Lys Thr Leu Pro Thr Phe Val Ser Leu Phe Pro Asn Lys Asn Ser 65 70 75 80 Asn Gly Leu Gly Phe Val Asp Val Pro Gly Leu Asn Phe Asp Ala Ser 85 90 95 Leu Leu Leu His Gly Gln Gln Tyr Ile Glu Ile Tyr Arg Pro Ile Pro 100 105 110 Ser Tyr Val Ser Val Val Asn Arg Val Lys Val Val Gly Leu His Asp 115 120 125 Lys Gly Lys Ala Thr Ile Leu Glu Leu Glu Thr Thr Thr Ser Leu Lys 130 135 140 Glu Ser Gly Glu Ile Leu Cys Met Asn Arg Ser Thr Ile Tyr Leu Arg 145 150 155 160 Gly Ala Gly Gly Phe Ser Asp Ser Ser Arg Pro Tyr Ser Tyr Ala Thr 165 170 175 Tyr Pro Ala Asn Gln Val Ser Arg Ile Ser Ile Pro Asn Ser Ala Pro 180 185 190 Ser Ala Val Cys Asp Asp Gln Thr Lys Gln Ser Gln Ala Leu Leu Tyr 195 200 205 Arg Leu Ser Gly Asp Tyr Asn Pro Leu His Ser Asp Pro Asp Ile Ala 210 215 220 Gln Leu Ala Gly Phe Thr Arg Pro Ile Leu His Gly Leu Cys Thr Leu 225 230 235 240 Gly Phe Ala Ala Arg Ala Val Ile Lys Ser Phe Cys Asn Gly Glu Pro 245 250 255 Thr Ala Val Lys Ser Ile Phe Gly Arg Phe Leu Leu His Val Tyr Pro 260 265 270 Gly Glu Thr Leu Ser Thr Glu Met Trp Leu Asp Gly Gln Lys Val His 275 280 285 Tyr Gln Thr Lys Ala Lys Glu Arg Asn Arg Ala Val Leu Ser Gly Tyr 290 295 300 Val Leu Leu Gln His Ile Pro Ser Ser Leu 305 310 3 3252 DNA Saccharomyces cerevisiae 3 tagaactctc ggcggttatt tgccaatttt tactccaacg gggatcaaca tcagcaagaa 60 agcaaaggag gatggggtaa aaaaacaagg aagacaagta aagaaattat ataagaagtc 120 gtttctggct gctttactcc cagtgttgaa aggtagaagg acttatcagc taatttatta 180 agcaaggtac acatatacga gctaaacaaa cattcgattt atttcttatt tgagtaagcc 240 atgcctggaa atttatcctt caaagataga gttgttgtaa tcacgggcgc tggagggggc 300 ttaggtaagg tgtatgcact agcttacgca agcagaggtg caaaagtggt cgtcaatgat 360 ctaggtggca ctttgggtgg ttcaggacat aactccaaag ctgcagactt agtggtggat 420 gagataaaaa aagccggagg tatagctgtg gcaaattacg actctgttaa tgaaaatgga 480 gagaaaataa ttgaaacggc tataaaagaa ttcggcaggg ttgatgtact aattaacaac 540 gctggaatat taagggatgt ttcatttgca aagatgacag aacgtgagtt tgcatctgtg 600 gtagatgttc atttgacagg tggctataag ctatcgcgtg ctgcttggcc ttatatgcgc 660 tctcagaaat ttggtagaat cattaacacc gcttcccctg ccggtctatt tggaaatttt 720 ggtcaagcta attattcagc agctaaaatg ggcttagttg gtttggcgga aaccctcgcg 780 aaggagggtg ccaaatacaa cattaatgtt aattcaattg cgccattggc tagatcacgt 840 atgacagaaa acgtgttacc accacatatc ttgaaacagt taggaccgga aaaaattgtt 900 cccttagtac tctatttgac acacgaaagt acgaaagtgt caaactccat ttttgaactc 960 gctgctggat tctttggaca gctcagatgg gagaggtctt ctggacaaat tttcaatcca 1020 gaccccaaga catatactcc tgaagcaatt ttaaataagt ggaaggaaat cacagactat 1080 agggacaagc catttaacaa aactcagcat ccatatcaac tctcggatta taatgattta 1140 atcaccaaag caaaaaaatt acctcccaat gaacaaggct cagtgaaaat caagtcgctt 1200 tgcaacaaag tcgtagtagt tacgggtgca ggaggtggtc ttgggaagtc tcatgcaatc 1260 tggtttgcac ggtacggtgc gaaggtagtt gtaaatgaca tcaaggatcc tttttcagtt 1320 gttgaagaaa taaataaact atatggtgaa ggcacagcca ttccagattc ccatgatgtg 1380 gtcaccgaag ctcctctcat tatccaaact gcaataagta agtttcagag agtagacatc 1440 ttggtcaata acgctggtat tttgcgtgac aaatcttttt taaaaatgaa agatgaggaa 1500 tggtttgctg tcctgaaagt ccaccttttt tccacatttt cattgtcaaa agcagtatgg 1560 ccaatattta ccaaacaaaa gtctggattt attatcaata ctacttctac ctcaggaatt 1620 tatggtaatt ttggacaggc caattatgcc gctgcaaaag ccgccatttt aggattcagt 1680 aaaactattg cactggaagg tgccaagaga ggaattattg ttaatgttat cgctcctcat 1740 gcagaaacgg ctatgacaaa gactatattc tcggagaagg aattatcaaa ccactttgat 1800 gcatctcaag tctccccact tgttgttttg ttggcatctg aagaactaca aaagtattct 1860 ggaagaaggg ttattggcca attattcgaa gttggcggtg gttggtgtgg gcaaaccaga 1920 tggcaaagaa gttccggtta tgtttctatt aaagagacta ttgaaccgga agaaattaaa 1980 gaaaattgga accacatcac tgatttcagt cgcaacacta tcaacccgag ctccacagag 2040 gagtcttcta tggcaacctt gcaagccgtg caaaaagcgc actcttcaaa ggagttggat 2100 gatggattat tcaagtacac taccaaggat tgtatcttgt acaatttagg acttggatgc 2160 acaagcaaag agcttaagta cacctacgag aatgatccag acttccaagt tttgcccacg 2220 ttcgccgtca ttccatttat gcaagctact gccacactag ctatggacaa tttagtcgat 2280 aacttcaatt atgcaatgtt actgcatgga gaacaatatt ttaagctctg cacgccgaca 2340 atgccaagta atggaactct aaagacactt gctaaacctt tacaagtact tgacaagaat 2400 ggtaaagccg ctttagttgt tggtggcttc gaaacttatg acattaaaac taagaaactc 2460 atagcttata acgaaggatc gttcttcatc aggggcgcac atgtacctcc agaaaaggaa 2520 gtgagggatg ggaaaagagc caagtttgct gtccaaaatt ttgaagtgcc acatggaaag 2580 gtaccagatt ttgaggcgga gatttctacg aataaagatc aagccgcatt gtacaggtta 2640 tctggcgatt tcaatccttt acatatcgat cccacgctag ccaaagcagt taaatttcct 2700 acgccaattc tgcatgggct ttgtacatta ggtattagtg cgaaagcatt gtttgaacat 2760 tatggtccat atgaggagtt gaaagtgaga tttaccaatg ttgttttccc aggtgatact 2820 ctaaaggtta aagcttggaa gcaaggctcg gttgtcgttt ttcaaacaat tgatacgacc 2880 agaaacgtca ttgtattgga taacgccgct gtaaaactat cgcaggcaaa atctaaacta 2940 taatacaaaa aaagatttga ataatataaa aaatagcgat tatattcttt tcatttaaca 3000 gctttgttaa gccatatcct tacatacatc tttccctaca taactaacct acccatttta 3060 agtacttttt ctttacggac gcaacttttt tgtcatgtgt aatattaaca gttttaatct 3120 atatagagga agaggatgga taatattaca aagtgtatat aggttgtata tagatacatg 3180 catatgatgg gaagactatg aagagagaga tagtcatcat ggtaagacat ttatccagaa 3240 attcatgaat tc 3252 4 1566 DNA Saccharomyces cerevisiae CDS (1)..(1563) Nucleotides 4-1566 of SEQ ID NO 4 corresponds to nucleotides 1381-2943 of SEQ ID NO 3. 4 atg gtc acc gaa gct cct ctc att atc caa act gca ata agt aag ttt 48 Met Val Thr Glu Ala Pro Leu Ile Ile Gln Thr Ala Ile Ser Lys Phe 1 5 10 15 cag aga gta gac atc ttg gtc aat aac gct ggt att ttg cgt gac aaa 96 Gln Arg Val Asp Ile Leu Val Asn Asn Ala Gly Ile Leu Arg Asp Lys 20 25 30 tct ttt tta aaa atg aaa gat gag gaa tgg ttt gct gtc ctg aaa gtc 144 Ser Phe Leu Lys Met Lys Asp Glu Glu Trp Phe Ala Val Leu Lys Val 35 40 45 cac ctt ttt tcc aca ttt tca ttg tca aaa gca gta tgg cca ata ttt 192 His Leu Phe Ser Thr Phe Ser Leu Ser Lys Ala Val Trp Pro Ile Phe 50 55 60 acc aaa caa aag tct gga ttt att atc aat act act tct acc tca gga 240 Thr Lys Gln Lys Ser Gly Phe Ile Ile Asn Thr Thr Ser Thr Ser Gly 65 70 75 80 att tat ggt aat ttt gga cag gcc aat tat gcc gct gca aaa gcc gcc 288 Ile Tyr Gly Asn Phe Gly Gln Ala Asn Tyr Ala Ala Ala Lys Ala Ala 85 90 95 att tta gga ttc agt aaa act att gca ctg gaa ggt gcc aag aga gga 336 Ile Leu Gly Phe Ser Lys Thr Ile Ala Leu Glu Gly Ala Lys Arg Gly 100 105 110 att att gtt aat gtt atc gct cct cat gca gaa acg gct atg aca aag 384 Ile Ile Val Asn Val Ile Ala Pro His Ala Glu Thr Ala Met Thr Lys 115 120 125 act ata ttc tcg gag aag gaa tta tca aac cac ttt gat gca tct caa 432 Thr Ile Phe Ser Glu Lys Glu Leu Ser Asn His Phe Asp Ala Ser Gln 130 135 140 gtc tcc cca ctt gtt gtt ttg ttg gca tct gaa gaa cta caa aag tat 480 Val Ser Pro Leu Val Val Leu Leu Ala Ser Glu Glu Leu Gln Lys Tyr 145 150 155 160 tct gga aga agg gtt att ggc caa tta ttc gaa gtt ggc ggt ggt tgg 528 Ser Gly Arg Arg Val Ile Gly Gln Leu Phe Glu Val Gly Gly Gly Trp 165 170 175 tgt ggg caa acc aga tgg caa aga agt tcc ggt tat gtt tct att aaa 576 Cys Gly Gln Thr Arg Trp Gln Arg Ser Ser Gly Tyr Val Ser Ile Lys 180 185 190 gag act att gaa ccg gaa gaa att aaa gaa aat tgg aac cac atc act 624 Glu Thr Ile Glu Pro Glu Glu Ile Lys Glu Asn Trp Asn His Ile Thr 195 200 205 gat ttc agt cgc aac act atc aac ccg agc tcc aca gag gag tct tct 672 Asp Phe Ser Arg Asn Thr Ile Asn Pro Ser Ser Thr Glu Glu Ser Ser 210 215 220 atg gca acc ttg caa gcc gtg caa aaa gcg cac tct tca aag gag ttg 720 Met Ala Thr Leu Gln Ala Val Gln Lys Ala His Ser Ser Lys Glu Leu 225 230 235 240 gat gat gga tta ttc aag tac act acc aag gat tgt atc ttg tac aat 768 Asp Asp Gly Leu Phe Lys Tyr Thr Thr Lys Asp Cys Ile Leu Tyr Asn 245 250 255 tta gga ctt gga tgc aca agc aaa gag ctt aag tac acc tac gag aat 816 Leu Gly Leu Gly Cys Thr Ser Lys Glu Leu Lys Tyr Thr Tyr Glu Asn 260 265 270 gat cca gac ttc caa gtt ttg ccc acg ttc gcc gtc att cca ttt atg 864 Asp Pro Asp Phe Gln Val Leu Pro Thr Phe Ala Val Ile Pro Phe Met 275 280 285 caa gct act gcc aca cta gct atg gac aat tta gtc gat aac ttc aat 912 Gln Ala Thr Ala Thr Leu Ala Met Asp Asn Leu Val Asp Asn Phe Asn 290 295 300 tat gca atg tta ctg cat gga gaa caa tat ttt aag ctc tgc acg ccg 960 Tyr Ala Met Leu Leu His Gly Glu Gln Tyr Phe Lys Leu Cys Thr Pro 305 310 315 320 aca atg cca agt aat gga act cta aag aca ctt gct aaa cct tta caa 1008 Thr Met Pro Ser Asn Gly Thr Leu Lys Thr Leu Ala Lys Pro Leu Gln 325 330 335 gta ctt gac aag aat ggt aaa gcc gct tta gtt gtt ggt ggc ttc gaa 1056 Val Leu Asp Lys Asn Gly Lys Ala Ala Leu Val Val Gly Gly Phe Glu 340 345 350 act tat gac att aaa act aag aaa ctc ata gct tat aac gaa gga tcg 1104 Thr Tyr Asp Ile Lys Thr Lys Lys Leu Ile Ala Tyr Asn Glu Gly Ser 355 360 365 ttc ttc atc agg ggc gca cat gta cct cca gaa aag gaa gtg agg gat 1152 Phe Phe Ile Arg Gly Ala His Val Pro Pro Glu Lys Glu Val Arg Asp 370 375 380 ggg aaa aga gcc aag ttt gct gtc caa aat ttt gaa gtg cca cat gga 1200 Gly Lys Arg Ala Lys Phe Ala Val Gln Asn Phe Glu Val Pro His Gly 385 390 395 400 aag gta cca gat ttt gag gcg gag att tct acg aat aaa gat caa gcc 1248 Lys Val Pro Asp Phe Glu Ala Glu Ile Ser Thr Asn Lys Asp Gln Ala 405 410 415 gca ttg tac agg tta tct ggc gat ttc aat cct tta cat atc gat ccc 1296 Ala Leu Tyr Arg Leu Ser Gly Asp Phe Asn Pro Leu His Ile Asp Pro 420 425 430 acg cta gcc aaa gca gtt aaa ttt cct acg cca att ctg cat ggg ctt 1344 Thr Leu Ala Lys Ala Val Lys Phe Pro Thr Pro Ile Leu His Gly Leu 435 440 445 tgt aca tta ggt att agt gcg aaa gca ttg ttt gaa cat tat ggt cca 1392 Cys Thr Leu Gly Ile Ser Ala Lys Ala Leu Phe Glu His Tyr Gly Pro 450 455 460 tat gag gag ttg aaa gtg aga ttt acc aat gtt gtt ttc cca ggt gat 1440 Tyr Glu Glu Leu Lys Val Arg Phe Thr Asn Val Val Phe Pro Gly Asp 465 470 475 480 act cta aag gtt aaa gct tgg aag caa ggc tcg gtt gtc gtt ttt caa 1488 Thr Leu Lys Val Lys Ala Trp Lys Gln Gly Ser Val Val Val Phe Gln 485 490 495 aca att gat acg acc aga aac gtc att gta ttg gat aac gcc gct gta 1536 Thr Ile Asp Thr Thr Arg Asn Val Ile Val Leu Asp Asn Ala Ala Val 500 505 510 aaa cta tcg cag gca aaa tct aaa cta taa 1566 Lys Leu Ser Gln Ala Lys Ser Lys Leu 515 520 5 521 PRT Saccharomyces cerevisiae Nucleotides 4-1566 of SEQ ID NO 4 corresponds to nucleotides 1381-2943 of SEQ ID NO 3. 5 Met Val Thr Glu Ala Pro Leu Ile Ile Gln Thr Ala Ile Ser Lys Phe 1 5 10 15 Gln Arg Val Asp Ile Leu Val Asn Asn Ala Gly Ile Leu Arg Asp Lys 20 25 30 Ser Phe Leu Lys Met Lys Asp Glu Glu Trp Phe Ala Val Leu Lys Val 35 40 45 His Leu Phe Ser Thr Phe Ser Leu Ser Lys Ala Val Trp Pro Ile Phe 50 55 60 Thr Lys Gln Lys Ser Gly Phe Ile Ile Asn Thr Thr Ser Thr Ser Gly 65 70 75 80 Ile Tyr Gly Asn Phe Gly Gln Ala Asn Tyr Ala Ala Ala Lys Ala Ala 85 90 95 Ile Leu Gly Phe Ser Lys Thr Ile Ala Leu Glu Gly Ala Lys Arg Gly 100

105 110 Ile Ile Val Asn Val Ile Ala Pro His Ala Glu Thr Ala Met Thr Lys 115 120 125 Thr Ile Phe Ser Glu Lys Glu Leu Ser Asn His Phe Asp Ala Ser Gln 130 135 140 Val Ser Pro Leu Val Val Leu Leu Ala Ser Glu Glu Leu Gln Lys Tyr 145 150 155 160 Ser Gly Arg Arg Val Ile Gly Gln Leu Phe Glu Val Gly Gly Gly Trp 165 170 175 Cys Gly Gln Thr Arg Trp Gln Arg Ser Ser Gly Tyr Val Ser Ile Lys 180 185 190 Glu Thr Ile Glu Pro Glu Glu Ile Lys Glu Asn Trp Asn His Ile Thr 195 200 205 Asp Phe Ser Arg Asn Thr Ile Asn Pro Ser Ser Thr Glu Glu Ser Ser 210 215 220 Met Ala Thr Leu Gln Ala Val Gln Lys Ala His Ser Ser Lys Glu Leu 225 230 235 240 Asp Asp Gly Leu Phe Lys Tyr Thr Thr Lys Asp Cys Ile Leu Tyr Asn 245 250 255 Leu Gly Leu Gly Cys Thr Ser Lys Glu Leu Lys Tyr Thr Tyr Glu Asn 260 265 270 Asp Pro Asp Phe Gln Val Leu Pro Thr Phe Ala Val Ile Pro Phe Met 275 280 285 Gln Ala Thr Ala Thr Leu Ala Met Asp Asn Leu Val Asp Asn Phe Asn 290 295 300 Tyr Ala Met Leu Leu His Gly Glu Gln Tyr Phe Lys Leu Cys Thr Pro 305 310 315 320 Thr Met Pro Ser Asn Gly Thr Leu Lys Thr Leu Ala Lys Pro Leu Gln 325 330 335 Val Leu Asp Lys Asn Gly Lys Ala Ala Leu Val Val Gly Gly Phe Glu 340 345 350 Thr Tyr Asp Ile Lys Thr Lys Lys Leu Ile Ala Tyr Asn Glu Gly Ser 355 360 365 Phe Phe Ile Arg Gly Ala His Val Pro Pro Glu Lys Glu Val Arg Asp 370 375 380 Gly Lys Arg Ala Lys Phe Ala Val Gln Asn Phe Glu Val Pro His Gly 385 390 395 400 Lys Val Pro Asp Phe Glu Ala Glu Ile Ser Thr Asn Lys Asp Gln Ala 405 410 415 Ala Leu Tyr Arg Leu Ser Gly Asp Phe Asn Pro Leu His Ile Asp Pro 420 425 430 Thr Leu Ala Lys Ala Val Lys Phe Pro Thr Pro Ile Leu His Gly Leu 435 440 445 Cys Thr Leu Gly Ile Ser Ala Lys Ala Leu Phe Glu His Tyr Gly Pro 450 455 460 Tyr Glu Glu Leu Lys Val Arg Phe Thr Asn Val Val Phe Pro Gly Asp 465 470 475 480 Thr Leu Lys Val Lys Ala Trp Lys Gln Gly Ser Val Val Val Phe Gln 485 490 495 Thr Ile Asp Thr Thr Arg Asn Val Ile Val Leu Asp Asn Ala Ala Val 500 505 510 Lys Leu Ser Gln Ala Lys Ser Lys Leu 515 520 6 1887 DNA Saccharomyces cerevisiae CDS (1)..(1887) Nucleotides 1-1887 of SEQ ID NO 6 corresponds to nucleotides 241- 2127 of SEQ ID NO 3. 6 atg cct gga aat tta tcc ttc aaa gat aga gtt gtt gta atc acg ggc 48 Met Pro Gly Asn Leu Ser Phe Lys Asp Arg Val Val Val Ile Thr Gly 1 5 10 15 gct gga ggg ggc tta ggt aag gtg tat gca cta gct tac gca agc aga 96 Ala Gly Gly Gly Leu Gly Lys Val Tyr Ala Leu Ala Tyr Ala Ser Arg 20 25 30 ggt gca aaa gtg gtc gtc aat gat cta ggt ggc act ttg ggt ggt tca 144 Gly Ala Lys Val Val Val Asn Asp Leu Gly Gly Thr Leu Gly Gly Ser 35 40 45 gga cat aac tcc aaa gct gca gac tta gtg gtg gat gag ata aaa aaa 192 Gly His Asn Ser Lys Ala Ala Asp Leu Val Val Asp Glu Ile Lys Lys 50 55 60 gcc gga ggt ata gct gtg gca aat tac gac tct gtt aat gaa aat gga 240 Ala Gly Gly Ile Ala Val Ala Asn Tyr Asp Ser Val Asn Glu Asn Gly 65 70 75 80 gag aaa ata att gaa acg gct ata aaa gaa ttc ggc agg gtt gat gta 288 Glu Lys Ile Ile Glu Thr Ala Ile Lys Glu Phe Gly Arg Val Asp Val 85 90 95 cta att aac aac gct gga ata tta agg gat gtt tca ttt gca aag atg 336 Leu Ile Asn Asn Ala Gly Ile Leu Arg Asp Val Ser Phe Ala Lys Met 100 105 110 aca gaa cgt gag ttt gca tct gtg gta gat gtt cat ttg aca ggt ggc 384 Thr Glu Arg Glu Phe Ala Ser Val Val Asp Val His Leu Thr Gly Gly 115 120 125 tat aag cta tcg cgt gct gct tgg cct tat atg cgc tct cag aaa ttt 432 Tyr Lys Leu Ser Arg Ala Ala Trp Pro Tyr Met Arg Ser Gln Lys Phe 130 135 140 ggt aga atc att aac acc gct tcc cct gcc ggt cta ttt gga aat ttt 480 Gly Arg Ile Ile Asn Thr Ala Ser Pro Ala Gly Leu Phe Gly Asn Phe 145 150 155 160 ggt caa gct aat tat tca gca gct aaa atg ggc tta gtt ggt ttg gcg 528 Gly Gln Ala Asn Tyr Ser Ala Ala Lys Met Gly Leu Val Gly Leu Ala 165 170 175 gaa acc ctc gcg aag gag ggt gcc aaa tac aac att aat gtt aat tca 576 Glu Thr Leu Ala Lys Glu Gly Ala Lys Tyr Asn Ile Asn Val Asn Ser 180 185 190 att gcg cca ttg gct aga tca cgt atg aca gaa aac gtg tta cca cca 624 Ile Ala Pro Leu Ala Arg Ser Arg Met Thr Glu Asn Val Leu Pro Pro 195 200 205 cat atc ttg aaa cag tta gga ccg gaa aaa att gtt ccc tta gta ctc 672 His Ile Leu Lys Gln Leu Gly Pro Glu Lys Ile Val Pro Leu Val Leu 210 215 220 tat ttg aca cac gaa agt acg aaa gtg tca aac tcc att ttt gaa ctc 720 Tyr Leu Thr His Glu Ser Thr Lys Val Ser Asn Ser Ile Phe Glu Leu 225 230 235 240 gct gct gga ttc ttt gga cag ctc aga tgg gag agg tct tct gga caa 768 Ala Ala Gly Phe Phe Gly Gln Leu Arg Trp Glu Arg Ser Ser Gly Gln 245 250 255 att ttc aat cca gac ccc aag aca tat act cct gaa gca att tta aat 816 Ile Phe Asn Pro Asp Pro Lys Thr Tyr Thr Pro Glu Ala Ile Leu Asn 260 265 270 aag tgg aag gaa atc aca gac tat agg gac aag cca ttt aac aaa act 864 Lys Trp Lys Glu Ile Thr Asp Tyr Arg Asp Lys Pro Phe Asn Lys Thr 275 280 285 cag cat cca tat caa ctc tcg gat tat aat gat tta atc acc aaa gca 912 Gln His Pro Tyr Gln Leu Ser Asp Tyr Asn Asp Leu Ile Thr Lys Ala 290 295 300 aaa aaa tta cct ccc aat gaa caa ggc tca gtg aaa atc aag tcg ctt 960 Lys Lys Leu Pro Pro Asn Glu Gln Gly Ser Val Lys Ile Lys Ser Leu 305 310 315 320 tgc aac aaa gtc gta gta gtt acg ggt gca gga ggt ggt ctt ggg aag 1008 Cys Asn Lys Val Val Val Val Thr Gly Ala Gly Gly Gly Leu Gly Lys 325 330 335 tct cat gca atc tgg ttt gca cgg tac ggt gcg aag gta gtt gta aat 1056 Ser His Ala Ile Trp Phe Ala Arg Tyr Gly Ala Lys Val Val Val Asn 340 345 350 gac atc aag gat cct ttt tca gtt gtt gaa gaa ata aat aaa cta tat 1104 Asp Ile Lys Asp Pro Phe Ser Val Val Glu Glu Ile Asn Lys Leu Tyr 355 360 365 ggt gaa ggc aca gcc att cca gat tcc cat gat gtg gtc acc gaa gct 1152 Gly Glu Gly Thr Ala Ile Pro Asp Ser His Asp Val Val Thr Glu Ala 370 375 380 cct ctc att atc caa act gca ata agt aag ttt cag aga gta gac atc 1200 Pro Leu Ile Ile Gln Thr Ala Ile Ser Lys Phe Gln Arg Val Asp Ile 385 390 395 400 ttg gtc aat aac gct ggt att ttg cgt gac aaa tct ttt tta aaa atg 1248 Leu Val Asn Asn Ala Gly Ile Leu Arg Asp Lys Ser Phe Leu Lys Met 405 410 415 aaa gat gag gaa tgg ttt gct gtc ctg aaa gtc cac ctt ttt tcc aca 1296 Lys Asp Glu Glu Trp Phe Ala Val Leu Lys Val His Leu Phe Ser Thr 420 425 430 ttt tca ttg tca aaa gca gta tgg cca ata ttt acc aaa caa aag tct 1344 Phe Ser Leu Ser Lys Ala Val Trp Pro Ile Phe Thr Lys Gln Lys Ser 435 440 445 gga ttt att atc aat act act tct acc tca gga att tat ggt aat ttt 1392 Gly Phe Ile Ile Asn Thr Thr Ser Thr Ser Gly Ile Tyr Gly Asn Phe 450 455 460 gga cag gcc aat tat gcc gct gca aaa gcc gcc att tta gga ttc agt 1440 Gly Gln Ala Asn Tyr Ala Ala Ala Lys Ala Ala Ile Leu Gly Phe Ser 465 470 475 480 aaa act att gca ctg gaa ggt gcc aag aga gga att att gtt aat gtt 1488 Lys Thr Ile Ala Leu Glu Gly Ala Lys Arg Gly Ile Ile Val Asn Val 485 490 495 atc gct cct cat gca gaa acg gct atg aca aag act ata ttc tcg gag 1536 Ile Ala Pro His Ala Glu Thr Ala Met Thr Lys Thr Ile Phe Ser Glu 500 505 510 aag gaa tta tca aac cac ttt gat gca tct caa gtc tcc cca ctt gtt 1584 Lys Glu Leu Ser Asn His Phe Asp Ala Ser Gln Val Ser Pro Leu Val 515 520 525 gtt ttg ttg gca tct gaa gaa cta caa aag tat tct gga aga agg gtt 1632 Val Leu Leu Ala Ser Glu Glu Leu Gln Lys Tyr Ser Gly Arg Arg Val 530 535 540 att ggc caa tta ttc gaa gtt ggc ggt ggt tgg tgt ggg caa acc aga 1680 Ile Gly Gln Leu Phe Glu Val Gly Gly Gly Trp Cys Gly Gln Thr Arg 545 550 555 560 tgg caa aga agt tcc ggt tat gtt tct att aaa gag act att gaa ccg 1728 Trp Gln Arg Ser Ser Gly Tyr Val Ser Ile Lys Glu Thr Ile Glu Pro 565 570 575 gaa gaa att aaa gaa aat tgg aac cac atc act gat ttc agt cgc aac 1776 Glu Glu Ile Lys Glu Asn Trp Asn His Ile Thr Asp Phe Ser Arg Asn 580 585 590 act atc aac ccg agc tcc aca gag gag tct tct atg gca acc ttg caa 1824 Thr Ile Asn Pro Ser Ser Thr Glu Glu Ser Ser Met Ala Thr Leu Gln 595 600 605 gcc gtg caa aaa gcg cac tct tca aag gag ttg gat gat gga tta ttc 1872 Ala Val Gln Lys Ala His Ser Ser Lys Glu Leu Asp Asp Gly Leu Phe 610 615 620 aag tac act acc aag 1887 Lys Tyr Thr Thr Lys 625 7 629 PRT Saccharomyces cerevisiae Nucleotides 1-1887 of SEQ ID NO 6 corresponds to nucleotides 241- 2127 of SEQ ID NO 3. 7 Met Pro Gly Asn Leu Ser Phe Lys Asp Arg Val Val Val Ile Thr Gly 1 5 10 15 Ala Gly Gly Gly Leu Gly Lys Val Tyr Ala Leu Ala Tyr Ala Ser Arg 20 25 30 Gly Ala Lys Val Val Val Asn Asp Leu Gly Gly Thr Leu Gly Gly Ser 35 40 45 Gly His Asn Ser Lys Ala Ala Asp Leu Val Val Asp Glu Ile Lys Lys 50 55 60 Ala Gly Gly Ile Ala Val Ala Asn Tyr Asp Ser Val Asn Glu Asn Gly 65 70 75 80 Glu Lys Ile Ile Glu Thr Ala Ile Lys Glu Phe Gly Arg Val Asp Val 85 90 95 Leu Ile Asn Asn Ala Gly Ile Leu Arg Asp Val Ser Phe Ala Lys Met 100 105 110 Thr Glu Arg Glu Phe Ala Ser Val Val Asp Val His Leu Thr Gly Gly 115 120 125 Tyr Lys Leu Ser Arg Ala Ala Trp Pro Tyr Met Arg Ser Gln Lys Phe 130 135 140 Gly Arg Ile Ile Asn Thr Ala Ser Pro Ala Gly Leu Phe Gly Asn Phe 145 150 155 160 Gly Gln Ala Asn Tyr Ser Ala Ala Lys Met Gly Leu Val Gly Leu Ala 165 170 175 Glu Thr Leu Ala Lys Glu Gly Ala Lys Tyr Asn Ile Asn Val Asn Ser 180 185 190 Ile Ala Pro Leu Ala Arg Ser Arg Met Thr Glu Asn Val Leu Pro Pro 195 200 205 His Ile Leu Lys Gln Leu Gly Pro Glu Lys Ile Val Pro Leu Val Leu 210 215 220 Tyr Leu Thr His Glu Ser Thr Lys Val Ser Asn Ser Ile Phe Glu Leu 225 230 235 240 Ala Ala Gly Phe Phe Gly Gln Leu Arg Trp Glu Arg Ser Ser Gly Gln 245 250 255 Ile Phe Asn Pro Asp Pro Lys Thr Tyr Thr Pro Glu Ala Ile Leu Asn 260 265 270 Lys Trp Lys Glu Ile Thr Asp Tyr Arg Asp Lys Pro Phe Asn Lys Thr 275 280 285 Gln His Pro Tyr Gln Leu Ser Asp Tyr Asn Asp Leu Ile Thr Lys Ala 290 295 300 Lys Lys Leu Pro Pro Asn Glu Gln Gly Ser Val Lys Ile Lys Ser Leu 305 310 315 320 Cys Asn Lys Val Val Val Val Thr Gly Ala Gly Gly Gly Leu Gly Lys 325 330 335 Ser His Ala Ile Trp Phe Ala Arg Tyr Gly Ala Lys Val Val Val Asn 340 345 350 Asp Ile Lys Asp Pro Phe Ser Val Val Glu Glu Ile Asn Lys Leu Tyr 355 360 365 Gly Glu Gly Thr Ala Ile Pro Asp Ser His Asp Val Val Thr Glu Ala 370 375 380 Pro Leu Ile Ile Gln Thr Ala Ile Ser Lys Phe Gln Arg Val Asp Ile 385 390 395 400 Leu Val Asn Asn Ala Gly Ile Leu Arg Asp Lys Ser Phe Leu Lys Met 405 410 415 Lys Asp Glu Glu Trp Phe Ala Val Leu Lys Val His Leu Phe Ser Thr 420 425 430 Phe Ser Leu Ser Lys Ala Val Trp Pro Ile Phe Thr Lys Gln Lys Ser 435 440 445 Gly Phe Ile Ile Asn Thr Thr Ser Thr Ser Gly Ile Tyr Gly Asn Phe 450 455 460 Gly Gln Ala Asn Tyr Ala Ala Ala Lys Ala Ala Ile Leu Gly Phe Ser 465 470 475 480 Lys Thr Ile Ala Leu Glu Gly Ala Lys Arg Gly Ile Ile Val Asn Val 485 490 495 Ile Ala Pro His Ala Glu Thr Ala Met Thr Lys Thr Ile Phe Ser Glu 500 505 510 Lys Glu Leu Ser Asn His Phe Asp Ala Ser Gln Val Ser Pro Leu Val 515 520 525 Val Leu Leu Ala Ser Glu Glu Leu Gln Lys Tyr Ser Gly Arg Arg Val 530 535 540 Ile Gly Gln Leu Phe Glu Val Gly Gly Gly Trp Cys Gly Gln Thr Arg 545 550 555 560 Trp Gln Arg Ser Ser Gly Tyr Val Ser Ile Lys Glu Thr Ile Glu Pro 565 570 575 Glu Glu Ile Lys Glu Asn Trp Asn His Ile Thr Asp Phe Ser Arg Asn 580 585 590 Thr Ile Asn Pro Ser Ser Thr Glu Glu Ser Ser Met Ala Thr Leu Gln 595 600 605 Ala Val Gln Lys Ala His Ser Ser Lys Glu Leu Asp Asp Gly Leu Phe 610 615 620 Lys Tyr Thr Thr Lys 625 8 6455 DNA Pseudomonas oleovorans 8 gaattcctgc gcgtgcactc cccctccgcc gaggtccagg gccacggtaa ccccatcctg 60 cagttcggca agatcaacgt cggcctcagc ggcctggaac ctgccgggca atacgcactg 120 aaactgacct tcgacgacgg ccatgacagc ggcctgttca cctgggaata cctcgagcag 180 ctgtgcctgc gccaggaaca gctgtgggcc gagtacctcg acgaactgca caaggccggg 240 aaatcccgcg accctgccga gtcggtggtc aaactcatgc tctagcgcaa ggcctgcagg 300 atttagagcg cattttctaa aatcatctgt ttgaatgact tacagacagc ccagtgacgg 360 gctgtcttgc gcattacatg aaagtcgggt aaccaatggg ggtggcaagt tccctgcatc 420 aaattgcagg tagtcagaac cctcgcagca ccgctgttcc ttatcactgg tcacccgagt 480 agcagtaccg ggctcagaac tgtgcacccg ccacagcaac cggtactcgt ctcaggacaa 540 cggagcgtcg tagatgagta acaagaacaa cgatgagctg cagcggcagg cctcggaaaa 600 caccctgggg ctgaacccgg tcatcggtat ccgccgcaaa gacctgttga gctcggcacg 660 caccgtgctg cgccaggccg tgcgccaacc gctgcacagc gccaagcatg tggcccactt 720 tggcctggag ctgaagaacg tgctgctggg caagtccagc cttgccccgg aaagcgacga 780 ccgtcgcttc aatgacccgg catggagcaa caacccactt taccgccgct acctgcaaac 840 ctatctggcc tggcgcaagg agctgcagga ctggatcggc aacagcgacc tgtcgcccca 900 ggacatcagc cgcggccagt tcgtcatcaa cctgatgacc gaagccatgg ctccgaccaa 960 caccctgtcc aacccggcag cagtcaaacg cttcttcgaa accggcggca agagcctgct 1020 cgatggcctg tccaacctgg ccaaggacct ggtcaacaac ggtggcatgc ccagccaggt 1080 gaacatggac gccttcgagg tgggcaagaa cctgggcacc agtgaaggcg ccgtggtgta 1140 ccgcaacgat gtgctggagc tgatccagta caagcccatc accgagcagg tgcatgcccg 1200 cccgctgctg gtggtgccgc cgcagatcaa caagttctac gtattcgacc tgagcccgga 1260 aaagagcctg gcacgctact gcctgcgctc gcagcagcag accttcatca tcagctggcg 1320 caacccgacc aaagcccagc gcgaatgggg cctgtccacc tacatcgacg cgctcaagga 1380 ggcggtcgac gcggtgctgg cgattaccgg cagcaaggac ctgaacatgc tcggtgcctg 1440 ctccggcggc atcacctgca cggcattggt cggccactat gccgccctcg gcgaaaacaa 1500 ggtcaatgcc ctgaccctgc tggtcagcgt gctggacacc accatggaca accaggtcgc 1560 cctgttcgtc gacgagcaga ctttggaggc cgccaagcgc cactcctacc aggccggtgt 1620 gctcgaaggc agcgagatgg ccaaggtgtt cgcctggatg cgccccaacg acctgatctg 1680 gaactactgg gtcaacaact acctgctcgg caacgagccg ccggtgttcg acatcctgtt 1740 ctggaacaac gacaccacgc gcctgccggc cgccttccac ggcgacctga tcgaaatgtt 1800 caagagcaac ccgctgaccc gcccggacgc cctggaggtt tgcggcactc cgatcgacct 1860 gaaacaggtc aaatgcgaca tctacagcct tgccggcacc aacgaccaca tcaccccgtg 1920 gcagtcatgc taccgctcgg cgcacctgtt cggcggcaag atcgagttcg tgctgtccaa 1980 cagcggccac atccagagca tcctcaaccc gccaggcaac cccaaggcgc gcttcatgac 2040 cggtgccgat cgcccgggtg acccggtggc ctggcaggaa aacgccacca agcatgccga 2100 ctcctggtgg ctgcactggc aaagctggct gggcgagcgt gccggcgagc tggaaaaggc 2160

gccgacccgc ctgggcaacc gtgcctatgc cgctggcgag gcatccccgg gcacctacgt 2220 tcacgagcgt tgagctgcag cgccgtggcc acctgcggga cgccacggtg ttcatttcac 2280 cccatgagtc acgcgcatgc cgcaacccta catcttcagg accgtcgagc tggacaacca 2340 gtccatccgc accgccgtcc gcccgggcaa accgcacctg acgccgttgc tgatcttcaa 2400 cggcatcggt gccaacctgg agctggtgtt tccgttcatc gaggcactgg acccggacct 2460 ggaagtcatt gcctttgacg tacccggggt cggcggctcg tccacgccgc gccacccata 2520 ccgcttcccc gggttggcca agctgacggc acgcatgctc gactacctcg actacggcca 2580 ggtcaatgtc atcggtgtgt cttggggcgg cgccctggcc cagcagttcg cccacgatta 2640 ccccgaacgc tgcaagaaac tggtgctggc cgccaccgca gccggtgcgg tgatggtgcc 2700 aggcaagccc aaggtgttgt ggatgatggc cagcccacgg cgttacgtgc agccgtcgca 2760 tgtcatccgc attgcgccga cgatctatgg cggcggcttc cggcgtgacc ccgaactggc 2820 catgcagcac gcctccaagg tgcgctccgg cggcaagatg ggctactact ggcagctgtt 2880 cgccgggctc ggctggacca gcatccactg gctgcacaag atccagcaac cgaccctggt 2940 gctggccggc gacgacgacc cgctgatccc gctgatcaac atgcgcctgc tggcctggcg 3000 gattcccaat gcccagctac acattatcga cgacggtcat ttgttcctga tcacccgggc 3060 cgaggccgtc gccccgatca tcatgaagtt ccttcagcaa gaacgacagc gcgccgtcat 3120 gcaccctcgc ccggcttcgg gcgggtaaat cgatgcggcc ttcttcgcgg gcgcgcccgc 3180 tcccacaggg atggcgccga acctgtggga gcgggcatgc ccgcgaaggt ctcgacagcg 3240 aaatggctta gacgagggag tgttgccatg aaagacaaac cggccaaagg aacgccaacg 3300 cttcccgcca ccagcatgaa cgtgcagaac gccatcctcg gcctgcgcgg tcgtgacctg 3360 atttccacgc tgcgcaatgt cagccgccaa agcctgcgtc acccgctgca caccgcacat 3420 cacctgttgg ccctgggtgg ccagctgggc cgggtgatac tgggtgacac accgcttcag 3480 ccgaacccgc gcgatccgcg cttcagcgac ccgacatgga gccagaaccc gttctaccgg 3540 cgcggcctgc aagcctacct ggcctggcag aagcagaccc gcctgtggat cgaggaaagc 3600 cacctggacg acgatgaccg ggcccgtgcg cacttcctgt tcaacctgat caacgatgcc 3660 ctggcgccaa gcaactcgct gctcaacccg ctggcggtca aggaactgtt caacagcggt 3720 ggccagagcc tggtgcgcgg cgtggcccac ctgctcgatg acctgcgcca caatgacggc 3780 ctgccacgcc aggtcgacga gcgcgccttc gaagtgggcg gcaacctggc cgcgactgcc 3840 ggcgccgtgg tgtttcgcaa cgagctgctg gaactgatcc agtacaagcc gatgagcgaa 3900 aagcagcacg cccggccact gctggtggtg ccgccacaga tcaacaagtt ctacatcttc 3960 gacctcagct cgaccaacag cttcgtccag tacatgctca agaatggcct gcaggtgttc 4020 atggtcagct ggcgcaaccc cgacccgcgc caccgcgaat ggggcctgtc cagctacgtg 4080 caggccctgg aagaagcgct caacgcttgc cgcagcatta gcggcaaccg cgaccccaac 4140 ctgatgggcg cctgcgccgg cggcctgacc atggccgcac tgcagggcca cctgcaggcc 4200 aagcaccagc tgcgccgggt gcgcagcgcc acctacctgg tcagcttgct ggacagcaag 4260 ttcgaaagcc ccgccagcct gttcgccgac gagcagacca tcgaggccgc caagcgccgc 4320 tcctaccagc gcggtgtgct ggatggcgcc gaggtggcgc ggatcttcgc ctggatgcgg 4380 cccaacgacc tgatctggaa ctactgggtc aacaactacc tgctcggcaa gacaccacca 4440 gccttcgaca tcctgtactg gaacgccgac agcacgcgcc tgcccgccgc gctgcatggc 4500 gacctgctgg acttcttcaa gctcaacccg ctgacccacc cagccggcct ggaggtatgc 4560 ggcacaccca tcgacctgca gaaggtcgag ctggacagtt tcaccgtggc cggcagcaac 4620 gaccacatca ccccgtggga tgcggtgtac cgctcggcct tgctgctggg tggcgaccgg 4680 cgcttcgtgc tggccaacag cgggcacatc cagagcatca tcaacccgcc cggcaacccc 4740 aaggcctact acctggccaa ccccaagctg tccagcgacc cgcgtgcctg gctccacgat 4800 gccaagcgca gcgaaggcag ctggtggccg ttgtggctgg agtggatcac cgcgcgctcc 4860 ggcccgctca aggcaccgcg cagcgaactg ggcaatgcca cctacccacc gctgggcccc 4920 gcgccgggca cctacgtgct gacccgatga gcatgccgac tggatgaaga ctcgcgaccg 4980 tatcctcgaa tgtgccctgc agctgttcaa ccagcagggc gaaccgaacg tatccaccct 5040 ggaaattgcc aacgaactgg gcatcagccc tggcaacctc tactaccact tccacggcaa 5100 ggagccgttg gtgctggggt tgttcgagcg ctttgaagaa gcgctgatgc ccttgctcga 5160 cccgccgctg gaggtacgcc tggacgccga ggattactgg ctgttcctgc acctgatcgt 5220 cgaacgcatg gcgcagtacc gcttcctgtt ccaggacctg tcgaacctga ccgggcgcct 5280 gcccaaactg gcccgcggca tgcgcaacct gatcaacgcg ctcaagcgca cactggcggc 5340 gttgctggcc agcctcaagg gccaagggtt ggtagagagc gagacccagg cgctggggca 5400 actggtggag cagatcaccc tgacactgat gttctcgctg gattatcagc gggtactggg 5460 gcgcgagggg gatgtgggga ttgtggtgta ccaggtgatg atgctggtgg cgccgcatct 5520 gcaggcccag gcgcgggggg cggcggagca attggcggtg cggtatctgg aggggtaagc 5580 ctgttgattc ggtgtcgcgg ctttcgcggg catgcccgct cccacaggtg aaatgcagtg 5640 ctcgagtgca cacaggacct gtgggagcgg gcaagcccgc gaagatggcg acgcggtatc 5700 agatcagggt accggtgcct gtctgtgccg aaggcgggtt gctggccgga gccgcagtgg 5760 gcgccgaagc tgcgctagcg gccggagcgg cgctggccgc tggagctgcc ggcttggcgg 5820 ctgccggctt ggccggtgct ttcttcactg caggcttctt cgccacagcc ggtttggccg 5880 ctgccggttt gccgcaggct tggccgccgc agtcttggca gcaggtttag ccgctgcggc 5940 cttggctgcc ggcttggctg ccgcggtttt gccgcaggct tggccgctgc cgtcttggcc 6000 agtggcttgg ccgccgcctt gctcgcagcc ggtttggctg cagtgcgcga cgaaatcggg 6060 gtaaccgaag cgccggtgag tttctcgatc tgcttggtca ggctgtccac ctgctggtgc 6120 agggccttga tctcgttgcg gctcggcacg ccaaggcgcg agatggcact gttcaggcgc 6180 ttgtcgaagg cctcttcgag ttcgctccac ttgcctagcg cacggtcctt cacgcccgac 6240 acacgcgaag tggtcgacga cttggcagtt tcagcaacat cttctgcggt cttcttcgcc 6300 tgtttctcgg ccttctcgcc atcctttacc agcgagtcga acagcttcgg gccgtcctgg 6360 tcgatcttcg aatagatacc aagccccgcc agccagatct tgcgggagta cttctcgatc 6420 ccgccgccca ggagctgcct tctttctcgg aattc 6455 9 5054 DNA Pseudomonas putida 9 ctgcagtcga cctcgccttg ccagtgggcg gcgagcatca ggccctcttc atcgggcagg 60 atgaaaacct gcaaggccgg ctggcggcag tcgatctcga tgaccttgcc ctccagcgcg 120 gccagccgcg gcagggccgt gctgtccatg cgcaggacgc ggttcaggcc atgttcggcg 180 ctggcgagca gcccggccag cagcatcagg gcttgatccc ccgatgcacg gcaacgatgc 240 cgctggtcat gttgtggtag gtgacccggt cgaaaccggc ctcgaccatc atggccttca 300 gggtttcctg atcggggtgc atgcggatcg actcggcgag gtaacggtag ctctccgaat 360 cgttggtgat cagcttgccg gccagcggca tgaaggcgaa cgagtaggcg tcgtaggcct 420 tggacatcag cttgttggtc ggcttggaga actccagcac cagcaggcga ccgcctggct 480 tgagcacgcg cagcatcgag cggatggctt cgtccttgtg ggtgacgttg cgcaggccga 540 aggcgatggt cacgcagtcg aagtggttgt ccgggaatgg cagcttctcg gcgtcggcct 600 gaacgaactc gatgttgccg gccacgccac ggtcgagcag gcggtcacga ccgaccttga 660 gcatcgattc gttgatgtcg gccagcacta cctgaccggt cgggccgacc agccgcgaga 720 acttggcggc caggtcgccg gtaccgccgg cgatgtccag tacacgattg ccggcgcgta 780 cgcccgacag ctcgatggtg aagcgcttcc acaggcggtg catgccgccg gaaagcacat 840 cgttcatcag gtcgtacttg gccgctaccg agtggaacac ctcggcgact ttcttcgcct 900 tctggctttc agggacgtcc tggtaaccga aatgggtggt gggttcggcg tggtcgcctt 960 tgcgctggtc gttcatatcg cttcaccgga aaaaattacc gccattctag ggcgaacggg 1020 cagatttgtc ttggcggggt gtgccagtgg gcaggggcgg gcataatgcc cattatgtct 1080 tcatcttcag gaacacccgc atgacccaga tcagcgtcga acgcaaacat tccctcggcc 1140 gcgatgccgc ccgtgccaag gctgaagcgc tggtcgacaa actgacccgc gaatacgacc 1200 tcaaggccac ctggaacggc gacagggtcg acgtggcgcg cagcggtgcc aacggcagcg 1260 tgcacatctt cgatgaccgc atccgcgtcg aattgaagct gggcatgatg ctgtcgatga 1320 tgagcggcac catcaagggc gagatcgagc gggcgctgga caaagccctg gcctgacccc 1380 ttccgggggc catgctcgtt caccttaggg tgaagattct aatttccctc tctaccttgt 1440 gctcaagccc aacctccatg ggcggtttat cctccactca tgagcatgag gtacagagca 1500 tggccaaagt gactgtgaag aaaaaggacg acgccccggg cacgctgggc gaggtgcgcg 1560 gctacgcgcg caagatctgg ctggcgggca tcggcgccta tgcgcgtgtg ggccaggaag 1620 gctccgacta cttcaatgag ctggtcaagg ccggcgaagg cgtcgagaag cgtggcaaga 1680 agcgcatcga caaggagctc gatgctgcca acaatcagat cgaagaagcg acccaggaag 1740 tcagtcgcgt acgcggcaag gtcgaagttc aactggacaa gatcgaaaaa gctttcgacg 1800 cgcgggtagg tcgcgccttg aatcgccttg gcattccgtc taaacatgac gttgaggcgt 1860 tgtccatcaa gcttgaacag ctgcacgagc tgctcgagcg cgtcgcgcac aaaccataag 1920 gagagcagga tggctggcaa gaagaattcc gagaaagaag gcagctcctg ggtcggcggg 1980 atcgagaagt actcccgcaa gatctggctg gcggggcttg gtatctattc gaagatcgac 2040 caggacggcc cgaagctgtt cgactcgctg gtgaaggatg gcgagaaggc cgagaagcag 2100 gcgaagaaga ccgccgaaga tgttgctgaa actgccaagt cgtcgaccac ttcgcgtgtg 2160 tcgggcgtga aggaccgtgc gctgggcaag tggagcgaac tcgaagaggc cttcgacaag 2220 cgcctgaaca gcgccatctc gcgccttggc gtgccgagcc gcaacgagat caaggccctg 2280 caccagcagg tggacagcct gaccaagcag atcgagaaac ttaccggcgc ttcgcttacc 2340 ccgatttcgt cgcgcgctgc ggccaaaccg gctgcgagca aggcggcggc caagccactg 2400 gccaagacgg cagcggccaa gcctgcggcg aaaactgcgg cggccaagcc ggcggccaag 2460 actgctgcgg cgaaacctgc tgccaagact gcagcggcca agcctgcggc gaaaccggca 2520 gcggccaagc cggctgtggc gaagaagccg gcagtgaaga aagcaccggc caagccagca 2580 gccgccaagc cggcagctcc agcggcgagc gccgctccga ccgctagcgc agctcctgca 2640 ccaaccgcgg ctccggccag caatccgcct tcggcaccga caggcaccgg taccctgatc 2700 tgataccgcg tcgccttctt cgcgggcttg cccgctccca caggtcctgt gtgcactcga 2760 gcactgcatt tcacctgtgg gagcgggcat gcccgcgaaa gccgcgacac cgaaacaaca 2820 ggcttacccc tccagatacc tcaccgccaa ctgctccgcc gccccccgcg cctgggcctg 2880 cagatgcggc gccaccagca tcatcacctg gtacaccaca atccccacat ccccctcgcg 2940 ccccagtacc cgctggtaat ccagcgagaa catcagtgtc agggtgatct gctccaccag 3000 ttgccccagc gcctgggtct cgctctctac cagcccctgg cccttgaggc tggccagcaa 3060 cgccgccagt gtgcgcttga gtgcgttgat caggctgcgc atgccgcggg ccagtttggg 3120 caggcgcccg gtcaggttcg acaggtcctg gaacaggaag cggtactgcg ccatgcgttc 3180 gacgatcagg tgcaggaaca gccagtaatc ctcggcatcc aggcgtacct ccagtggcgg 3240 gtcgagcaag ggcattagcg cttcttcaaa gcgctcgaac agccccagca ccaacggctc 3300 cttgccgtgg aagtggtagt agaggttgcc ggggctgatg cccagttcgt tggcaatttc 3360 cagggtggat acgttcggtt cgccctgctg gttgaacagc tgcagggcac attccaggat 3420 acggtcgcga gtcttcatcc agtcggcatg ctcatcgggt cagcacgtag gtgcccggcg 3480 cagggcccag cggtgggtag gtggcattgc ccagttcgct gcgtggtgcc ttgagcgggc 3540 cggagcgtgg tgtgatccac tccagccaca acggccacca gctgccttcg ctgcgcttgg 3600 cgtcgtggaa ccaggcacgg gggtcgctgg acagcttggg gttggccagg taataggcct 3660 tggggttgcc gggcgggttg atgatgctct ggatgtgccc gctgttggcc agcacgaagc 3720 gacggtcgcc gcccagcagc aaggccgagc ggtacaccgc atcccacggg gtgatgtggt 3780 cgttgctgcc ggccacggtg aaactgtcca gatcgacctt ctgcaggtcg atgggggtgc 3840 cgcatacctc caggccggct gggtgagtca gcgggttgag cttgaagaag tccagcaggt 3900 cgccatgcag cgcggcgggc aggcgcgtgc tgtcggcgtt ccagtacagg atgtcgaaag 3960 ctggtggtgt cttgccgagc aggtagttgt tgacccagta gttccagatc aggtcgttgg 4020 gccgcatcca ggcgaagatc cgcgccacct cggcgccatc cagcacgcct cgttggtagg 4080 agcggcgctt ggcggcttcg atggtctgct cgtcggcgaa caggctggcg gggctttcga 4140 acttgctgtc cagaaggctg accaggtagg tggcgctgcg cacccgacgc agctggtgct 4200 tggcctgcag gtgaccctgt agcgcggcca tggtcaggcc gccggcgcag gcccccatca 4260 ggttggggtc gcggttgccg ctgatgctgc ggcaagcgtt gagcgcttct tccagggcct 4320 gcacgtagct cgacaggccc cattcgcggt ggcgcgggtc ggggttgcgc cagctcacca 4380 tgaacacctg caggccattc ttgagcatgt actggacgaa gctgttggtc gagctgaggt 4440 cgaagatgta gaacttgttg atctgtggcg gcactaccag cagtggccgg gcgtgctgct 4500 tttcgctcat cggcttgtac tggatcagct ccagcagctc gttgcgaaac accacggcgc 4560 cggcggttgc agccaggttg ccgcccactt cgaaggcgcg ctcgtcgacc tggcgtggta 4620 ggccatcgtt gtggcgcagg tcatcgagca ggtgggccac gccgcgtacc aggctctggc 4680 caccgctgtt gaacagttcc ttgaccgcca gcgggttgag cagcgagttg ctcggcgcca 4740 gtgcatcgtt gatcaggttg aacaggaagt gcgcacgggc ccggtcatcg tcgtccaggt 4800 ggctttcctc gatccacagg cgggtctgct tctgccaggc caggtaggct tgcaggccgc 4860 gccggtagaa cgggttctgg ctccatgtcg ggtcgctgaa gcgcggatcg cgcgggttcg 4920 gctgaaacgg tgtgtcaccc agcatcaccc tgcccagctg accacccagg gccaacaggt 4980 gatgcgcggt gtgcagcggg tgacgcaggc tttggcggct gacattgcgc agcgtggaaa 5040 tcaggtcacg gccg 5054 10 7311 DNA Pseudomonas aeruginosa 10 ctgcagggcc gcctgccgat ccgcgtggag ctcaaggccc tcagtccgaa cgatttcgag 60 cgcatcctca ccgagccgca tgcctcgctc accgagcagt accgcgagct gctgaagacc 120 gaggggctgg ccatcgagtt cgccgaggac ggcatcaagc gccttgccga gatcgcctgg 180 caggtcaacg agaagaccga gaacatcggt gcccgccgcc tgcatacgct gctcgagcgg 240 ctgctggaag aggtctcgtt cagcgccgcc gacctggcca gcgagcatag cgacaagccg 300 atcctgatcg atgccggcta cgtcaacagc cacctcggcg agctggccga ggacgaggac 360 ctgtcccgct acatcctttg accaccgccg ccgccacggc caacaccgtg gcggcgggcc 420 gcgcggtccc gaaccggggt atccttgtac tccgcccttc cgcgagtccg atgcgatgcg 480 tatcccttcc gccatccagc tgcacaaagc ctcgaagacc ctgaccctgc gctacggcga 540 ggatagctac gacctgcctg ccgagttcct ccgcgtgcat tcgccctcgg ccgaggtcca 600 gggccacggc aacccggtat tgcagtacgg caagctgaac gtcggcctgg tcggcgtcga 660 acccgccggc cagtacgcac tgaagctgag cttcgacgac ggccacgaca gcggcctgtt 720 cacctgggac tacctgtacg agctggcgac ccgcaaggac cagctatggg ccgactacct 780 ggcggagctg gccagcgccg gcaagtcgcg cgaccccgac gagtcggtcg tcaagctgat 840 gctctgaagc gccgggccga gtgccccttc gcggcacacg gcatccaggc gatttttctc 900 cgctgccccg aacgagggca cgtcatttgc aacattcgcc ccgcagactg ccagaccaga 960 catccggtcg gcgcggtcgc cacggcgaat gtcctaccct gcacggcagc aacgctctag 1020 tccgcaggtc ggccgctcgc cgggccagcc acgccggcca ccgatgcttc tgccagcttg 1080 cacgccagcg gaaactcgcg taaccaagac ggtgcagccc cttgtccggg ttcccttccc 1140 tgacgtgcgt ccgccccgcc ctgaaacgtg cgggtgggct ggtagtcgag cagtagccgg 1200 ccggacacct agtccccggt ttcgttgcgg tcccctgttt cgcctcgaac aatggagcgt 1260 tgccgatgag tcagaagaac aataacgagc ttcccaagca agccgcggaa aacacgctga 1320 acctgaatcc ggtgatcggc atccggggca aggacctgct cacctccgcg cgcatggtcc 1380 tgctccaggc ggtgcgccag ccgctgcaca gcgccaggca cgtggcgcat ttcagcctgg 1440 agctgaagaa cgtcctgctc ggccagtcgg agctacgccc aggcgatgac gaccgacgct 1500 tttccgatcc ggcctggagc cagaatccac tgtacaagcg ctacatgcag acctacctgg 1560 cctggcgcaa ggagctgcac agctggatca gccacagcga cctgtcgccg caggacatca 1620 gtcgtggcca gttcgtcatc aacctgctga ccgaggcgat gtcgccgacc aacagcctga 1680 gcaacccggc ggcggtcaag cgcttcttcg agaccggcgg caagagcctg ctggacggcc 1740 tcggccacct ggccaaggac ctggtgaaca acggcgggat gccgagccag gtggacatgg 1800 acgccttcga ggtgggcaag aacctggcca ccaccgaggg cgccgtggtg ttccgcaacg 1860 acgtgctgga actgatccag taccggccga tcaccgagtc ggtgcacgaa cgcccgctgc 1920 tggtggtgcc gccgcagatc aacaagttct acgtcttcga cctgtcgccg gacaagagcc 1980 tggcgcgctt ctgcctgcgc aacggcgtgc agaccttcat cgtcagttgg cgcaacccga 2040 ccaagtcgca gcgcgaatgg ggcctgacca cctatatcga ggcgctcaag gaggccatcg 2100 aggtagtcct gtcgatcacc ggcagcaagg acctcaacct cctcggcgcc tgctccggcg 2160 ggatcaccac cgcgaccctg gtcggccact acgtggccag cggcgagaag aaggtcaacg 2220 ccttcaccca actggtcagc gtgctcgact tcgaactgaa tacccaggtc gcgctgttcg 2280 ccgacgagaa gactctggag gccgccaagc gtcgttccta ccagtccggc gtgctggagg 2340 gcaaggacat ggccaaggtg ttcgcctgga tgcgccccaa cgacctgatc tggaactact 2400 gggtcaacaa ctacctgctc ggcaaccagc cgccggcgtt cgacatcctc tactggaaca 2460 acgacaccac gcgcctgccc gccgcgctgc acggcgagtt cgtcgaactg ttcaagagca 2520 acccgctgaa ccgccccggc gccctggagg tctccggcac gcccatcgac ctgaagcagg 2580 tgacttgcga cttctactgt gtcgccggtc tgaacgacca catcaccccc tgggagtcgt 2640 gctacaagtc ggccaggctg ctgggtggca agtgcgagtt catcctctcc aacagcggtc 2700 acatccagag catcctcaac ccaccgggca accccaaggc acgcttcatg accaatccgg 2760 aactgcccgc cgagcccaag gcctggctgg aacaggccgg caagcacgcc gactcgtggt 2820 ggttgcactg gcagcaatgg ctggccgaac gctccggcaa gacccgcaag gcgcccgcca 2880 gcctgggcaa caagacctat ccggccggcg aagccgcgcc cggaacctac gtgcatgaac 2940 gatgaaaagc gaccagcctg aagaacagcc gcaggagcga tccgcggcct ccgccgacga 3000 aaccccgcca gcgcctccgg cgcggccccg tgccgcgcgg aagccggcca ggccccgtat 3060 cgccgagccc gcggctgcgc cgccgaggac cccgagcatg ccccagccct tcgtcttccg 3120 gaccatcgac ctcgacggcc agaccatccg caccgcagtg cggccgggca aggaaggaag 3180 cactccgctg ctgatcttca acggcatagg cgccaacctg gaactggtgt tccccttcgt 3240 ccaggcgctc gacccggaac tggaggtgat cgccttcgac gttcccggcg tcggcggttc 3300 ctcgacgccc agcgtgccct accgctttcc cggcctggcc aagctggcgg cgcggatgct 3360 cgactacctg gactacggcc aggtcaacgc gatcggcgtg tcctggggcg gcgcgctggc 3420 ccagcagttc gcccacgact atccggaacg ctgcaagaag ctgatcctcg ccgccacttc 3480 ggctggcgcg gtgatggtgc cgggcaagcc gaaggtactg atgcgcatgg ccagcccgcg 3540 gcgctacatc cagccctcct atggcgtaca catcgccccg gacatctacg gcggggcctt 3600 ccgccgcgac cccaagctgg ccatggcgca tgccagcaag gtgcgttcgt cgggcaagct 3660 gggctactac tggcaactgt tcgccgggct cggctggacc agcatccact ggctgcatag 3720 gatccgccag ccgaccctgg tgctggccgg cgacgacgac ccgatcatcc cgctgatcaa 3780 catgcgtgtg ctggcctggc gcattcccaa cgccgaactg cacgtgatcg acgacggcca 3840 cctgttcctg gtgacccgcg ccgaatcggt ggcgccgatc atcatgaagt tcctcgccga 3900 ggagcgccgt cgcgccgtca tgcaccctcg tccgttcctg cccaagaccg gctgaactgc 3960 tgcacgggca acaccagggc agcagacact gcttcgtcat ggtgcaggtg catagtcaat 4020 gttccgcaac ggcgcatggc gcctggcttc gccgcgacag cgcaagctct gcgacgcccc 4080 tcccggcgtt gtggtacagc gcttgctgcc ccactgtcgg gcggcaacct ccctgttcag 4140 gcaggtgtgc ggttctgccc cgggtgaaac ggaattcaca ggctataact gagtattccg 4200 agccagggga cgatcgcccg tggcatccag actgtggtcc tacgacggag tgtggtccat 4260 gcgagaaaag caggaatcgg gtagcgtgcc ggtgcccgcc gagttcatga gtgcacagag 4320 cgccatcgtc ggcctgcgcg gcaaggacct gctgacgacg gtccgcagcc tggctgtcca 4380 cggcctgcgc cagccgctgc acagtgcgcg gcacctggtc gccttcggag gccagttggg 4440 caaggtgctg ctgggcgaca ccctgcacca gccgaaccca caggacgccc gcttccagga 4500 tccatcctgg cgcctcaatc ccttctaccg gcgcaccctg caggcctacc tggcgtggca 4560 gaaacaactg ctcgcctgga tcgacgaaag caacctggac tgcgacgatc gcgcccgcgc 4620 ccgcttcctc gtcgccttgc tctccgacgc cgtggcaccc agcaacagcc tgatcaatcc 4680 actggcgtta aaggaactgt tcaataccgg cgggatcagc ctgctcaatg gcgtccgcca 4740 cctgctcgaa gacctggtgc acaacggcgg catgcccagc caggtgaaca agaccgcctt 4800 cgagatcggt cgcaacctcg ccaccacgca aggcgcggtg gtgttccgca acgaggtgct 4860 ggagctgatc cagtacaagc cgctgggcga gcgccagtac gccaagcccc tgctgatcgt 4920 gccgccgcag atcaacaagt actacatctt cgacctgtcg ccggaaaaga gcttcgtcca 4980 gtacgccctg aagaacaacc tgcaggtctt cgtcatcagt tggcgcaacc ccgacgccca 5040 gcaccgcgaa tggggcctga gcacctatgt cgaggccctc gaccaggcca tcgaggtcag 5100 ccgcgagatc accggcagcc gcagcgtgaa cctggccggc gcctgcgccg gcgggctcac 5160 cgtagccgcc ttgctcggcc acctgcaggt gcgccggcaa ctgcgcaagg tcagtagcgt 5220 cacctacctg gtcagcctgc tcgacagcca gatggaaagc ccggcgatgc tcttcgccga 5280 cgagcagacc ctggagagca gcaagcgccg ctcctaccag catggcgtgc tggacgggcg 5340 cgacatggcc aaggtgttcg cctggatgcg ccccaacgac ctgatctgga actactgggt 5400 caacaactac ctgctcggca ggcagccgcc ggcgttcgac atcctctact ggaacaacga 5460 caacacgcgg ctgcccgcgg cgttccacgg cgaactgctc gacctgttca agcacaaccc 5520 gctgacccgc ccgggcgcgc tggaggtcag cgggaccgcg gtggacctgg gcaaggtggc

5580 gatcgacagc ttccacgtcg ccggcatcac cgaccacatc acgccctggg acgcggtgta 5640 tcgctcggcc ctcctgctgg gcggccagcg ccgcttcatc ctgtccaaca gcgggcacat 5700 ccagagcatc ctcaaccctc ccggaaaccc caaggcctgc tacttcgaga acgacaagct 5760 gagcagcgat ccacgcgcct ggtactacga cgccaagcgc gaagagggca gctggtggcc 5820 ggtctggctg ggctggctgc aggagcgctc gggcgagctg ggcaaccctg acttcaacct 5880 tggcagcgcc gcgcatccgc ccctcgaagc ggccccgggc acctacgtgc atatacgctg 5940 aaagatccgg cccgggcgcc tggagccggg cacctccatc cccagaagaa gtccggatga 6000 agacacgcga ccgaatcctc gaatgctcgc tgttgctgtt caacgaacag ggcgagccca 6060 acgtctcgac actggagatc gccaacgaac tgggcatcag cccgggcaat ctctactacc 6120 acttccatgg caaggaaccg ctggtgatgg cgctgttcga gcggttccag gccgagttgg 6180 cgccgctgct cgatccgccc gaggaggtgc gcctgggcgc ggaggactac tggctgttcc 6240 tgcacctgat cgtcgagcgc ctcgcccact accgcttcct gttccaggac ctgtccaacc 6300 tgaccgggcg cctgccacgg ctggctcgcg gcatccgtac ctggctcggc gcgctgaagc 6360 ggaccctggc caccctgctg gcccgcctca aggccgaccg gcagttgcgc agcgacgcgc 6420 cggcactcgg gcaactggtc gaagagatca ccctgaccct gctgttctcc ctggattacc 6480 agcgggtact gggcagcaag ggcgaggtgc gcacggtggt ctaccagatc atgatgctgg 6540 tcgccccgca tctgcgcagc gaggcccaac gctcggcgga aagcctggcg cagcgctacc 6600 tgggaccgga atgaaaaacg cccggcgagt gccgggcgtg tgccttgccg ccaggactca 6660 gccctggctg ctcggcgtcg ccggagcgtt cgctgccgga gcgctggcag ccggtgtggc 6720 ggcgggggca gcgggcgcgc tcgaagacgc ggcgggagcg gccggtttcg ccgcggcggg 6780 cttggctgcc ggcttcttcg ccgcaggctt tttcgcagcc ggcttggcgg caggtttcgc 6840 cgcgggcttg gcggcggtcg cagcggccgg cttggctgcc ggcttcgcgg caggcttggc 6900 tgcggcaggt ttcgcagcag gcttcgccgc ggccttggct gccggcttgg tcgccggttt 6960 cgctgcggcc ttcgcagtcg gtttggcggc gggcttggct gccggcttgg cggccgcggt 7020 tttcgccgcg gtttttttcg ccgcgggttt cgccgcaggc ttggcggcgg ccttggctgc 7080 cggtttagct gccggcttgg ccgctgcggt tttcgccgcg ggcttggcag ccggtttcgc 7140 cgcaggcttg gccgctgcct tcgccgccgg cttgacgctg acgccggtga gtttctcgat 7200 ctgcttggtc agcgtatcga ccttgctgtg cagctccttc acctcgttgc ggctcggcac 7260 gccgagacgc gagatggcgc tgttcaggcg cttgtcgaaa gcttcctcga g 7311 11 5051 DNA Aeromonas caviae 11 gatatccatc agcccctgac agagagcact gatatgaccg gcaggcatcg ccatcccctg 60 ccatcccgcc gcgtactggg tgtgattgga aaactgcacc gtattgacgg gccagacctc 120 catgccgaga cggcgcatgg ggaagacggc ggcgctgttg ccggcacagc cgaaaaccac 180 gtgggactgg atagagagaa tgcgtttcat cgacaccacc actgaagaag catgaggaaa 240 gcgggcaata tatcggatgc atagcgggct ctgccagcga ttcccggcaa ccctcgccat 300 ttatgcggca aaaagcccat tcaatggagt gttacaccaa cttcccccca cgcctgctca 360 acctggcaag ccaagagcgc ctcggccccg tcggtgtgat atgggctctt cacactcttg 420 tttgatggca tatgagggca tttttaatac aaggtggcgg atggcgggag aaagcgtcga 480 ggaagagcaa tgaggcgcgg cggcaggcaa gccggggccg acgccccggg ctttgtactt 540 aaggcttaag gcctgacgaa gtgatagacc cagccttcgt ccagggggaa atcggagggc 600 gcagagacaa gtgccggcag ggaggcgacg gcccctccgg cggcgtattg cagcgccatc 660 agggcgcaga gggcggcgtc gtaacgatcc gtcccggcca ccacatcagg tggcaaggag 720 gcggctatct ccgccctggc cggtgaggcc ttgcctgcta ccttggcaaa gagccgggat 780 agacctccag cacggcgcga tcggcgtggg gctgctgcct cgggcacaag ctgcagatcc 840 ccgaggcgcg gcaagatgga gagcgccagg gtggcgttgt tgcctagcct gtcgaaggtg 900 gccgacagcg gcttcttgcc gtattgctgc tgcagccagc gctcgcagtc ccgataggca 960 taggggttgt ctatctcccg ctggggcacg gcgcactcga ctcccctccc cgccagcaga 1020 tcgccgaggg ctcggggaaa ggccaggggg gcatcgatgc cgagcgccag tcgcgggcag 1080 gccagcacct gcatcagcgc catctcgtct tgcagcgccg ggcgcagcaa ggcgtccaga 1140 tccggcgcca cccgggagct caaccgaaac aggggggaga ccccgagcca gtgcaactgc 1200 tgcgtgcggg cctgccagcc caccacggcc accgcctggg cattgccctg ccagcccctt 1260 acatcccaac cgatgccgat ggcatccaag tgttccgttg tcatgcccct gtcacctctg 1320 cgcactattg atggagcaca gggtaatggc tgcgcccctc aaagcaaggc tggcggcacc 1380 gcttagaaag atttcttgaa gttgatcagc ggcaagatgg ggaagatgcc gttctctgcg 1440 tagttggcga gccctatctg caggccatcg aggtgcttgg tggcattgat gaagccaagc 1500 tggaaggtgg tgcgctcggc atagttgacg aagccgaggt tggccagggc attgcccttg 1560 gcgatgttga ccgcccccca gttcagcccc tgcacgttgt tggtcaggtt gacgaaaccc 1620 aggttgaccc cggtgtcctg cccctcatgc cagttgacgg cgttgatggc gaccccgccg 1680 aactgatggc gcacccgggc ggcgccgaag aagatgccaa gctgcaggcc ggtgaactga 1740 tccacgtcgg agagggcgaa caccggcaga tctatgccct tcacctgacc ggtgcggcca 1800 tacaggaagg aggcgcgcgc cccttccacc tggtgggagg aaggcaggtt gatgccgggc 1860 agggagatct ggaccggggt gctggcctgg gccacgccgg cgagggccag cgcggagcaa 1920 ccgagcagca gggcgagagg tttcatcggg attccttggc agtctgaatg acgtgccagc 1980 ctatcagcgc ggcgccggtg cggcgagggc gcgccggacc cagtgcgtca cctctcgtct 2040 gatccgcctc cctcgacggg cgtcgctgac aaaaaaattc aaacagaaat taacatttat 2100 gtcatttaca ccaaaccgca tttggttgca gaatgctcaa acgtgtgttt gaacagagca 2160 agcaacacgt aaacagggat gacatgcagt acccgtaaga agggccgatt ggcccacaac 2220 aacactgttc tgccgaactg gagaccgatg atgaatatgg acgtgatcaa gagctttacc 2280 gagcagatgc aaggcttcgc cgcccccctc acccgctaca accagctgct ggccagcaac 2340 atcgaacagc tgacccggtt gcagctggcc tccgccaacg cctacgccga actgggcctc 2400 aaccagttgc aggccgtgag caaggtgcag gacacccaga gcctggcggc cctgggcaca 2460 gtgcaactgg agaccgccag ccagctctcc cgccagatgc tggatgacat ccagaagctg 2520 agcgccctcg gccagcagtt caaggaagag ctggatgtcc tgaccgcaga cggcatcaag 2580 aaaagcacgg gcaaggcctg ataacccctg gctgcccgtt cgggcagcca catctcccca 2640 tgactcgacg ctacgggcta gttcccgcct cgggtgtggg tgaaggagag cacatgagcc 2700 aaccatctta tggcccgctg ttcgaggccc tggcccacta caatgacaag ctgctggcca 2760 tggccaaggc ccagacagag cgcaccgccc aggcgctgct gcagaccaat ctggacgatc 2820 tgggccaggt gctggagcag ggcagccagc aaccctggca gctgatccag gcccagatga 2880 actggtggca ggatcagctc aagctgatgc agcacaccct gctcaaaagc gcaggccagc 2940 cgagcgagcc ggtgatcacc ccggagcgca gcgatcgccg cttcaaggcc gaggcctgga 3000 gcgaacaacc catctatgac tacctcaagc agtcctacct gctcaccgcc aggcacctgc 3060 tggcctcggt ggatgccctg gagggcgtcc cccagaagag ccgggagcgg ctgcgtttct 3120 tcacccgcca gtacgtcaac gccatggccc ccagcaactt cctggccacc aaccccgagc 3180 tgctcaagct gaccctggag tccgacggcc agaacctggt gcgcggactg gccctcttgg 3240 ccgaggatct ggagcgcagc gccgatcagc tcaacatccg cctgaccgac gaatccgcct 3300 tcgagctcgg gcgggatctg gccctgaccc cgggccgggt ggtgcagcgc accgagctct 3360 atgagctcat tcagtacagc ccgactaccg agacggtggg caagacacct gtgctgatag 3420 tgccgccctt catcaacaag tactacatca tggacatgcg gccccagaac tccctggtcg 3480 cctggctggt cgcccagggc cagacggtat tcatgatctc ctggcgcaac ccgggcgtgg 3540 cccaggccca aatcgatctc gacgactacg tggtggatgg cgtcatcgcc gccctggacg 3600 gcgtggaggc ggccaccggc gagcgggagg tgcacggcat cggctactgc atcggcggca 3660 ccgccctgtc gctcgccatg ggctggctgg cggcgcggcg ccagaagcag cgggtgcgca 3720 ccgccaccct gttcactacc ctgctggact tctcccagcc cggggagctt ggcatcttca 3780 tccacgagcc catcatagcg gcgctcgagg cgcaaaatga ggccaagggc atcatggacg 3840 ggcgccagct ggcggtctcc ttcagcctgc tgcgggagaa cagcctctac tggaactact 3900 acatcgacag ctacctcaag ggtcagagcc cggtggcctt cgatctgctg cactggaaca 3960 gcgacagcac caatgtggcg ggcaagaccc acaacagcct gctgcgccgt ctctacctgg 4020 agaaccagct ggtgaagggg gagctcaaga tccgcaacac ccgcatcgat ctcggcaagg 4080 tgaagacccc tgtgctgctg gtgtcggcgg tggacgatca catcgccctc tggcagggca 4140 cctggcaggg catgaagctg tttggcgggg agcagcgctt cctcctggcg gagtccggcc 4200 acatcgccgg catcatcaac ccgccggccg ccaacaagta cggcttctgg cacaacgggg 4260 ccgaggccga gagcccggag agctggctgg caggggcgac gcaccagggc ggctcctggt 4320 ggcccgagat gatgggcttt atccagaacc gtgacgaagg gtcagagccc gtccccgcgc 4380 gggtcccgga ggaagggctg gcccccgccc ccggccacta tgtcaaggtg cggctcaacc 4440 ccgtgtttgc ctgcccaaca gaggaggacg ccgcatgagc gcacaatccc tggaagtagg 4500 ccagaaggcc cgtctcagca agcggttcgg ggcggcggag gtagccgcct tcgccgcgct 4560 ctcggaggac ttcaaccccc tgcacctgga cccggccttc gccgccacca cggcgttcga 4620 gcggcccata gtccacggca tgctgctcgc cagcctcttc tccgggctgc tgggccagca 4680 gttgccgggc aaggggagca tctatctggg tcaaagcctc agcttcaagc tgccggtctt 4740 tgtcggggac gaggtgacgg ccgaggtgga ggtgaccgcc cttcgcgagg acaagcccat 4800 cgccaccctg accacccgca tcttcaccca aggcggcgcc ctcgccgtga cgggggaagc 4860 cgtggtcaag ctgccttaag caccggcggc acgcaggcac aatcagcccg gcccctgccg 4920 ggctgattgt tctcccccgc tccgcttgcc ccctttttcg gggcaatttg gcccaggccc 4980 tttccctgcc ccgcctaact gcctaaaatg gccgccctgc cgtgtaggca ttcatccagc 5040 tagaggaatt c 5051 12 2849 DNA Thiococcus pfennigii 12 ggatcctggt cgcgagcgcg ccgcccagcc acctgccggc gcgccccgcc gggaccgctc 60 gaggacgcct cgcgaaggct ctaggggctg tatcttcaag agtctacgcc cctttgttgc 120 agtgcacaaa tttccgtgct agcttcatgc tatcacgccc cagacgagga agattcaccg 180 tgaacgatac ggccaacaag accagcgact ggctggacat ccaacgcaag tactgggaga 240 cctggtcgga gctcggccgc aagaccttgg gtctggagaa gaccccggcc aatccttggg 300 ccggcgccct cgatcattgg tggcagacgg tctcgcccgc cgcccccaac gacctggttc 360 gcgacttcat ggagaagctc gccgagcagg gcaaggcctt cttcggcctc accgactact 420 tcacgaaggg cctcggcggc agtagcggta cgcagggctg ggacaccctc tcgaagacca 480 tcgacgacat gcaaaaggcc ttcgccagcg gccggatcga aggcgacgag accttccgcc 540 gcctgatggc cttctgggag atgccgctcg acaactggca gcgcaccatg tcctcgctgt 600 ccccggtgcc cggcgacctg ctgcgcaaca tgccgcacga ccaagtcagg gacagcgtcg 660 accgcatcct ctcggcaccc gggctcggct acacgcgcga ggagcaggcc cgctaccagg 720 atctgatccg ccgctcgctg gagtaccagt cggccctgaa cgaatacaac ggcttcttcg 780 gccagctcgg tgtcaagtcc ctcgagcgga tgcgcgcctt cctgcaggga caggccgaga 840 agggcgtcgc catcgagtcg gcgcgcaccc tctacgacgc ctgggtcggc tgctgcgaag 900 aggtctatgc cgaggaggtc agctccgccg actacgcgca catccacggc cgcctcgtca 960 acgcccagat ggccctcaag cagcgcatgt cgaccatggt cgacgaggtc ctcggcgcga 1020 tgccgctgcc gacccgcagc gagctgcgca cgctccagga tcggctccag gagtcgcgcg 1080 gcgagggcaa gcgccagcgc caagagatcg agacgctgaa gcggcaggtc gcggccttgg 1140 ccggcggcgc ccagcccgcg ccccaggcct ccgcccagcc cagcacccgg cccgcgccgg 1200 cgacggcccc ggcggcgagc gcggcgccca agcgcagcac cacgacccgc cgcaagacca 1260 ccaagcccac caccggccag tgatgtcggc cgcccgtcca tcgccaccag gagagagtgc 1320 cgtgtcccca ttcccgatcg acatccggcc cgacaagctg accgaggaga tgctggagta 1380 cagccgcaag ctcggcgagg gtatgcagaa cctgctcaag gccgaccaga tcgacacagg 1440 cgtcaccccc aaggacgtcg tccaccgcga ggacaagctg gtcctctacc gctaccggcg 1500 cccggcgcag gtggcgaccc agacgatccc gctgctgatc gtctacgccc tcgtcaatcg 1560 gccctacatg accgacatcc aggaggatcg ctcgacgatc aagggcctgc tcgccaccgg 1620 tcaggacgtc tatctgatcg actggggcta cccggatcag gccgaccggg cgctgaccct 1680 cgatgactac atcaacggct acatcgaccg ctgcgtcgac tacctgcgcg agacccacgg 1740 cgtcgaccag gtcaacctgc tcgggatctg ccagggcggg gccttcagcc tctgctacac 1800 ggccctgcac tccgagaagg tcaaaaacct cgtcaccatg gtcacgccgg tcgacttcca 1860 gaccccgggc aacctgctct cggcctgggt ccagaacgtc gacgtcgacc tggccgtcga 1920 caccatgggc aacatcccgg gcgaactgct caactggacc ttcctgtcgc tcaagccctt 1980 cagcctgacc ggccagaagt acgtcaacat ggtcgacctg ctcgacgacg aggacaaggt 2040 caagaacttc ctgcggatgg agaagtggat cttcgacagc ccggaccagg ccggcgagac 2100 cttccgccag ttcatcaagg acttctacca gcgcaacggc ttcatcaacg gcggcgtcct 2160 gatcggcgat caggaggtcg acctgcgcaa catccgctgc ccggtcctga acatctaccc 2220 gatgcaggac cacctggtgc cgccggatgc ctccaaggcc ctcgcgggac tgacctccag 2280 cgaggactac acggagctcg ccttccccgg cgggcacatc ggcatctacg tcagcggcaa 2340 ggcgcaggaa ggagtcaccc cggcgatcgg ccgctggctg aacgaacgcg gctgagccgg 2400 gtcgacccac ccgctcgacg ggcgcggccg gcggcatcga aggccgccgg ccggcgccca 2460 tgagccatcc gcgccgctgg cgcccgcccc ccgaccttcg ccgccgcacc cgcatcgccc 2520 ccgcggctgg cgtacaatga cggtcttcgc gagcgagccc cgcatcgtca acggaggctg 2580 catgggcgcc gaccaccaac tgctggccgc gtacgacgcg ctggccgaga cctacgacgc 2640 ccaccgcggc ctcttcgaca tgcgcgccgt gctcgaggac atcttcgccc gcctgccggc 2700 ctgcggcacc ctcctcgacc tcggctgcgg cgccggggag ccgtgcgcgc gcgccttcct 2760 cgaccgcggc tggcgggtga ccggggtgga cttctgcccg gccatgctcg ccctcgcggc 2820 gcgctacgtc cccgagatgg agcggatcc 2849 13 2768 DNA Ralstonia eutropha 13 cccgggcaag taccttgccg acatctatgc gctggcgcgc acgcgcctgg cgcgcgccgg 60 ctgtaccgag gtctacggcg gcgacgcctg caccgtggcc gacgccggtc gcttctactc 120 ctatcggcgc gatggcgtga ccggccgcat ggccagcctg gtctggctgg cggactgagc 180 ccgccgctgc ctcactcgtc cttgcccctg gccgcctgcg cgcgctcggc ttcagccttg 240 cgtcggcggc ggccgggcgt gcccatgatg tagagcacca cgccaccggc gccatgccat 300 acatcaggaa ggtggcaacg cctgccacca cgttgtgctc ggtgatcgcc atcatcagcg 360 ccacgtagag ccagccaatg gccacgatgt acatcaaaaa ttcatccttc tcgcctatgc 420 tctggggcct cggcagatgc gagcgctgca taccgtccgg taggtcggga agcgtgcagt 480 gccgaggcgg attcccgcat tgacagcgcg tgcgttgcaa ggcaacaatg gactcaaatg 540 tctcggaatc gctgacgatt cccaggtttc tccggcaagc atagcgcatg gcgtctccat 600 gcgagaatgt cgcgcttgcc ggataaaagg ggagccgcta tcggaatgga cgcaagccac 660 ggccgcagca ggtgcggtcg agggcttcca gccagttcca gggcagatgt gccggcagac 720 cctcccgctt tgggggaggc gcaagccggg tccattcgga tagcatctcc ccatgcaaag 780 tgccggccag ggcaatgccc ggagccggtt cgaatagtga cggcagagag acaatcaaat 840 catggcgacc ggcaaaggcg cggcagcttc cacgcaggaa ggcaagtccc aaccattcaa 900 ggtcacgccg gggccattcg atccagccac atggctggaa tggtcccgcc agtggcaggg 960 cactgaaggc aacggccacg cggccgcgtc cggcattccg ggcctggatg cgctggcagg 1020 cgtcaagatc gcgccggcgc agctgggtga tatccagcag cgctacatga aggacttctc 1080 agcgctgtgg caggccatgg ccgagggcaa ggccgaggcc accggtccgc tgcacgaccg 1140 gcgcttcgcc ggcgacgcat ggcgcaccaa cctcccatat cgcttcgctg ccgcgttcta 1200 cctgctcaat gcgcgcgcct tgaccgagct ggccgatgcc gtcgaggccg atgccaagac 1260 ccgccagcgc atccgcttcg cgatctcgca atgggtcgat gcgatgtcgc ccgccaactt 1320 ccttgccacc aatcccgagg cgcagcgcct gctgatcgag tcgggcggcg aatcgctgcg 1380 tgccggcgtg cgcaacatga tggaagacct gacacgcggc aagatctcgc agaccgacga 1440 gagcgcgttt gaggtcggcc gcaatgtcgc ggtgaccgaa ggcgccgtgg tcttcgagaa 1500 cgagtacttc cagctgttgc agtacaagcc gctgaccgac aaggtgcacg cgcgcccgct 1560 gctgatggtg ccgccgtgca tcaacaagta ctacatcctg gacctgcagc cggagagctc 1620 gctggtgcgc catgtggtgg agcagggaca tacggtgttt ctggtgtcgt ggcgcaatcc 1680 ggacgccagc atggccggca gcacctggga cgactacatc gagcacgcgg ccatccgcgc 1740 catcgaagtc gcgcgcgaca tcagcggcca ggacaagatc aacgtgctcg gcttctgcgt 1800 gggcggcacc attgtctcga ccgcgctggc ggtgctggcc gcgcgcggcg agcacccggc 1860 cgccagcgtc acgctgctga ccacgctgct ggactttgcc gacacgggca tcctcgacgt 1920 ctttgtcgac gagggccatg tgcagttgcg cgaggccacg ctgggcggcg gcgccggcgc 1980 gccgtgcgcg ctgctgcgcg gccttgagct ggccaatacc ttctcgttct tgcgcccgaa 2040 cgacctggtg tggaactacg tggtcgacaa ctacctgaag ggcaacacgc cggtgccgtt 2100 cgacctgctg ttctggaacg gcgacgccac caacctgccg gggccgtggt actgctggta 2160 cctgcgccac acctacctgc agaacgagct caaggtaccg ggcaagctga ccgtgtgcgg 2220 cgtgccggtg gacctggcca gcatcgacgt gccgacctat atctacggct cgcgcgaaga 2280 ccatatcgtg ccgtggaccg cggcctatgc ctcgaccgcg ctgctggcga acaagctgcg 2340 cttcgtgctg ggtgcgtcgg gccatatcgc cggtgtgatc aacccgccgg ccaagaacaa 2400 gcgcagccac tggactaacg atgcgctgcc ggagtcgccg cagcaatggc tggccggcgc 2460 catcgagcat cacggcagct ggtggccgga ctggaccgca tggctggccg ggcaggccgg 2520 cgcgaaacgc gccgcgcccg ccaactatgg caatgcgcgc tatcgcgcaa tcgaacccgc 2580 gcctgggcga tacgtcaaag ccaaggcatg acgcttgcat gagtgccggc gtgcgtcatg 2640 cacggcgccg gcaggcctgc aggttccctc ccgtttccat tgaaaggact acacaatgac 2700 tgacgttgtc atcgtatccg ccgcccgcac cgcggtcggc aagtttggcg gctcgctggc 2760 caagatcc 2768 14 1980 DNA Acinetobacter sp. 14 ctgaattcaa atcggaattt gaaaagttag ttagcgaatc tatgcctaac aataaataac 60 actgctctga aaaccatgcg ttatcaggac gaatgttacg gggaagtgtg aaaatttccc 120 cgttttagtt tcagccctgc actcaatttg attgctaaaa gccatgtgct atggagcgat 180 gaaatgaacc cgaactcatt tcaattcaaa gaaaacatac tacaattttt ttctgtacat 240 gatgacatct ggaaaaaatt acaagaattt tattatgggc aaagcccaat taatgaggct 300 ttggcgcagc tcaacaaaga agatatgtct ttgttctttg aagcactatc taaaaaccca 360 gctcgcatga tggaaatgca atggagctgg tggcaaggtc aaatacaaat ctaccaaaat 420 gtgttgatgc gcagcgtggc caaagatgta gcaccattta ttcagcctga aagtggtgat 480 cgtcgtttta acagcccatt atggcaagaa cacccaaatt ttgacttgtt gtcacagtct 540 tatttactgt ttagccagtt agtgcaaaac atggtagatg tggtcgaagg tgttccagac 600 aaagttcgct atcgtattca cttctttacc cgccaaatga tcaatgcgtt atctccaagt 660 aactttctgt ggactaaccc agaagtgatt cagcaaactg tagctgaaca aggtgaaaac 720 ttagtccgtg gcatgcaagt tttccatgat gatgtcatga atagcggcaa gtatttatct 780 attcgcatgg tgaatagcga ctctttcagc ttgggcaaag atttagctta cacccctggt 840 gcagtcgtct ttgaaaatga cattttccaa ttattgcaat atgaagcaac tactgaaaat 900 gtgtatcaaa cccctattct agtcgtacca ccgtttatca ataaatatta tgtgctggat 960 ttacgcgaac aaaactcttt agtgaactgg ttgcgccagc aaggtcatac agtcttttta 1020 atgtcatggc gtaacccaaa tgccgaacag aaagaattga cttttgccga tctcattaca 1080 caaggttcag tggaagcttt gcgtgtaatt gaagaaatta ccggtgaaaa agaggccaac 1140 tgcattggct actgtattgg tggtacgtta cttgctgcga ctcaagccta ttacgtggca 1200 aaacgcctga aaaatcacgt aaagtctgcg acctatatgg ccaccattat cgactttgaa 1260 aacccaggca gcttaggtgt atttattaat gaacctgtag tgagcggttt agaaaacctg 1320 aacaatcaat tgggttattt cgatggtcgt cagttggcag ttaccttcag tttactgcgt 1380 gaaaatacgc tgtactggaa ttactacatc gacaactact taaaaggtaa agaaccttct 1440 gattttgata ttttatattg gaacagcgat ggtacgaata tccctgccaa aattcataat 1500 ttcttattgc gcaatttgta tttgaacaat gaattgattt caccaaatgc cgttaaggtt 1560 aacggtgtgg gcttgaatct atctcgtgta aaaacaccaa gcttctttat tgcgacgcag 1620 gaagaccata tcgcactttg ggatacttgt ttccgtggcg cagattactt gggtggtgaa 1680 tcaaccttgg ttttaggtga atctggacac gtagcaggta ttgtcaatcc tccaagccgt 1740 aataaatacg gttgctacac caatgctgcc aagtttgaaa ataccaaaca atggctagat 1800 ggcgcagaat atcaccctga atcttggtgg ttgcgctggc aggcatgggt cacaccgtac 1860 actggtgaac aagtccctgc ccgcaacttg ggtaatgcgc agtatccaag cattgaagcg 1920 gcaccgggtc gctatgtttt ggtaaattta ttctaatcgg tcatataaca acagccatgc 1980 15 4936 DNA Alcaligenes latus 15 ggttcttctc gttccggcgg gaccgggtca cggggcggca ggctgccgcc gtctggctgc 60 gcggatgaag cggtgtcctc ggcgcgcttg cgcgcccgtc gccgcgccgg cgtccccagg 120 aagtacagga cgatggacaa gggcagtacg ccatacagca gcagcgtgaa caccgcgccg 180 agcaaggtgc cgttgggcgc catggcttcg gccacggcca tcatcagcac cacgtacagc 240 catgccagag caaccaagta catagcaaaa acccgcaatt acgcagaatg acgtatttcg 300 tacaatgaaa actgttgtca tgatgcggta agacacgaag cctacaacgc gatccagcaa 360 cggttttcgt gaaaaagtcc tcaggagacg

agcgtgacac tgcatcccat tcccgcactg 420 caacagcttg gcgacaacgc cacggcgctg agtgccgcca tctcggaagc gctgcgcgcg 480 atgtcgggcc tgaacctgcc gatgcaggcc atgaccaagc tgcagggcga gtacctcaac 540 gaggcgacgg cgctgtggaa ccagacgctg ggccgcctgc agcccgacgg cagcgcccaa 600 ccggccaagc tgggcgaccg gcgcttctcg gccgaggact gggccaagaa ccccgccgcg 660 gcctacctgg cgcaggtcta cctgctcaat gcccgcacgc tgatgcagat ggccgagtcc 720 atcgagggcg acgccaaggc caaggcgcgc gtgcgcttcg ccgtgcagca gtggatcgac 780 gccgcggcgc cgagcaactt cctggcgctc aatcccgagg cgcagcgcaa ggcgctggag 840 accaaggggg agagcatcag ccagggcctg cagcagctgt ggcatgacat ccagcagggc 900 cacgtgtcgc agacggacga gagcgtgttc gaggtgggca agaacgtcgc caccaccgag 960 ggcgcggtcg tgtacgagaa cgacctgttc cagctcatcg agtacaagcc gctgacgccc 1020 aaggtgcacg agaagccgat gctgttcgtg ccgccgtgca tcaacaagta ctacatcctg 1080 gacctgcagc cggacaacag cctcatccgc tacaccgtcg cccagggcca ccgggtgttc 1140 gtggtgagct ggcgcaaccc cgacgcctcc gtcgccggca agacctggga cgactacgtg 1200 gagcagggcg tgatccgcgc catccgcgtg atgcagcaga tcacggggca cgagaaggtc 1260 aacgcgctgg gcttctgcgt cggcggcacc atcctgagca cggcgctggc ggtgctggcc 1320 gcgcgcggcg agcagcccgc ggcgagcctg acgctgctga ccacgctgct ggacttcagc 1380 aacaccggcg tgctggacct gttcatcgac gaggccggcg tgcgcctgcg cgagatgacc 1440 atcggcgaga aggcgcccaa cggcccgggc ctgctcaacg gcaaggagct ggccaccacc 1500 ttcagcttcc tgcgcccgaa cgacctggtc tggaactacg tggtgggcaa ctacctcaag 1560 ggcgaggcgc cgccgccctt cgacctgctg tactggaact ccgacagcac caacatggcc 1620 gggcccatgt tctgctggta cctgcgcaac acctacctgg agaacaagtt gcgcgttccc 1680 ggtgccctga ccatctgcgg cgagaaggtg gacctctcgc gcatcgaggc gccggtgtac 1740 ttctacggtt cgcgcgagga ccacatcgtg ccctgggaat cggcctacgc cggcacgcag 1800 atgctgagcg gccccaagcg ctatgtcctg ggtgcgtctg gccacatcgc cggcgtgatc 1860 aaccccccgc agaagaagaa gcgcagctac tggaccaacg agcagctcga cggcgacttc 1920 aaccagtggc tggaaggctc caccgagcat cctggcagct ggtggaccga ctggagcgac 1980 tggctcaagc agcacgcggg caaggaaatc gccgcaccca agactcccgg caacaagacc 2040 cacaagccca tcgagcccgc ccccgggcgt tacgtgaagc agaaggcctg agccgcggcc 2100 cctgagcctt ctttaacccg accttgacaa acgaggagat aagcatgacc gacatcgtca 2160 tcgtcgccgc agcccgcacc gccgtgggca agttcggcgg cacgctggcc aagacccccg 2220 ctccggagct gggcgccgtg gtcatcaagg ccctgctgga gaagacgggc gtcaagcccg 2280 accagatcgg tgaagtcatc atgggccagg tgctggccgc cggcgcgggc cagaaccccg 2340 cgcgccaggc gatgatgaag gcgggcatcg ccaaggaaac gccggcgctg accatcaacg 2400 ccgtgtgcgg ctccggcctc aaggccgtga tgctggccgc ccaggccatc gcctggggcg 2460 acagcgacat cgtcatcgcc ggcggccagg agaacatgag cgccagcccg cacgtgctga 2520 tgggcagccg cgacggccag cgcatgggcg actggaagat ggtcgacacc atgatcaacg 2580 acggcctgtg ggacgtgtac aacaagtacc acatgggcat cacggccgag aacgtcgcca 2640 aggaacacga catcagccgc gaccagcagg acgccctggc cctggccagc cagcagaagg 2700 ccaccgccgc gcaggaagcc ggccgcttca aggacgagat cgttccggtc tcgatcccgc 2760 agcgcaaggg cgacccggtg ctgttcgaca ccgacgagtt catcaacaag aagaccaccg 2820 ccgaagcgct ggcgggcctg cgcccggcct tcgacaaggc cggcagcgtg accgcgggca 2880 acgcctcggg catcaacgac ggcgccgctg cggtgatggt gatgtccgcc gccaaggcga 2940 aggagctggg cctgacgccc atggcgcgca tcaagagctt cggcaccagc ggcctggatc 3000 cggccaccat gggcatgggc ccggtgccgg cctcgcgcaa ggcgctggag cgcgccggct 3060 ggcaggtcgg tgacgtggac ctgttcgagc tcaacgaagc cttcgccgcc caggcctgcg 3120 cggtgaacaa ggagctgggc gtggatccgg ccaaggtcaa cgtcaacggc ggtgccatcg 3180 ccatcggcca ccccatcggc gcctccggct gccgcgtgct ggtgacgctg ctgcacgaga 3240 tgcagcgccg ggacgccaag aagggcctgg ccgcgctgtg catcggcggc ggcatgggcg 3300 tgtcgctgac cgtcgagcgc tgatcagaag aaccgggcgg ccccgcgccg cccgcccggc 3360 gttccacgcg ggtgcgccgg gataccagac gaaccaaacc accaagggct tcgagacggc 3420 ccgaagaagg agagacagat ggcacagaaa ctggcttacg tgaccggcgg catgggcggc 3480 atcggcacct cgatgtgcca gcgcctgcac aaggacggct tcaaggtgat cgccggctgc 3540 ggtccgagcc gcgaccacca gaagtggatc gatgaacagg ccgcgctggg ctataccttc 3600 tacgcctccg tgggcaacgt ggccgactgg gactccaccg tggccgcctt cgagaaggtc 3660 aaggccgagc acggcaccgt ggacgtgctg gtgaacaacg ccggcatcac gcgtgacggg 3720 cagttccgca agatgagcaa ggccgattgg caggccgtga tgtcgaccaa cctcgacagc 3780 atgttcaacg tcaccaagca ggtgatcgag ggcatgctgg acaagggctg gggccggatc 3840 atcaacatct cctcggtcaa cggcgagaag ggccagttcg gccagaccaa ctactccgcc 3900 gccaaggccg gcatgcacgg cttctcgatg gcgctggcgc aggaagtggc ggccaagggc 3960 gtgacggtga acaccgtgag cccgggctac atcgccacgg acatggtcaa ggccatccgc 4020 caggacgtgc tggacaagat catcgccacc attcccatcc gtcgcctggg tacgccggag 4080 gagatcgcct ccatcgtcgc ctggctggcc ggcgaggagt cgggcttcac caccggtgcc 4140 gacttcagct gcaacggcgg cctgcacatg ggctgaggcc cgcggctcca tgcccacctg 4200 cgtgggcatg gacgggccga aggacccgag ctctgcgagg gtgcggcctg caaggctgag 4260 gcctgctgcg ccgcgtgccc gcgagggcac gtgccgaagc accaaaaggc cgcgcattgc 4320 gcggcctttt cctttctgga tcggtgcgga cgggtgccgc gtcaggcagg gcagggcccc 4380 cgggccttca ctccaccatg cccgacatga agtacttgat cagccccttg gccgcgaagc 4440 ccagcatgcc gaagcccagc gccaggaaca gcacgaaggt gccgaacttg ccggccttcg 4500 actcgcgcgc gagctgaaag atgatgaatg ccatgtagag catgaaggcc gtgacgccga 4560 cggtcaggcc cagctgggca atgttttcct cgttgatttc gaacatcgtt tgttgtctca 4620 ggctgctgca cgcggctgac gtgctcgccg cgcggccggg ccccaactgc ccgcagcggt 4680 tctcgatcag gttctcaagg catctcgtgc cactgggagg tgtccaccag gtcgcggtag 4740 gcgtgccagc tcgaatgcgc cagccacggc actaccacga tcaggcccag cagcagcgtg 4800 gccatgccca gcagcgtcag cgccatgatc agcgccgccc acagcgccag cggcagtggg 4860 tgctgcatca ccacgcgcca gctcgtgagc accgccacca gcacgcccac gtggcggtcc 4920 agcagcatcg ggatcc 4936 16 1752 DNA Azorhizobium caulinodans 16 atggaggcgt tcgcccagaa ccttgcgaag atggtggagg aaggcggcaa ggccgtcgcc 60 gcttacatgc gtccgcgcga ggagggcaag ccggacgaca tgacggacga tatcgccgat 120 gcgctcaaga ccatcggcga ggtggccaat tactggatgt ccgatccaaa gcgctccttc 180 gaggcccagt cgcgcctcat gatgggctac atgggcgtct gggcgggggc gctccagaag 240 ctctccggcg agaaggccga gcccatcgcg aaggccgacc ccaaggacgg ccgcttcaag 300 gacccggaat gggaaagccc gttcttcgac gccctgaagc agacctatct cgtgaccagc 360 aactgggccg agtccatggt caaggaggcc gaggggctcg atccccacac caagcacaag 420 gccgaattcc tggtgcgcca gctctcgaat gcggtggcgc cctcgaactt cctcatgacc 480 aacccggagc tgatccgcga aacgctctcc tccagcggcg agaacctcgt gcgcggcatg 540 aagaatctgg ccgaggatct ggtggagggc aaaggcgatc tcaagatccg ccagacggac 600 atgagcgcct tcgaggtggg ccgcaatctg gcgctcagcc ccggcaaggt catcttcgag 660 accgagctga tgcagctcat ccagtatgcg ccctcgacgc ccagcgtgaa gaagacgccg 720 gtgctgatcg tgccgccctg gatcaacaag ttctacatcc tcgacctgac gcccgagaaa 780 tccctcatca agtggatggt ggatcagggg ctgacggtct tcgtcatctc gtgggtcaat 840 ccggacgccc gtctcgccga caagggcttc gacgactaca tgcgcgatgg catcttcgcc 900 gcgctcgatg cggtggagaa ggcgaccggc gagcatcagg cgcacaccat cggctattgc 960 gtgggcggca cgctgctcgc ggtcacgctt gcctacatgg cggcgaccgg cgatgaccgg 1020 gtggcgagct ccaccttcct caccacccag atcgacttca cccacgcggg cgatctcaag 1080 gtcttcgtgg acgaggcgca gctctcggtc atcgagcgtc gcatgaagga gatgggctat 1140 cttgaaggac gcaagatggc cgacgccttc aacatgctgc ggtccaacga cctgatctgg 1200 ccctatgtgg tgaacaatta tctcaagggg aagcagccgt tccccttcga tcttctgttc 1260 tggaatgccg attccacccg catgccggcg gcgaaccact cctattacct gcgcaactgc 1320 tatctccaga acaacatcgc caaggggctg gcggagatcg cgggcgtcaa gatcgacatg 1380 ggcaaggtga cgatcccggt ctattcgctc gccacccgcg aggaccatat cgcgccgccc 1440 aactccgctt atatcggtgc aggcctgctc ggcggccccg tgcgcttcgt gctggccggg 1500 tccggccata ttgcgggcgt ggtcaatccg ccggtgaagc acaagtacca gtactggacc 1560 ggcggtccga ccggcgggga ctatgacgtc tggctgaagg gggcgcagga gcacaagggc 1620 tcgtggtggc cggattgggc gcagtggttc agcgccctgc atccggacga ggtccctgcc 1680 cgcgagccag gcggcagcgc gttcaatccc atcgaggatg cccccggccg ctacgtacgc 1740 gagaagtcct ga 1752 17 3973 DNA Comamonas acidovorans 17 cccgggcaag cggcgcagca gaccctggcc tggctgggac cctgcatcgg tccgcgctcc 60 ttcgaagtgg gagccgaggt gcgcgcggcc ttcatcgcgc atgacgcggc ggccggggcg 120 catttcaccg ccgtggcgca agagggcggg ggcgcaccca agtacctggc caacctggcc 180 gggcttgcgc gccagcgcct ggcggcgctg ggcatcaccg ccatctacgg caatgacggc 240 ggcgacgcat ggtgcacggt gctcaacggc tcacggttct tttcgcaccg gcgcgacgct 300 gcccgcctcg gcagcagcgg gcggttcgcc gcctgcatct ggaaggactg agcgcgcctg 360 cgtgccgggg ccgttttgcg aggcctgctg ctgcgcgagc caggcctcgc gctcggccaa 420 ttcgcgtgca tggcgttttc gcttgcgcgc gggcgtgccc atgatgtaga tcacgatcga 480 cagcggcagc agtccgtaga gtgcgaacgt gatcacggcg ccgagtatgc tgcccgtggg 540 gctggcggcc tcggccactg ccatcatcag agtgacatac agccaggcga taacaacgag 600 atacatggtc gaaccaaaaa aacgagcggg gcacgggttt tcgtggaggt ctgtgccggg 660 caaggcgggg ctcgcctggg tcacaatcaa aacggatgcc ccgcaagccc caggctattg 720 catggcatcc acgaacaagg atgcagcatg aattttgacc cactcgctgg cctgtcaggc 780 caatccgtcc agcaattctg gaacgagcaa tggagccgca cgctgcagac actgcagcag 840 atggggcaac caggccttcc gggaatccag ggcatgccag gcatgccgga catggcccag 900 gcctggaaag cggccgtccc cgaacccggc gccctgcccg agaacgcgct gtcgctggac 960 cccgaaaagc tgctggagct gcagcgccag tacctggatg gcgccaaggc catggccgaa 1020 cagggcggcg cccaggcgct gctggccaag gacaagcgct tcaataccga atcgtgggcc 1080 ggcaacccgc tgacggcggc cacggccgcc acctatctgc tcaacagccg catgctcatg 1140 ggcctggccg atgccgtgca ggccgatgac aagacgcgca accgcgtgcg cttcgccatc 1200 gagcaatggc tggcggccat ggcgcccagc aacttcctgg cactcaatgc cgaggcccag 1260 aagaaggcca tcgaaaccca gggcgagagc ctggcccagg gcgtggccaa cctgctggcc 1320 gacatgcgcc agggccatgt gtccatgacc gacgagagcc tgttcaccgt gggcaagaac 1380 gtcgccacca ccgaaggcgc cgtggtgttc gagaacgagc tgttccagct catcgaatac 1440 aagccgctga cggacaaggt gcacgagcgg cccttcctca tggtgccgcc ctgcatcaac 1500 aagttctaca tcctggacct gcagcctgac aactcgctga tccgctacgc cgtcagccag 1560 ggccatcgca ccttcgtgat gagctggcgc aaccccgacg aaagcctggc gcgcaagacc 1620 tgggacaact acatcgagga cggcgtgctc accggcatcc gcgtggcgcg cgagatcgcg 1680 ggagccgagc agatcaatgt gctgggcttt tgcgtgggcg gcaccatgct gtccaccgcg 1740 ctggcggtgc tgcaggcacg ccacgaccgc gagcatggcg cagttgctgc tccggcggcc 1800 aaggcgcctg cggccaagcg cgcggctggc agccgcagcg ccgcccgcac atccacagcc 1860 cgcgccacgg cgccggccgg cgtgccgttt cccgtggcca gcgtcacgct gctgaccacc 1920 ttcatcgact tcagcgacac cggcatcctc gatgtcttca tcgacgaatc cgtggtgcgc 1980 ttccgcgaga tgcagatggg cgagggcggc ctcatgaagg gccaggacct ggcgtccacc 2040 ttcagctttc tgcggcccaa cgacctggtc tggaactacg tggtgggcaa ctacctcaag 2100 ggcgagacgc ccccgccgtt cgacctgctg tactggaaca gcgactccac caacctgccc 2160 ggcccctact acgcctggta cctgcgcaac ctctacctgg aaaacaggct ggcccagccc 2220 ggcgcgctga ccgtctgcgg cgagcgcatc gacatgcacc agctgcgcct gccggcctat 2280 atctacggct cgcgcgagga ccacatcgtg cccgtgggcg gctcctatgc gtccacccag 2340 gtgctgggcg gcgacaagcg ttttgtgatg ggcgcgtcgg gccacatcgc gggcgtgatc 2400 aatccgccgg ccaagaaaaa gcgcagctac tggctgcgcg aggacggcca gctgcccgcc 2460 acgctcaagg agtggcaggc cggcgccgac gagtacccgg gcagctggtg ggccgactgg 2520 agcccctggc tggccgagca cggcggcaag ctggtcgcgg cgccaaagca gtacggcaag 2580 ggcagggaat acacggccat cgagccggcc ccgggccgct acgtactggt caaggcctga 2640 ggcacaatcg tttcgatgct gcagcgcaat aacgcttcgg cttgaatgca gggtgccgct 2700 gccgcgactg cggccgcgtg cgcccaggca tcccttcaag agtccaaaga aaggtccgtc 2760 atcatggaag acatcgtcat cgtttccgct gtccgtactg ccgttggcaa gtttggcggc 2820 acccttgcca agatccccgc caccgaactg ggctccatcg tgatccgcga ggcactgaac 2880 cgtgccaagg tcggcaccga tcaggtcggt gaagtcatca tgggccaggt gctggccgct 2940 ggcgcaggcc agaaccccgc acgccaggcc atgatgaagg ccggcgttgc caaggaaacc 3000 ccggccctca ccatcaacgc cgtctgcggc tcgggcctga aggccgtgat gctggcagcc 3060 caggccgtgg ccacgggcga cagcgagatc gtcgtggccg gcggccagga gaacatgagc 3120 ctgtcgcccc acgtgctgcc gggctcgcgc gacggccagc gcatgggcga ctggaagatg 3180 gccgacacca tgatcgtgga cggcctgtgg gacgtctaca accagtacca catgggcatc 3240 acggccgaga acgtggccaa ggaaaagagc gtcagccgcg agcagcagga tgccctggcc 3300 ctggccagcc agcaaaaggc cacggccgcg caggacgccg gcaagttcgt ggacgaaatc 3360 gttcccgtca gcattcccca gcgcaagggc gatcccgtgg tgttcgctgc cgacgagtac 3420 atcaaccgca agaccaatgc cgacgccctg gcaggcctgc gcccggcctt cgacaaggcc 3480 ggctcggtga cggccggcaa cgcctcgggc ctgaacgacg gcgcggccgc cgtggtggtg 3540 atgagcgcct ccaaggccaa ggccctgggc ctgacgccgc tggcgcgcat cgtctcctac 3600 gccaccagcg gcctggaccc cgccaccatg ggcctgggcc cggtgttcgc ctcgcgcaag 3660 gcgctggagc gtgcgggctg gaccgcgcag gacgtggacc tgttcgagct gaacgaagcc 3720 ttcgcggccc aggcctgtgc ggtgaaccag gagctgggca tcgacccggc caaggtcaac 3780 gtcaacggcg gcgccattgc catcggccac cccatcggcg catcgggcgc ccgcatcctg 3840 gtgaccctgc tgcacgagat gcagcgcagc ggcgccaaga agggcctggc cggcctgtgc 3900 atcggcggcg gcatgggcgt ggccatggcg gtggagcgcg tttgagaggc tccgggaaaa 3960 cgttagcaag ctt 3973 18 3690 DNA Methylobacterium extorquens 18 cgtgtccggg ttgaggctca ccccgaagcg ccgctggtag tagccggcct gggcgcggcg 60 caggccggca atgcccttgg aggcggagta gcggtcggtg cgcggcttgc cggccgtctc 120 gacgagcttc tcgatgacgt ggcggggcgc gtcgagatcg ggattgccca tgccgagatc 180 gatgatgtcg gcgccgcggg ctcgggcggc ggccttgatc cggttcacct gctcgaagac 240 gtaaggcgga aggcgcttga tgcggtggaa atcggtcatg gcgttggtct gtccggtgtc 300 gcggaactga tcctcccgcg aggatgcgtc ggcggtgagg agcctgcctg tttaccatcg 360 acgcgttcac gcgccgactt aaaaatcaac atcgcgaggc cccgccacga tcggggtgga 420 acgatcactc caccgggttg ggcgcgagcc cctgcgcggc attcttggcc gccccttccg 480 ccgcgcgccc tttggactcg ttgcggttgc gcgcgccttc cagctcggcc tgcagagcgc 540 tttgtccggc cgaattgcgg gcgcggacgg cgcgccgggg cgccgattcg cctaccggca 600 tgtagctcgc atcgctcttg cgggattgcg gccacgaaat cgggcgcctc caccggcttg 660 atgccggagc ccgccccgac catcgccgcc ttcagcggat tcacgtcgcc ggagcagccg 720 ccggtcgccg ccgcggcggc gaaaaccggc aaggcgatgg cgatggcgcg gacggtgcgg 780 cgcacgttca tgacaacggt ctgaaaatgg agtctgggat gggcgacttc tgctgggtgg 840 agcgttaatt tgtcggaaac gaggctcggg ccgccccacg gggcgggatg cgggcagacc 900 tttgggagga cgtgtcgcgt gggcaccgag cggacgaacc cggcagcgcc ggatttcgag 960 accatcgcgc gcaacgcgaa tcagctcgcg gaggtgttcc ggcaatcggc cgccgcctcg 1020 ctgaagccgt tcgagccggc gggccaggga gccctgctcc cgggcgcgaa cctccagggc 1080 gccagcgaga tcgacgagat gacccggacc ctcacgcggg tcgcggagac atggctgaag 1140 gatcccgaga aggcgcttca ggcccagacc aagctcggcc agtccttcgc cgcgctctgg 1200 gcctcgaccc tgacccggat gcagggggcc gtcaccgagc ccgtcgtcca gcccccgccc 1260 acggacaagc gcttcgccca tgccgattgg agcgcgaacc cggtcttcga cctgatcaag 1320 cagagctacc tgctccttgg ccgctgggcc gaggagatgg tcgagacggc cgaaggcatc 1380 gatgagcaca cccgccacaa ggcggagttc tacctgcgcc agctcctctc ggcctactcg 1440 ccctcgaact tcgtgatgac gaaccccgag ctcctgcgcc agacgctgga ggaggggggc 1500 gccaacctga tgcgcggcat gaagatgctg caggaggatc tggaagccgg cggcggtcag 1560 ctccgggtgc ggcagacgga cctgtccgcc ttcaccttcg gcaaggacgt ggcggtgacc 1620 cccggcgagg tcatcttccg caacgatctg atggagttga tccagtacgc gcccacgacc 1680 gagacggtgc tgaagcgtcc gctgctgatc gtgccgccct ggatcaacaa gttctacatc 1740 ctcgatctca acccgcagaa gagcctcatc ggctggatgg tgtctcaagg gatcacggtg 1800 ttcgtgatct cctgggtgaa cccggacgag cgccaccgcg acaaggactt cgagtcctac 1860 atgcgggaag gcatcgagac cgccatcgac atgatcggcg tggcgaccgg cgagaccgat 1920 gtcgcggcgg cgggctactg cgtcggcggc acgctgctcg ccgtcacgct ggcctaccag 1980 gcggcgaccg gcaaccgccg gatcaagagc gcgaccttcc tcaccacgca ggtcgatttc 2040 acccatgcgg gcgatctcaa ggtcttcgcc gacgaggggc agatcaaggc gatcgaggag 2100 cggatggccg agcacggcta cctggagggc gcgcgcatgg ccaacgcctt caacatgctc 2160 aggcccaacg acctgatctg gtcctacgtc gtcaacaact acgtacgcgg caaggcgccg 2220 gccgccttcg acctgctcta ctggaacgcc gacgccacgc ggatgcccgc ggccaaccac 2280 tcgttctacc tgcgcaactg ctacctcaac aacacgctcg ccaaggggca gatggtgctc 2340 ggcaacgtgc gcctcgacct caagaaggtg aaggtgccgg tcttcaacct cgccacccgc 2400 gaggaccaca tcgccccggc gctctcggtc ttcgaagggt cggccaagtt cggcggcaag 2460 gtcgattacg tgctggcggg ctcgggccac atcgccggcg tcgtcgcccc gccgggcccc 2520 aaggccaaat acggctttcg caccggcggc ccggcccgag gccggttcga ggattgggtc 2580 gcggcggcga cggagcatcc gggctcgtgg tggccctact ggtacaagtg gctcgaggag 2640 caggcgcccg agcgcgtgcc cgcccgcatt cctggaacgg gggccctgcc ttccctggcg 2700 ccagcaccgg gcacctatgt ccgcatgaag gcgtgagggc atgaaggtgt gagggatcga 2760 caggaaccgt gcacgcactg catcgctgtt gcggacacgg gaccgtgatt tgccttatct 2820 gatcctgggg ccggctcctg ccccaggagg atagaagcgc gttgcaacac gccaccgatc 2880 tcatttcgat catcgcgctg gggctggtct gcgcgttcat cggcggcatg ctggcccagc 2940 gaatgcggct tcccccgctc gtcggctacc tcgtcgccgg catcgccatc ggcccgttca 3000 cgccgggttt cgtcggcgat ccggctttag cgagccagct cgccgaactc ggcgtcatcc 3060 tgctgatgtt cggcgtcggg cttcacttct ccatcggtga cctactcgcg gtccgcacca 3120 tcgccctgcc cggtgcgatc gtgcagatca ccgtggcgac cgccatgggc gcgggtctcg 3180 cctggggctt cggctggggg gccggggcgg gcctcgtgtt cgggctggcg ctctcggtgg 3240 cgagcactgt cgtgctgttg cgggcgctcg aagggcaggg gcttctcgac tcggacaagg 3300 gccgcatcgc cgtcggctgg ctgatcgtcg aggatctcgc catggtggtg gccctggtgc 3360 tgctgccggc gttggcgccc tcgctcggcg gcgaggcgat gcaggccgcc ggccatcacg 3420 gcccgccgga gcacggcctg tgggtgacgc tcgggctgac gctcgccaag gtcggcgtgt 3480 tcgtcgccgt gatgcttgtc ggcgggcggc gcctcattcc ctacctgctg ggtctggccg 3540 cccgcaccgg ctcgcgcgag ctgttcaccc tggccgtgct ggcgagcgcc gtcggtatcg 3600 ccttcgcctc ctccgagctg ttcggcgtgt ccttcgccct cggcgcgttc ttcgccggga 3660 tggtgctcgc cgaatccgac ctcagccatc 3690 19 2712 DNA Paracoccus denitrificans 19 gggcccgcgc gctggcgccc atccattcat tggtgttgcg gatcgtctcc atcgcatcat 60 agagagatga tccctctcaa acccttgtac gtcgtcattg caaccttctc ccatgaaccc 120 gccggattcg gccttgccgg ggaaggacac ggctttacct gcgtattgtg ctgggcataa 180 tgattttacg gttcagggag gattgtcaat tatggcgggc aaggacgaaa aaccggaagc 240 tgcgggcgcg gccgaagccg ggggcaaacc acgcaggggc cgcggggcag gggccaaggg 300 ccgtaccggg gacagcgaaa caggagccga agcctgccga ttcggaaaag ccggcgcgac 360 cgcggcgcga aaaggccgcg gtgaagaccc cgcccgagcc cgtgccggag cccgcggcgc 420 ctgccaggtc caaggcggcc gcggcctccg atcccgccaa gcctgccgcc aaggccggca 480 cacgcaaacg ccgcgttgcg cgcgggcggg ggccggtcgc agcgatccgc gccccggtcc 540 gcgccgaaag ccctcgggca aggccgccgc gaaggcgcag gccgagaccg cctcgctcag 600 tatggccgac gaggcgctgc gtccgctggg cgccgtcgga ccgggcggcg gcgtgccgcc 660 catggccgcg ccccgcgccc aggctgccgc cccggcgggg accggccagt ctgccggact 720 cgctgccgag ccgcatccag cccgaacacc gccgccttcg tcgaggcggc

cttcggtccc 780 ggcagccgcc tcccaacagc tggcccagaa catcgagcgc atcgaatcgc tgacccagcg 840 cctgatcagc gcgctggcgc agcgccgtcc ctcgaatccc ggcgtcgaga tgccgggccc 900 cgaccttttc gccaccgcga cctcggcctg gatcaagctt ctggccgagc agcccgagcg 960 ggtgatcggc cagcaggtca gctattgggg cgaaaccttg cgccatttcg ccgaggccca 1020 ggccgccttt gcccgcggca ccgtgacgcc gccgcccagc gaagggccgc gggaccggcg 1080 ctttgccaac ccgctgtggg aggcgcatcc cttcttcaac ttcatcaagc ggcaatacca 1140 gatcaacgcc caggccctgc aggaggcggc cagcacactg gacctgcccg agatgaccga 1200 ccggcgccgg atcgaatggt tcacccgcca gatgatcgac atgatggcgc cgacgaattt 1260 tctggccacc aatcccgacg acagctggaa aaggcgctgg agaccgaggg acgaaagcct 1320 ggtcaggggc cttgagaacc tggtgcgcga cgtcgagcag aacagcggcg agctgatcgt 1380 gtcgctggcc gaccgcgatg ccttccgtgt gggcgagaac atcggcacca ccgagggcac 1440 ggtggtcgcg cgcaccaagc tttacgagct gatccagtac aagcccacca ccgcgcaggt 1500 gcatgagatc ccgctggtga tctttccgcc ctggatcaac aaattctaca tcctcgacct 1560 caagccgcag aacagcctga tcaaatggat cgtggaccag ggccatacgc tgttcgtggt 1620 ggcctggaag aaccccgacc ccagctatgg cgacaccggc atggacgatt acgtcagcgc 1680 ctatctggag gtgatggacc gggttctgga tctgaccgac cagaaaaagc tgaatgcggt 1740 gggctattgc atcgccggca ccaccctggc gctgacccct gtcgtgctga agcagcgcgg 1800 cgacgaccgg gtgaacgcgg ccaccttctt caccgcgctg accgatttcg ccgaccaggg 1860 cgagttcact gcctatctgc aggaggattt cgtctcaggc atcgaggagg aggcggcgcg 1920 gaccggcatc ctgggcgcgc agctgatgac gcgcaccttc agcttcctgc gcgccaacga 1980 cctggtctgg gggccggcga tccgcagcta catgctgggc gagacgccgc cggccttcga 2040 cctgctgttc tggaacggcg acggcaccaa cctgcccggg cgcatggccg tggaatacct 2100 gcgcggcctg tgccagcaga accgcttcgt caaggagggg ttcgatctga tgggccaccg 2160 cctgcatgtc ggcgacgtga ccgtgccgct ttgcgccatc gcctgcgaga ccgaccatat 2220 cgcgccctgg aaggacagct ggcgcggcat cgcgcagatg ggctccaggg acaagacctt 2280 catcctgtcc gaatcgggcc atatcgccgg catcgtcaac ccgcccagca agaagaaata 2340 cggccattat acctcggacg ccggtttcgg tcagggcgag cagcactggc tggacaaggc 2400 cagccatcac gagggcagct ggtggggccg ctggggcgaa tggctggccc ggcgggcggg 2460 gggcatggtc gatgcccgcg acccgggcga gggcttcggc cctgcgccgg gcctttacgt 2520 ccacgagcgg gcgtaaaatt tttctgcacc gcagcaagaa aaccttgcaa tgctgcggtg 2580 cagaaagtat atgttggaca gaacagttca accggtgccg aagcttcgcg ccgacctagg 2640 agagagcaaa tggccaagac ccctgacttt accaagacca tgcaagaagt tatggcgaaa 2700 ttcccggtcg ac 2712 20 2587 DNA Zoogloea ramigera 20 gtcgacctgg gcaaggtcgg gccgcagcaa gtgcccgagg cggacgccag catgaccacc 60 atcgccggca aggtgtgctg gtgatgacgg ccgactgcct gccggtgctg ttttgcgaca 120 cgcgcggcac cgttcgtggc tgccgcccac gccggtcggc gcggcctggc cgctggcgtg 180 ctggaaaaca cctacgcgag cagtgcgcgt ccgcggcgcc ggcgactgat ggcctggatg 240 gggccggcca tcggcgccta ccagttcgag gtgggcggcg atgtgcgcca ggcctttctg 300 caaaccgcac ctgacgattc aagcgtgcgg cacgtgacgg cagcgtttac gccactcgaa 360 caccggccgg gcaagttcct ggccgacatc tacgcgctgg cccggcatcg catgctgcgc 420 gccggcgtgg cgcaagtgca tggcggcgaa tactgcacgg tggccaattc cgggcgcttc 480 tattcgttcc gtcgcgacgg cgtcaccgga cgccaggcaa gtttgatctg gctcaaataa 540 gtatcaggta gggggcgctg gactttttag cgtctcgcgc cgcctgtcgc gctttgcggc 600 gtgctttctc ttgcccgttc ccgcgatgta aatcaaggtc cccagcggca gcacgcaata 660 gaacaaaaac gcaatagcaa caatcagcat gtagcaaccc aggttaagag atattcaata 720 tttttttagg gaatcacaca tgaatttgcc cgatccgcaa gccattgcca acgcctggat 780 gtcccaggtg ggcgacccca gccaatggca atcctggttc agcaaggcgc ccaccaccga 840 ggcgaacccg atggccacca tgttgcagga tatcggcgtt gcgctcaaac cggaagcgat 900 ggagcagctg aaaaacgatt atctgcgtga cttcaccgcg ttgtggcagg attttttggc 960 tggcaaggcg ccagccgtcc agcgaccgcg cttcagctcg gcagcctggc agggcaatcc 1020 gatgtcggcc ttcaatgccg catcttacct gctcaacgcc aaattcctca gtgccatggt 1080 ggaggcggtg gacaccgcac cccagcaaaa gcagaaaata cgctttgccg tgcagcaggt 1140 gattgatgcc atgtcgcccg cgaacttcct cgccaccaac ccggaagcgc agcaaaaact 1200 gattgaaacc aagggcgaga gcctgacgcg tggcctggtc aatatgctgg gcgatatcaa 1260 tatgctgggc gatatcaaca acggccatat ctcgctgtcg gacgaatcgg cctttgaagt 1320 gggccgcaac ctggccatta ccccgggcac cgtgatttac gaaaatccgc tgttccagct 1380 gatccagtac acgccgacca cgccgacggt cagccagcgc ccgctgttga tggtgccgcc 1440 gtgcatcaac aagttctaca tcctcgacct gcaaccggaa aattcgctgg tgcgctacgc 1500 ggtggagcag ggcaacaccg tgttcctgat ctcgtggagc aatccggaca agtcgctggc 1560 cggcaccacc tgggacgact acgtggagca gggcgtgatc gaagcgatcc gcatcgtcca 1620 ggacgtcagc ggccaggaca agctgaacat gttcggcttc tgcgtgggcg gcaccatcgt 1680 tgccaccgca ctggcggtac tggcggcgcg tggccagcac ccggcggcca gcctgaccct 1740 gctgaccacc ttcctcgact tcagcgacac cgggtgctcg acgtcttgtc gagaaaccca 1800 ggtcgcgctg cgtgaacagc aattgcgcga tggcggcctg atgccgggcc gtgacctggc 1860 ctcgaccttc tcgagcctgc gtccgaacga cctggtatgg aactatgtgc agtcgaacta 1920 cctcaaaggc aatgagccgg cggcgtttga cctgctgttc tggaattcgg acagcaccaa 1980 tttgccgggc ccgatgttct gctggtacct gcgcaacacc tacctggaaa acagcctgaa 2040 agtgccgggc aagctgacgg tggccggcga aaagatcgac ctcggcctga tcgacgcccc 2100 ggccttcatc tacggttcgc gcgaagacca catcgtgccg tggatgtcgg cgtacggttc 2160 gctcgacatc ctgaaccagg gcaagccggg cgccaaccgc ttcgtgctgg gcgcgtccgg 2220 ccatatcgcc ggcgtgatca actcggtggc caagaacaag cgcacgtact ggatcaacga 2280 cggtggcgcc gccgatgccc aggcctggtt cgatggcgcg caggaagtgc cgggcagctg 2340 gtggccgcaa tgggccgggt tcctgaccca gcatggcggc aagaaggtca agcccaaggc 2400 caagcccggc aacgcccgct acaccgcgat cgaggcggcg cccggccgtt acgtcaaagc 2460 caagggctga ccacggggaa ctgccagact accggactcg gctattggaa gcgttgcaat 2520 aaatgccagc actattgcga gtttctgcaa tctttctact ttcccctcta taataggatc 2580 cgtcgac 2587 21 3187 DNA Aeromonas caviae 21 agatctggac cggggtgctg gcctgggcca cgccggcgag ggccagcgcg gagcaaccga 60 gcagcagggc gagaggtttc atcgggattc cttggcagtc tgaatgacgt gccagcctat 120 cagcgcggcg ccggtgcggc gagggcgcgc cggacccagt gcgtcacctc tcgtctgatc 180 cgcctccctc gacgggcgtc gctgacaaaa aaattcaaac agaaattaac atttatgtca 240 tttacaccaa accgcatttg gttgcagaat gctcaaacgt gtgtttgaac agagcaagca 300 acacgtaaac agggatgaca tgcagtaccc gtaagaaggg ccgattggcc cacaacaaca 360 ctgttctgcc gaactggaga ccgatgatga atatggacgt gatcaagagc tttaccgagc 420 agatgcaagg cttcgccgcc cccctcaccc gctacaacca gctgctggcc agcaacatcg 480 aacagctgac ccggttgcag ctggcctccg ccaacgccta cgccgaactg ggcctcaacc 540 agttgcaggc cgtgagcaag gtgcaggaca cccagagcct ggcggccctg ggcacagtgc 600 aactggagac cgccagccag ctctcccgcc agatgctgga tgacatccag aagctgagcg 660 ccctcggcca gcagttcaag gaagagctgg atgtcctgac cgcagacggc atcaagaaaa 720 gcacgggcaa ggcctgataa cccctggctg cccgttcggg cagccacatc tccccatgac 780 tcgacgctac gggctagttc ccgcctcggg tgtgggtgaa ggagagcaca tgagccaacc 840 atcttatggc ccgctgttcg aggccctggc ccactacaat gacaagctgc tggccatggc 900 caaggcccag acagagcgca ccgcccaggc gctgctgcag accaatctgg acgatctggg 960 ccaggtgctg gagcagggca gccagcaacc ctggcagctg atccaggccc agatgaactg 1020 gtggcaggat cagctcaagc tgatgcagca caccctgctc aaaagcgcag gccagccgag 1080 cgagccggtg atcaccccgg agcgcagcga tcgccgcttc aaggccgagg cctggagcga 1140 acaacccatc tatgactacc tcaagcagtc ctacctgctc accgccaggc acctgctggc 1200 ctcggtggat gccctggagg gcgtccccca gaagagccgg gagcggctgc gtttcttcac 1260 ccgccagtac gtcaacgcca tggcccccag caacttcctg gccaccaacc ccgagctgct 1320 caagctgacc ctggagtccg acggccagaa cctggtgcgc ggactggccc tcttggccga 1380 ggatctggag cgcagcgccg atcagctcaa catccgcctg accgacgaat ccgccttcga 1440 gctcgggcgg gatctggccc tgaccccggg ccgggtggtg cagcgcaccg agctctatga 1500 gctcattcag tacagcccga ctaccgagac ggtgggcaag acacctgtgc tgatagtgcc 1560 gcccttcatc aacaagtact acatcatgga catgcggccc cagaactccc tggtcgcctg 1620 gctggtcgcc cagggccaga cggtattcat gatctcctgg cgcaacccgg gcgtggccca 1680 ggcccaaatc gatctcgacg actacgtggt ggatggcgtc atcgccgccc tggacggcgt 1740 ggaggcggcc accggcgagc gggaggtgca cggcatcggc tactgcatcg gcggcaccgc 1800 cctgtcgctc gccatgggct ggctggcggc gcggcgccag aagcagcggg tgcgcaccgc 1860 caccctgttc actaccctgc tggacttctc ccagcccggg gagcttggca tcttcatcca 1920 cgagcccatc atagcggcgc tcgaggcgca aaatgaggcc aagggcatca tggacgggcg 1980 ccagctggcg gtctccttca gcctgctgcg ggagaacagc ctctactgga actactacat 2040 cgacagctac ctcaagggtc agagcccggt ggccttcgat ctgctgcact ggaacagcga 2100 cagcaccaat gtggcgggca agacccacaa cagcctgctg cgccgtctct acctggagaa 2160 ccagctggtg aagggggagc tcaagatccg caacacccgc atcgatctcg gcaaggtgaa 2220 gacccctgtg ctgctggtgt cggcggtgga cgatcacatc gccctctggc agggcacctg 2280 gcagggcatg aagctgtttg gcggggagca gcgcttcctc ctggcggagt ccggccacat 2340 cgccggcatc atcaacccgc cggccgccaa caagtacggc ttctggcaca acggggccga 2400 ggccgagagc ccggagagct ggctggcagg ggcgacgcac cagggcggct cctggtggcc 2460 cgagatgatg ggctttatcc agaaccgtga cgaagggtca gagcccgtcc ccgcgcgggt 2520 cccggaggaa gggctggccc ccgcccccgg ccactatgtc aaggtgcggc tcaaccccgt 2580 gtttgcctgc ccaacagagg aggacgccgc atgagcgcac aatccctgga agtaggccag 2640 aaggcccgtc tcagcaagcg gttcggggcg gcggaggtag ccgccttcgc cgcgctctcg 2700 gaggacttca accccctgca cctggacccg gccttcgccg ccaccacggc gttcgagcgg 2760 cccatagtcc acggcatgct gctcgccagc ctcttctccg ggctgctggg ccagcagttg 2820 ccgggcaagg ggagcatcta tctgggtcaa agcctcagct tcaagctgcc ggtctttgtc 2880 ggggacgagg tgacggccga ggtggaggtg accgcccttc gcgaggacaa gcccatcgcc 2940 accctgacca cccgcatctt cacccaaggc ggcgccctcg ccgtgacggg ggaagccgtg 3000 gtcaagctgc cttaagcacc ggcggcacgc aggcacaatc agcccggccc ctgccgggct 3060 gattgttctc ccccgctccg cttgccccct ttttcggggc aatttggccc aggccctttc 3120 cctgccccgc ctaactgcct aaaatggccg ccctgccgtg taggcattca tccagctaga 3180 ggaattc 3187 22 2535 DNA Rattus norvegicus 22 gagcagtacc ccggccactg ccagtgtgtg cgtggtgcag gatagactca tggcttcgcc 60 tctgaggttc gacgggcgtg tggtcctggt caccggcgcc gggggagggt tgggcagagc 120 ttatgccctg gcttttgcag aaagaggagc attagttgtt gtgaatgact taggagggga 180 cttcaaaggc gttgggaaag gctcttctgc cgcagacaag gtcgtggaag aaataagaag 240 gagaggcggg aaagcggtgg ccaattacga ttcaggcgaa gcaggcgaga agcttgtgaa 300 gacagcactg gacacattcg gcagaataga tgttgtggtg aacaatgctg ggatcctgag 360 ggaccgttcc ttctctagga taagtgatga agactgggat ataattcaaa gagttcattt 420 gcggggctcc ttccaagtga cccgggcagc atgggatcat atgaagaagc agaattatgg 480 aagaatcatt atgacggcct cagcttctgg aatatacagc aactttggcc aggcaaatta 540 tagtgctgca aagctgggcc ttctgggtct cgccaatact ctcgtgattg aaggcaggaa 600 gaacaacatt cattgtaaca ccattgcccc aaacgctggg tcacggatga cagagacggt 660 gatgccagaa gacctcgttg aagccctgaa gccagagtat gtggcaccgc tggtcctttg 720 gctttgccat gagagctgtg aggaaaatgg tggcttgttt gaggttggag caggatggat 780 tggaaaattg cgctgggaga ggaccctggg agccattgtc aggaagcgga atcagcccat 840 gactcccgag gcagtgaggg acaactgggt gaagatctgt gacttcagca atgccagcaa 900 gccgaagagc attcaagagt ccacaggtgg tataatcgaa gttttacata aaatagattc 960 agaaggaatc tcacaaaatc acaccggtca agtggcatct gcagatgcat caggatttgc 1020 tggcgtcgtt ggccacaaac ttccttcatt ttcttcttca tatacggaac tgcagtgcat 1080 tatgtatgcc ctcggagtag gagcttcagt caaaaatcca aaggacttga agtttgttta 1140 tgaagggagt gctgacttct cctgtttgcc tacatttgga gtcattgtcg ctcagaagtc 1200 cttgacgagt ggaggcttag cagaggttcc tgggctgtca atcaactttg caaaggttct 1260 tcatggggag cagtacttgg agttgtataa gccacttccc cgatcagggg aattaaaatg 1320 tgaagcagtt attgctgaca tcctggataa aggctctggc atagtgattg ttatggacgt 1380 ctattcttat tctggcaagg aacttatatg ctataatcag ttctctgtct tcgttgttgg 1440 ctctggaggc tttggtggaa aacggacatc agaaaaactc aaagcagctg tagccgtacc 1500 ggatcggcct ccagatgctg tactgagaga taccacttca ctgaatcagg ccgctctgta 1560 ccgcctcagt ggagactcga atcctttaca cattgacccg agctttgcga gcattgccgg 1620 ttttgagaaa cccatattac acggattatg tacttttggg ttttctgcaa ggcatgtttt 1680 acagcagttt gcggataatg atgtgtcaag attcaaggcc attaaggttc gttttgccaa 1740 accagtgtat ccaggacaaa ctctacaaac tgagatgtgg aaggaaggaa acagaattca 1800 ttttcaaacc aaggtccaag agactggaga cattgtcatt tccaatgcat atgtggatct 1860 tgttcctaca tctggagttt ccgctcagac accttctgag ggtggagcac tgcagagtgc 1920 tcttgtattt ggggaaatag gtcgacgcct caaggatgtt ggacgtgagg tggtaaagaa 1980 agtaaatgct gtatttgaat ggcatatcac gaaaaatggg aatgttgcag ccaagtggac 2040 cattgacctg aagaacggct ctggagaggt ttaccaaggc cctgccaaag gctctgctga 2100 cacgaccatc acaatttctg atgaggattt catggaagtg gtcctgggca agcttaaccc 2160 acagaatgcc ttcttcagtg gcagactgaa ggcccgagga aacatcatgc tgagccagaa 2220 gctacagatg attctgaaag actatgccaa gctctgaagg acccactgcg tgctttaata 2280 aaaccagaat cattacgttc tgtctacgca gtcatgctcc agccttcttt gaaacgatcc 2340 acggtaatgt gcagcagaaa tcgcttaaca ttttcagatt cagataactt tcagattttc 2400 attttctact aatttttcac atattatttt tataaggaac tgtaatctag ctagcaaata 2460 attgttctgt tcatagatct gtatcttaat aaaaaaaaag tcaaccgaaa aaaaaaaaaa 2520 aaaaaaaaaa aaaaa 2535 23 2328 DNA Ralstonia eutropha 23 ctgcaggttc cctcccgttt ccattgaaag gactacacaa tgactgacgt tgtcatcgta 60 tccgccgccc gcaccgcggt cggcaagttt ggcggctcgc tggccaagat cccggcaccg 120 gaactgggtg ccgtggtcat caaggccgcg ctggagcgcg ccggcgtcaa gccggagcag 180 gtgagcgaag tcatcatggg ccaggtgctg accgccggtt cgggccagaa ccccgcacgc 240 caggccgcga tcaaggccgg cctgccggcg atggtgccgg ccatgaccat caacaaggtg 300 tgcggctcgg gcctgaaggc cgtgatgctg gccgccaacg cgatcatggc gggcgacgcc 360 gagatcgtgg tggccggcgg ccaggaaaac atgagcgccg ccccgcacgt gctgccgggc 420 tcgcgcgatg gtttccgcat gggcgatgcc aagctggtcg acaccatgat cgtcgacggc 480 ctgtgggacg tgtacaacca gtaccacatg ggcatcaccg ccgagaacgt ggccaaggaa 540 tacggcatca cacgcgaggc gcaggatgag ttcgccgtcg gctcgcagaa caaggccgaa 600 gccgcgcaga aggccggcaa gtttgacgaa gagatcgtcc cggtgctgat cccgcagcgc 660 aagggcgacc cggtggcctt caagaccgac gagttcgtgc gccagggcgc cacgctggac 720 agcatgtccg gcctcaagcc cgccttcgac aaggccggca cggtgaccgc ggccaacgcc 780 tcgggcctga acgacggcgc cgccgcggtg gtggtgatgt cggcggccaa ggccaaggaa 840 ctgggcctga ccccgctggc cacgatcaag agctatgcca acgccggtgt cgatcccaag 900 gtgatgggca tgggcccggt gccggcctcc aagcgcgccc tgtcgcgcgc cgagtggacc 960 ccgcaagacc tggacctgat ggagatcaac gaggcctttg ccgcgcaggc gctggcggtg 1020 caccagcaga tgggctggga cacctccaag gtcaatgtga acggcggcgc catcgccatc 1080 ggccacccga tcggcgcgtc gggctgccgt atcctggtga cgctgctgca cgagatgaag 1140 cgccgtgacg cgaagaaggg cctggcctcg ctgtgcatcg gcggcggcat gggcgtggcg 1200 ctggcagtcg agcgcaaata aggaaggggt tttccggggc cgcgcgcggt tggcgcggac 1260 ccggcgacga taacgaagcc aatcaaggag tggacatgac tcagcgcatt gcgtatgtga 1320 ccggcggcat gggtggtatc ggaaccgcca tttgccagcg gctggccaag gatggctttc 1380 gtgtggtggc cggttgcggc cccaactcgc cgcgccgcga aaagtggctg gagcagcaga 1440 aggccctggg cttcgatttc attgcctcgg aaggcaatgt ggctgactgg gactcgacca 1500 agaccgcatt cgacaaggtc aagtccgagg tcggcgaggt tgatgtgctg atcaacaacg 1560 ccggtatcac ccgcgacgtg gtgttccgca agatgacccg cgccgactgg gatgcggtga 1620 tcgacaccaa cctgacctcg ctgttcaacg tcaccaagca ggtgatcgac ggcatggccg 1680 accgtggctg gggccgcatc gtcaacatct cgtcggtgaa cgggcagaag ggccagttcg 1740 gccagaccaa ctactccacc gccaaggccg gcctgcatgg cttcaccatg gcactggcgc 1800 aggaagtggc gaccaagggc gtgaccgtca acacggtctc tccgggctat atcgccaccg 1860 acatggtcaa ggcgatccgc caggacgtgc tcgacaagat cgtcgcgacg atcccggtca 1920 agcgcctggg cctgccggaa gagatcgcct cgatctgcgc ctggttgtcg tcggaggagt 1980 ccggtttctc gaccggcgcc gacttctcgc tcaacggcgg cctgcatatg ggctgacctg 2040 ccggcctggt tcaaccagtc ggcagccggc gctggcgccc gcgtattgcg gtgcagccag 2100 cgcggcgcac aaggcggcgg gcgtttcgtt tcgccgcccg tttcgcgggc cgtcaaggcc 2160 cgcgaatcgt ttctgcccgc gcggcattcc tcgctttttg cgccaattca ccgggttttc 2220 cttaagcccc gtcgcttttc ttagtgcctt gttgggcata gaatcagggc agcggcgcag 2280 ccagcaccat gttcgtgcag cgcggccctc gcgggggcga ggctgcag 2328 24 1462 DNA Raphanus sativus 24 gactagtcgc gcgctctctg aactgaagta caagatttcc cgatggccca ttcagcagat 60 tcctccgaca atcccagaga tgtttgcatc gtgggtgttg cacgcactcc tatgggtggc 120 tttctcggat ctctctcctc cttacccgcc acaaagcttg gatcccttgc catcacagct 180 gctctgaaga gagaaatgtt gacccgtctc tggtccaagg aagttgtgtt tgggaatgtt 240 ctcagtgcta atttgggtca agctcccgct cgtcaggccg ctttaggtgc tgggatctct 300 aactctgtta tctgtaccac tgtcaacaag gtctgtgcct caggcatgaa agctgtgatg 360 attgctgctc agagtatcca gctggggatc aatgatgtag tcgtggcggg tggtatggaa 420 agcatgtcta atacaccaaa gtatcttgca gaagcaagaa aaggatctag gtttggtcat 480 gattctctcg tagatgggat gcttaaggat ggactgtggg atgtctataa cgactgtggg 540 atgggaagct gtgcagagtt atgcgctgag aagtttgaga taaccaggga gcagcaagat 600 gattacgctg ttcagagctt tgagcgtggt attgctgctc aggaatctgg cgccttcaca 660 tgggagatcg tcccggttga agtttctgga ggaaggggta ggccatcaac cattgttgac 720 aaggatgaag gtcttgggaa gtttgatgct gcaaaactga ggaaactccg tccgagtttc 780 aaggagaatg gaggcacagt tacagctgga aatgcctcta gcataagtga tggtgcagct 840 gctattgtcc tagtgagtgg agagaaggcg cttcagctag gacttcaagt acttgcaaaa 900 gttaaaggtt atggtgatgc agctcaggag ccagagtttt tcactactgc tcctgctctg 960 gcaataccaa aagctattgc acccaattcg ccctatagtg agtcgtatca agttgattac 1020 tatgagatca atgaagcatt tgcagttgta gcacttgcaa atcaaaagct acttgggatt 1080 agtccggaga aggtgaatgt aaatggagga gccgtctcct taggacatcc tctaggctgc 1140 agtggagccc gtattctaat cacattgctt gggatactga agaagagaaa cggcaagtac 1200 ggtgtgggag gagtgtgcaa cggaggagga ggtgcttctg ctcttgttct tgaagtcgtg 1260 tgatgcattt atatgaatcc caggttgttg aactatatag agcgtatcta ctatcattct 1320 accaacttgc acttcaagtt tgatattggt tggtctctct caataaatga gtgatgatga 1380 tctttgatgt tgttaagttt atttagttat attatatgaa aactatgttt ctgttaaaaa 1440 aaaaaaaaaa aaaaaaaaaa ac 1462 25 1440 DNA Pichia membranifaciens 25 atggcactcg tcctgcgcag gttcttctcc ggctccgtgg ccagggccac ggcgccagcc 60 agcctgatca agctccaccc ggtctcgcag ctcaaacaga gacagctgcc cacctacgtg 120 ggccagtcga actcgatcgt gaagtcgctg gtgtggacca cgccgcccag caacgtgctc 180 attgtgaaga agccgtggca ctccaaggtg ctcgacgccg ccatcacctt catcaagcat 240 ctccacgcaa actacccgtc cgtgaacatc atcgtggtgc ccgaggtcgc cgaggagctc 300 aactcgatcg aacgcaagag ctccgaccca gacacgccca tcggcatcta cacaggcccg 360 ctcaacgaga tcatctccaa gacagacctc attgtctccc tcggcggaga cggcaccatc 420 ctccggggcg tgtcgctctt ctccaacacg acggtcccac ccgtcttgtc cttctccctc 480 ggcacactag ggttccttct cccgttcgac ttcaacaact acgcggaggc gttcaaacag 540 atgttcgagt cccgctccag catcctcaaa agagaacgca

tagagtgcca catcgtcaag 600 gctagcccgc aatcggaggc gctcaaccag cagcggaagg acctcgaaac gtcctaccag 660 aacacacgct ccctaaacgc acaagaagag gtggaaaggt tgaagcgctt gtccgcagcc 720 atggatgctc cgttcgacaa tctgacagtc tcctccgagc tcgaggccct caagaaattg 780 aaaatccacg ccatgaacga cattgtcctc cacagaggct ctctcccggg attggtcaac 840 ctcgacgtct acatcaacgg caacctactc acacggacaa ccgcagacgg cctcatcttt 900 gccaccccaa caggctccac agcgtactct ctttcggcag gcggttccat cgtccaccca 960 gtcgtcaagt gcatccttct caccccgatc tgtccgcgaa gcttgtcctt caggcccttg 1020 atcctcccac taaactccca tatcttgatc aaggtcatcg gcaaggaaaa cgtgaagatc 1080 gactacacca agtgcaacgc caaattgagc atagacggaa ttccgcaact gaaaatggtc 1140 cccggcgacg agatccacat catctccgag tccgtctcca gacttaactc cgtaaacgac 1200 gacgaagacg acatcgcctc cggaacaact gcagacgcac cggactgcgt caatgcttcc 1260 actactgtct cgaaggaatc taagacgaag tctctgggaa gacgccgtgg cgtccaaaag 1320 agaaccgccg aacgaagtgg cgtctggtgt gttgtccaga gtaagggcga ctgggtcaac 1380 ggcatcaacg gaatgttggg attcaaccta ggattcaagt cttccaagtc caacaaatga 1440 26 3967 DNA Saccharomyces cerevisiae 26 gatcaattct gttaagctct ttacgcatgc ttttattttc ttcactttgg cacattcgct 60 aaagagaaag cgtttgatag ccgcttttgc gatttggtcc tatggtatct ttacactatt 120 catcaatcaa aaaatgaaaa atctcccctt taataatatt gctatcatcc taagtcccat 180 ggatcaatgg tataagggta tcgttcctcg atgggatttt tttttcaatt ttacattatt 240 gcgtttgtta agttactcca tggatttttt ggaaagatgg catgaacaat tgagccgcca 300 accttcgata gattacgatg atagacgacc tgaattcaga aaaagtttat ctggttctac 360 tctacaaacc atttatgagt caggtaagaa tgttctggag gaaaaggaac gactggtagc 420 agaacatcac atccaggatt acaactttat caattttatc gcttatatta cttacgcgcc 480 attgttttta gtgggcccaa ttatcacttt taatgactac ctttatcaat cagaaaataa 540 gcttccttcg ctaacgaaaa aaaacatagg cttctatgcc ctcaaagtat tttcgagttt 600 gcttttgatg gaaattatcc tacattatat ctatgtgggt gcaatagcaa ggaccaaggc 660 atggaacaat gatacaccct tgcaacaggc tatgatcgcg ctgttcaact tgaacattat 720 gtatttaaaa cttttgatcc catggaggct ctttcggctg tgggccatgg tcgatggtat 780 tgatgcacct gaaaatatgc tacgatgtgt ggataataat tatagtacag tgggattctg 840 gagagcctgg catacaagtt ttaacaagtg ggtaatccgt tacatctatg ttccatttgg 900 cgggtccaat aacaaaatat taacgagctt tgccgtattc tcatttgtag caatatggca 960 tgacatccaa ttacgagtgt tgttttgggg gtggttaaca gtccttttat tattaggcga 1020 aacctacatt actaactgtt ttagtagata tagattcaga agctggtaca ggtttgtttg 1080 cggtatcggt gctgcaataa atatttgcat gatgatgatt attaatgtct atggattttg 1140 cttgggtgca gagggaacga agcttctatt gaagggcata tttaacaatt cacatagtcc 1200 ggagtttttg actgcggtaa tggtaagcct atttattgct gttcaggtaa tgtttgagat 1260 tagagaagaa gaaaaaagac atggcatcaa cttgaaatgt tgatctagtt attagataag 1320 ctatgaaagt caatcctttt aatcgagaat gtaaatatgt ggaatacaca attttaacca 1380 aagtactata tatgcgttac aagtaattta aatttaagtt caccgaagta aaactaactg 1440 caagattgtt acaaagaaca atgcactatt taaatcacac aatggctatt gaaaactgta 1500 actgtcagaa atgctgcatg tatctatatg catcactaag ttgcgacttt taagaaactt 1560 ccacagttct caactcttct ttgtgctttt cacacatttt cacaattttc cgaaatctcc 1620 aaattgaaaa aaaaataaaa ataaaaaaag gcaggagaag actaagtatt cattattcgc 1680 tgtttcataa ataaaaggat aaaaaggtta aggatactga ttaaaatgtt tgtcagggtt 1740 aaattgaata aaccagtaaa atggtatagg ttctatagta cgttggattc acattcccta 1800 aagttacaga gcggctcgaa gtttgtaaaa ataaagccag taaataactt gaggagtagt 1860 tcatcagcag atttcgtgtc cccaccaaat tccaaattac aatctttaat ctggcagaac 1920 cctttacaaa atgtttatat aactaaaaaa ccatggactc catccacaag agaagcgatg 1980 gttgaattca taactcattt acatgagtca taccccgagg tgaacgtcat tgttcaaccc 2040 gatgtggcag aagaaatttc ccaggatttc aaatctcctt tggagaatga tcccaaccga 2100 cctcatatac tttatactgg tcctgaacaa gatatcgtaa acagaacaga cttattggtg 2160 acattgggag gtgatgggac tattttacac ggcgtatcaa tgttcggaaa tacgcaagtt 2220 cctccggttt tagcatttgc tctgggcact ctgggctttc tatcaccgtt tgattttaag 2280 gagcataaaa aggtctttca ggaagtaatc agctctagag ccaaatgttt gcatagaaca 2340 cggctagaat gtcatttgaa aaaaaaggat agcaactcat ctattgtgac ccatgctatg 2400 aatgacatat tcttacatag gggtaattcc cctcatctca ctaacctgga cattttcatt 2460 gatggggaat ttttgacaag aacgacagca gatggtgttg cattggccac tccaacgggt 2520 tccacagcat attcattatc agcaggtgga tctattgttt ccccattagt ccctgctatt 2580 ttaatgacac caatttgtcc tcgctctttg tcattccgac cactgatttt gcctcattca 2640 tcccacatta ggataaagat aggttccaaa ttgaaccaaa aaccagtcaa cagtgtggta 2700 aaactttctg ttgatggtat tcctcaacag gatttagatg ttggtgatga aatttatgtt 2760 ataaatgagg tcggcactat atacatagat ggtactcagc ttccgacgac aagaaaaact 2820 gaaaatgact ttaataattc aaaaaagcct aaaaggtcag ggatttattg tgtcgccaag 2880 accgagaatg actggattag aggaatcaat gaacttttag gattcaattc tagctttagg 2940 ctgaccaaga gacagactga taatgattaa acgctctgaa tgcaaagatt caatgagatt 3000 ctctaagaat tctattgata agatttaaag gtatttgaca agtagagatc tttatttttt 3060 cttgcatttt gtctagagaa atctcaactg acatactcga catgaaattt ttggtattgt 3120 gtcttttatt ctattgcttt aagaaaactg tgacatatag ggaagacatg cttaacaaga 3180 agatataatt atataatata tatattatta ataataacat ccttactgca gtcctgttgt 3240 gggagaaaat ggagagagac tatgtttcgt atcaattcct aaaatcaaaa aaaaaaaaaa 3300 aaaaaagtta aacaagcact cgctgttcat ttgttttaca agtattcata ctctaatagg 3360 tcattgagct tcttttcttg aggagagatc caatttgaag tcggaataag atttgctttc 3420 attagcgtag gcaataatta tgagataaat ggtgcagcac tattaagtag tgtggatttc 3480 aataatttcc gaattaggaa taaatgcgct aaatagacat cccgttctct ttggtaatct 3540 gcataattct gatgcaatat ccaacaacta tttgtgcaat tatttaacaa aatccaatta 3600 actttcctaa ttagtccttc aatagaacat ctgtattcct tttttttatg aacaccttcc 3660 taattaggcc atcaacgaca gtaaattttg ccgaatttaa tagcttctac tgaaaaacag 3720 tggaccatgt gaaaagatgc atctcattta tcaaacacat aatattcaag tgagccttac 3780 ttcaattgta ttgaagtgca agaaaaccaa aaagcaacaa caggttttgg ataagtacat 3840 atataagagg gccttttgtt cccatcaaaa atgttactgt tcttacgatt catttacgat 3900 tcaagaatag ttcaaacaag aagattacaa actatcaatt tcatacacaa tataaacgat 3960 taaaaga 3967 27 1728 DNA Saccharomyces cerevisiae 27 gttattagat aagctatgaa agtcaatcct tttaatcgag aatgtaaata tgtggaatac 60 acaattttaa ccaaagtact atatatgcgt tacaagtaat ttaaatttaa gttcaccgaa 120 gtaaaactaa ctgcaagatt gttacaaaga acaatgcact atttaaatca cacaatggct 180 attgaaaact gtaactgtca gaaatgctgc atgtatctat atgcatcact aagttgcgac 240 ttttaagaaa cttccacagt tctcaactct tctttgtgct tttcacacat tttcacaatt 300 ttccgaaatc tccaaattga aaaaaaaata aaaataaaaa aaggcaggag aagactaagt 360 attcattatt cgctgtttca taaataaaag gataaaaagg ttaaggatac tgattaaaat 420 gtttgtcagg gttaaattga ataaaccagt aaaatggtat aggttctata gtacgttgga 480 ttcacattcc ctaaagttac agagcggctc gaagtttgta aaaatagagg cagtaaataa 540 cttgaggagt agttcatcag cagatttcgt gtccccacca aattccaaat tacaatcttt 600 aatctggcag aaccctttac aaaatgttta tataactaaa aaaccatgga ctccatccac 660 aagagaagcg atggttgaat tcataactca tttacatgag tcataccccg aggtgaacgt 720 cattgttcaa cccgatgtgg cagaagaaat ttcccaggat ttcaaatctc ctttggagaa 780 tgatcccaac cgacctcata tactttatac tggtcctgaa caagatatcg taaacagaac 840 agacttattg gtgacattgg gaggtgatgg gactatttta cacggcgtat caatgttcgg 900 aaatacgcaa gttcctccgg ttttagcatt tgctctgggc actctgggct ttctattacc 960 gtttgatttt aaggagcata aaaaggtctt tcaggaagta atcagctcta gagccaaatg 1020 tttgcataga acacggctag aatgtcattt gaaaaaaaag gatagcaact catctattgt 1080 gacccatgct atgaatgaca tattcttaca taggggtaat tcccctcatc tcactaacct 1140 ggacattttc attgatgggg aatttttgac aagaacgaca gcagatggtg ttgcattggc 1200 cactccaacg ggttccacag catattcatt atcagcaggt ggatctattg tttccccatt 1260 agtccctgct attttaatga caccaatttg tcctcgctct ttgtcattcc gaccactgat 1320 tttgcctcat tcatcccaca ttaggataaa gataggttcc aaattgaacc aaaaaccagt 1380 caacagtgtg gtaaaacttt ctgatgatgg tattcctcaa caggatttag atgttggtga 1440 tgaaagttat gttataaatg aggtcggcac tatatacata gatggtactc agcttccgac 1500 gacaagaaaa actgaaaatg actttaataa ttcaaaaaag cctaaaaggt cagggattta 1560 ttgtgtcgcc aagaccgaga atgactggat tagaggaatc aatgaacttt gtaggattca 1620 ttctagcttt aggctgacca agagacagac tgataatgat taaacgctct gaatgcaaag 1680 attcaatgag attctctaag aattctattg ataagattta aaggtacc 1728

Claims

1. A plant genetically manipulated to produce polyhydroxyalkanoate in its peroxisomes, said plant comprising in its genome: a stably integrated first DNA construct comprising a promoter that drives expression in a plant cell operably linked to a first coding sequence, wherein said first coding sequence encodes a polyhydroxyalkanoate synthase and is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal; and a stably integrated second DNA construct comprising a promoter that drives expression in a plant cell operably linked to a second coding sequence, wherein said second coding sequence is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal and said second coding sequence is selected from the group consisting of: (a) a nucleotide sequence encoding a 2-enoyl-CoA hydratase that is capable of catalyzing the synthesis of R-(-)-3-hydroxyacyl-CoA; (b) a nucleotide sequence set forth in SEQ ID NO: 21; (c) a nucleotide sequence comprising the 2-enoyl-CoA hydratase domain of a multifunctional protein-2. (d) a nucleotide sequence set forth in SEQ ID NO: 4; (e) a nucleotide sequence set forth in SEQ ID NO: 1; and (f) a nucleotide sequence encoding a multifunctional protein-2, wherein the dehydrogenase activity of said multifunctional protein has been eliminated.

2. The plant of claim 1, wherein said promoters are selected from the group consisting of seed-preferred promoters, chemical-regulatable promoters, germination-preferred promoters, and leaf-preferred promoters.

3. The plant of claim 1, wherein said polyhydroxyalkanoate synthase is capable of catalyzing the synthesis of polyhydroxyalkanoate copolymers.

4. The plant of claim 3, wherein said polyhydroxyalkanoate synthase is encoded by a nucleotide sequence selected from the group consisting SEQ ID NOs: 8-12.

5. The plant of claim 1 further comprising in its genome a stably integrated third DNA construct comprising a promoter that drives expression in a plant cell operably linked to a third coding sequence, wherein said third coding sequence encodes a 3-ketoacyl-CoA reductase and is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal.

6. The plant of claim 1, wherein said third coding sequence comprises at least a portion of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a 3-ketoacyl-CoA reductase that is capable of utilizing NADH; (b) a nucleotide sequence set forth in SEQ ID NO: 3. (c) a nucleotide sequence set forth in SEQ ID NO: 22; (d) the nucleotide sequence set forth in SEQ ID NO: 1; (e) a nucleotide sequence encoding a multifunctional protein-2, wherein the hydratase activity of said multifunctional protein has been eliminated; (f) the nucleotide sequence set forth in SEQ ID NO: 6; and (g) a nucleotide sequence set forth in SEQ ID NO: 23.

7. The plant of claim 5 further comprising in its genome a stably integrated fourth DNA construct comprising a promoter that drives expression in a plant cell operably linked to fourth coding sequence, wherein said fourth coding sequence encodes an acetyl-CoA:acetyl transferase and is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal.

8. The plant of claim 7, wherein said fourth coding sequence comprises at least a portion of the nucleotide sequence set forth in SEQ ID NO: 24.

9. The plant of claim 7 further comprising in its genome a fifth DNA construct comprising a promoter that drives expression in a plant cell operably linked to a fifth coding sequence, wherein said fifth coding sequence encodes an NADH kinase or an NAD.sup.+ kinase and said fifth coding sequence is operably linked to a nucleotide sequence encoding a peroxisome-targeting signal

10. The plant of claim 9, wherein said fifth coding sequence comprises at least a portion of a nucleotide sequence selected from the group consisting SEQ ID NOs: 25-27.

11. The plant of claim 1, wherein said plant is an oilseed plant.

12. The plant of claim 11, wherein said oilseed plant has been genetically manipulated for the enhanced production of short-chain or modified fatty acids.

13. The plant of claim 11, wherein said oilseed plant comprises in its genome a DNA construct comprising a coding sequence for an acyl-CoA oxidase operably linked to a promoter that drives expression in a plant.