AU770120B2 - Plant multi-gene expression constructs - Google Patents

Description

WO 00/78985 PCT/US00/17197 PLANT MULTI-GENE EXPRESSION CONSTRUCTS Background of the Invention The present invention generally relates to constructs for transforming.

plants, in particular plants useful in the production of polymers.

Genetic engineering of plant crops to produce stacked input traits, such as tolerance to herbicides and insect resistance, or value added products, such as polyhydroxyalkanoates (PHAs), requires the expression of multiple foreign genes. The traditional breeding methodology used to assemble more than one gene within a plant requires repeated cycles of producing and crossing homozygous lines, a process that contributes significantly to the cost and time for generating transgenic plants suitable for field production (Hitz, Current Opinion in Plant Biology, 2:135-38 (1999). This cost could be drastically reduced by the insertion of multiple genes into a plant in a single transformation event.

The creation of a single vector containing cassettes of multiple genes, each flanked by a promoter and polyadenylation sequence, allows for a single transformation event but can lead to gene silencing if any of the promoter or polyadenylation sequences are homologous (Matzke, et al., in Homologous Recombination and Gene Silencing in Plants (Paszkowski, ed.) pp. 271-300 (Kluwer Academic Publishers, Netherlands, 1994)). Multiple unique promoters can be employed, but coordinating the expression is difficult. Researchers have coordinated the expression of multiple genes from one promoter by engineering ribozyme cleavage sites into multi-gene constructs such that a polycistronic RNA is produced that can subsequently be cleaved into a monocistronic RNA (see U.S. Patent No. 5,519,164).

Multiple genes have also been expressed as a polyprotein in which coding regions are joined by protease recognition sites (Dasgupta, et al., The Plant Journal, 16:107-16 (1998)). A co-expressed protease releases the individual enzymes but often leaves remnants of the protease cleavage site that may affect the activity of the enzymes.

WO 00/78985 PCT/US00/17197 The majority of eukaryotic mRNAs are thought to be translated by a scanning ribosome mechanism in which ribosomes can only gain access to translation initiation sites via the 5'-end of the mRNA (Bailey-Serres, Trends in Plant Science, 4:142-47 (1999)). A cap structure at the extreme 5' end of the mRNA plays an important role in the initial binding of the ribosome.

Several RNAs of viral origin do not contain a 5' cap structure yet can be translated efficiently by the eukaryotic host This mechanism of capindependent initiation of protein synthesis is referred to as internal initiation and the sequence promoting the initiation is called an internal ribosome entry site (IRES) (Belsham Sonenberg, Microbiol. Rev., 60:499-511 (1996)).

The internal initiation of protein synthesis is independent of the location of the IRES sequence within the mRNA and does not require the presence of any viral proteins The sequences used by these viruses therefore can be manipulated to create artificial expression cassettes capable of capindependent translation of multiple gene coding regions in plants. The use of IRES sequences in plants for the expression of multiple genes to enhance input traits, or for multi-gene expression for the formation of natural or novel plant products has not been demonstrated. In vitro studies with bicistronic constructs containing the 5'-UTR of the crucifer-infecting Tobamovirus have resulted in the successful translation of both coding sequences (Ivanov, et al., Virology, 232:32-43 (1997)). Constructs containing several viral sequences for the enhanced expression of a single foreign gene in plants also have been disclosed in PCT WO 98/55636, wherein an IRES signal is used to promote expression of a marker gene for selection of the transgenic plant.

The expression of multiple enzymes is useful for altering the metabolism of plants to increase, for example, the levels of nutritional amino acids (Falco, et al., Bio/Technology 13:577 (1995)), to modify lignin metabolism, to modify oil compositions (Murphy, TIBTECH 14:206-13 (1996)), to modify starch biosynthesis, or to produce polyhydroxyalkanoate polymers (Huisman Madison, Microbiol. Mol. Biol. Rev. 63:21-53 (1999)). In order to produce PHAs, it is desirable to divert carbon from normal plant metabolism by the expression of two or more recombinant proteins. PHAs of different compositions and properties can be produced in plants depending on the substrate specificity of the engineered enzymes, the tissues in which these enzymes are expressed, and the pathways from which carbon is diverted (Huisman Madison, Microbiol. Mol. Biol. Rev. 63: 21-53 (1999)).

Prior to producing PHAs from plants on an industrial scale, polymer production in crops of agronomic value must be optimized. Preliminary studies in some crops of agronomic value have been performed including PHB production in maize cell suspension cultures and in the peroxisomes of intact tobacco plants (Hahn, Ph. D.

Thesis, University of Minnesota (Feb. 1998)), as well as PHB production in transgenic canola and soybean seeds (PCT WO 98/00557). In these studies, however, the levels of polymer observed were too low for economical production of the polymer.

It is a preferred embodiment of the present invention to provide DNA constructs for the improved insertion of multiple genes into a plant in a single transformation event.

It is another preferred embodiment of this invention to provide enhanced, cost effective methods for developing transgenic plants for the production of polymers, S"particularly polyhydroxyalkanoates.

".Summary of the Invention !•Methods and constructs are provided for the introduction of multiple genes into 20 plants using a single transformation event. In particular, the construction of multi-gene expression cassettes containing a single promoter and a single polyadenylation signal is provided. Coordinated expression of genes in the cassette, producing proteins with native amino acid sequences, is achieved by production of one polycistronic mRNA that contains separate translation initiation signals for each enzyme coding region. The 25 described arrangement of genes allows the insertion of multiple genes into a plant using a single transformation event. This methodology for the insertion and expression of multiple genes encoding metabolic pathways is useful for producing value added *o products, as well as for engineering plants to express multiple input traits.

Creation of polycistronic internal ribosome entry site (IRES) containing vectors is useful for reducing the complexity of the traditional breeding methodology required to make the transgenic plant agronomically useful. Bicistronic constructs contain a single gene 1, an internal ribosome entry site (IRES), gene 2, and a single 3'polyadenylation sequence. For polycistronic constructs, additional cassettes of genes, in which each coding region is preceded by an IRES, can be inserted between gene 2 and ~v WI MMI X MM1~ the polyadenylation sequence. The methods and constructs are useful for creating plants with stacked input traits glyphosate tolerant plants producing BT toxin) and/or value added products the production of PHAs in plants).

In one aspect, this invention resides in an expression cassette comprising a single plant promoter at the 5'-end of the construct, a first protein encoding sequence 3' to the promoter, an internal ribosome entry site sequence 3' to the first protein encoding sequence, a second protein encoding sequence immediately 3' to the internal ribosome entry site, and a polyadenylation sequence 3' to the second protein encoding sequence.

wherein the encoded proteins function in the production of polyhydroxyalkanoates.

In another aspect, this invention resides in plant material transformed with an expression cassette comprising a single plant promoter at the 5'-end of the construct, a first protein encoding sequence 3' to the promoter, an internal ribosome entry site 3' to the first protein encoding sequence, a second protein encoding sequence immediately 3' to the internal ribosome entry site, and a polyadenylation sequence 3' to the second protein encoding sequence, wherein the encoded proteins function in the production of *polyhydroxyalkanoates.

In a still further aspect, this invention a method for expression of heterologous 25 genes in plant material comprising transforming the material with an expression cassette comprising a single plant promoter at the 5'-end of the construct, a first protein encoding sequence 3' to the promoter, an internal ribosome entry site 3' to the first protein encoding sequence, 30 a second protein.encoding sequence immediately 3' to the internal ribosome entry S. site, and a polyadenylation sequence 3' to the second protein encoding sequence, 4 ~~ih~h~u*U~iiK wherein the encoded proteins function in the production of polyhydroxyalkanoates.

In the specification the term "comprising" shall be understood to have a broad meaning similar to the term "including" and will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term "comprising" such as "comprise" and "comprises".

Brief Description of the Drawings Figures la-d are illustrations of multi-gene expression using internal ribosome entry sites, where Fig. la shows a cassette for multi-gene expression; bicistronic construct polycistronic construct, (n Fig. lb shows bi-or poly-cistronic mRNA with one 5' cap and one polyadenylation signal; Fig. Ic shows binding of ribosomes to bicistronic or polycistronic (n 1) mRNA constructs, where ribosomes initiate translation at 5'capped portion of mRNA and at all internal ribosome initiation sites downstream of the 5'cap; and Fig. Id shows translation of bi or polycistronic mRNA to produce proteins.

Figure 2 is an illustration of short and medium chain length PHA production from fatty acid-oxidation pathways.

Figure 3 is an illustration of bicistronic construct for testing in vivo cap independent translation in Arabidopsis protoplasts.

Figure 4 is an illustration of medium chain length PHA production from fatty acid biosynthesis pathways.

25 Figure 5 is an illustration of a construct for medium chain length PHA production in chloroplasts from fatty acids containing two promoters and two polyadenylation sequences.

*e 2' ~~?lZ~M'ka 2 l 0. -44444 1*A&l Al WO 00/78985 PCT/US00/17197 Figures 6a-c are illustrations of IRES Constructs for medium chain -length-PHA production,.wherein_Eig..6a..shows a cnstruct for medium chain length PHA production in chloroplasts from fatty acid biosynthesis; Fig. 6b shows a construct for medium chain length PHA production in the peroxisomes of leaves using fatty acid p-oxidation; and Fig. 6c shows a construct for medium chain length PHA production in the cytosol of oil seeds using fatty acid p-oxidation.

Figures 7a-f are illustrations of constructs for short chain PHA production from acetyl CoA in plant tissues, wherein Fig. 7a shows a construct expressing reductase and synthase in cytosol; Fig. 7b shows a construct expressing reductase, synthase, and thiolase in cytosol; Fig. 7c shows a construct targeting reductase and synthase to chloroplasts or plastids; Fig. 7d shows a construct targeting reductase, synthase, and thiolase to chloroplasts or plastids; Fig. 7e shows a construct for targeting reductase and synthase to peroxisomes; and Fig. 7f shows a construct for targeting reductase, synthase, and thiolase to peroxisomes.

Figures 8a-c are illustrations of constructs for cytosolic short chain PHA production in seeds from fatty acids using cytosolic fatty acid Poxidation, wherein Fig. 8a shows a construct for expressing acyl CoA oxidase, a synthase with specificity for short chain substrates, an a-subunit of P-oxidation, a p-subunit of P-oxidation, and a reductase; Fig. 8b shows a construct for expressing an acyl CoA oxidase, a synthase accepting short chain substrates, an a-subunit of P-oxidation, a p-subunit of P-oxidation, a reductase, and a catalase; and Fig. 8c shows a construct for expressing an acyl CoA oxidase, a synthase expressing short chain substrates, an a-subunit of p-oxidation, a P-subunit of P-oxidation, a reductase, and a thiolase.

Figure 9 is an illustration of a construct for producing a plant that is tolerant to glyphosate and that produces Bacillus thuringiensis toxin.

Detailed Description of the Invention Optimization of PHA production in crops of agronomic value requires the screening of multiple enzymes, targeting signals, and sites of production until a high yielding route to the polymer with the desired 'R i lmu% WO 00/78985 PCT/US00/17197 composition is obtained. This is a task which can be simplified if multiple genes are inserted in a single transformation event. The usefulness of bi- or poly-cistronic vectors to produce PHAs in plants is described herein.

Expression cassettes are provided in which viral IRES sequences are utilized for multi-gene translation. For example, the strategy for utilizing the expression cassettes for producing multiple proteins from a single polycistronic mRNA is outlined in Figure 1. The expression cassettes contain a single promoter at the 5'-end of the construct (Fig. la). The promoter may be inducible, constitutive, or tissue specific. The first gene to be expressed is placed directly behind the promoter. If necessary, this gene may be preceded or followed by a peptide signal that targets the protein to a particular compartment of the cell. A viral IRES sequence that is able to initiate cap-independent translation in plants is placed immediately behind the first gene. The source of the IRES sequence may be from any of the viruses capable of initiating cap-independent translation of mRNA. The IRES sequence is followed by a second gene. For targeting of this gene to a certain organelle, a peptide signal can be fused to the coding sequence of the gene. After the second gene, the IRES-gene sequence can be repeated as often as desired for expression of multiple proteins in the same cell (Fig. la, n For multi-IRES containing constructs, it may be useful to use IRES elements from different sources. After the sequence of the last gene to be expressed, a polyadenylation signal must be inserted. This arrangement of genetic information will allow the formation of one polycistronic mRNA (Fig. Ib). Ribosomes can bind independently at both the 5' end of the mRNA and at the IRES sequences allowing independent translation of all protein coding sequences in the polycistronic mRNA (Figs. Ic and ld).

I. Materials Plant Transformation Vectors The DNA constructs provided herein include transformation vectors capable of introducing transgenes into plants. There are many plant transformation vector options available Gene Transfer to Plants, Potrykus Spangenberg, eds., Springer-Verlag Berlin Heidelberg New York 6 iWW; A A '4WN iW' 1 1fti 1 Ma of k u 0r 1UV~ N 141 1 1 0 M0 i f 1 OA .W" WO 00/78985 PCT/US00/17197 (1995); Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins. Owen Pen, eds., John Wiley Sons Ltd.

England (1996); and Methods in Plant Molecular Biologv-a laboratory course manual, Maliga, et al., eds., Cold Spring Laboratory Press, New York (1995)). In general, plant transformation vectors comprise one or more coding sequences of interest under the transcriptional control of 5' and 3' regulatory sequences, including a promoter, a transcription termination and/or polyadenylation signal and a selectable or screenable marker gene.

The usual requirements for 5' regulatory sequences include a promoter, a transcription initiation site, and a RNA processing signal. 3' regulatory sequences include a transcription termination and/or a polyadenylation signal.

Plant Promoters A large number of plant promoters are known and result in either constitutive, or environmentally or developmentally regulated expression of the gene of interest. Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser Fraley, Science 244:1293-99 (1989)). Examples of suitable constitutive plant promoters include the cauliflower mosaic virus 35S promoter (CaMV) and enhanced CaMV promoters (Odell et al., Nature, 313:810 (1985)), actin promoter (McElroy et al., Plant Cell 2:163-71 (1990)), AdhI promoter (Fromm et al., Bio/Technology 8:833-39 (1990); Kyozuka et al., 1991, Mol.

Gen. Genet. 228:40-48), ubiquitin promoters, the Figwort mosaic virus promoter, mannopine synthase promoter, nopaline synthase promoter and octopine synthase promoter. Useful regulatable promoter systems include, for example, spinach nitrate-inducible promoter, heat shock promoters, small subunit of ribulose biphosphate carboxylase promoters and chemically inducible promoters (see U.S. Patents No. 5,364,780; No. 5,364,780; No.

5,777,200).

Fi~~rr~n in*n~~*unw lhdlarr!lr,;lrrh~llsa~s~~ i ~~sX WO 00/78985 PCT/US00/17197 Tissue Specific Promoters In some embodimentsitmaybepreferableto.express.the transgenes only in the developing seeds. Promoters suitable for this purpose include the napin gene promoter Patent No. 5,420,034 and No. 5,608,152), the acetyl-CoA carboxylase promoter Patent No. 5,420,034 and No.

5,608,152), 2S albumin promoter, seed storage protein promoter, phaseolin promoter (Slightom et. al., Proc. Natl. Acad. Sci. USA 80:1897-901 (1983)), oleosin promoter (Plant et al., Plant Mol. Biol. 25:193-205 (1994); Rowley et. al., 1997, Biochim. Biophys. Acta. 1345:1-4 (1997); U.S. Patent No.

5,650,554; PCT WO 93/20216), zein promoter, glutelin promoter, starch synthase promoter, and starch branching enzyme promoter.

Alternatively, for some constructs it may be preferable to express the transgene only in the leaf. A suitable promoter for this purpose would include the C4PPDK promoter preceded by the 35S enhancer (Sheen, J.

EMBO, 12:3497-505 (1993)) or any other promoter that is specific for expression in the leaf.

Targeting Sequences The 5' end of the transgene may be engineered to include sequences encoding plastid or other subcellular organelle targeting peptides linked inframe with the transgene. A chloroplast targeting sequence is any peptide sequence that can target a protein to the chloroplasts or plastids, such as the transit peptide of the small subunit of the alfalfa ribulose-biphosphate carboxylase (Khoudi, et al., Gene 197:343-51 (1997)). A peroxisomal targeting sequence refers to any peptide sequence, either N-terminal, internal, or C-terminal, that can target a protein to the peroxisomes, such as the plant C-terminal targeting tripeptide SKL (Banjoko Trelease, Plant Physiol. 107:1201-08 (1995)).

SEQ ID NO:10 is the sequence of the chloroplast targeting signal from the alfalfa rubisco protein fused to the coding sequence of PhaC from Pseudomonas aeruginosa. The underlined sequences "ccatgg", "gcatgc", and "gcggccgc" are Nco I, Sph I, and Not I restriction sites, respectively, introduced for cloning purposes. The Nco I site sequence contains the N- M AW '0M A, WO 00/78985 PCT/US00/17197 terminal ATG of the targeting signal. The Sph I site fuses the ATG of the protein to be targeted to the N-terminal targeting signal. N-terminal methionines of the targeting signal and PhaC are indicated in bold.

SEQ ID NO:11 is the sequence of the pea rubisco chloroplast targeting signal including 24 amino acids of the pea rubisco protein fused to the coding sequence of PhaG. The underlined sequences "ggatcc" and "tctaga" are BamHI and Xba I restriction sites, respectively, introduced.for cloning purposes. N-terminal methionines of the targeting signal and PhaG are indicated in bold.

SEQ ID NO: 12 is the sequence of the chloroplast targeting signal from the alfalfa rubisco protein fused to the coding sequence of the reductase from Alcaligenes eutrophus. The underlined sequences "ccatgg", "gcatgc", and "gcggccgc" are Nco 1, Sph I, and Not I restriction sites, respectively, introduced for cloning purposes. The Nco I site sequence contains the Nterminal ATG of the targeting signal. The Sph I site fuses the ATG of the protein to be targeted to the N-terminal targeting signal. N-terminal methionines of the targeting signal and PhaC are indicated in bold.

SEQ ID NO:13 is the sequence of the pea rubisco chloroplast targeting signal including 24 amino acids of the pea rubisco protein fused to the coding sequence of the synthase from A. eutrophus. The underlined sequences "ggatcc", "tctaga", are BamHI and Xba I restriction sites, respectively, introduced for cloning purposes. N-terminal methionines of the targeting signal and PhaG are indicated in bold.

Internal Ribosome Entry Sites (IRES) In the following examples, an IRES pertains to any sequence that can initiate cap-independent translation of mRNA in plants, such as the IRES of the Tobamovirus (Ivanov, et al., Virology 232:32-43 (1997)), the IRES of the turnip mosaic potyvirus (Basso, et al., J. General Virology, 75:3157-65 (1994)), the IRES of the cow pea mosaic virus (Thomas, et al., J. Virology, 65:2953-59 (1991)), or the potato virus Y (Levis Astier-Manifacier, Virus Genes 7:367-79 (1993)).

P~I U WO 00/78985 WO 0078985PCT/USOO/17197 The sequence encoding the potato virus Y IRES ((SEQ ID NO:7); Karchi, et al. Virus Gene.4:2l5-24{l199O)) can be isolated from _plasmid pTHC8 (American Type Culture Collection #45127) using primers PVY.c and PVY.r.

PVY.c 5'cgizatccacaacataagagaaaacaacgcaaaaa,-c (SEQ ID NO:l1) PVY.r 5'catgccatugagtatgctagtaaatgaaggaaat (SEQ ID NO:2) The sequence encoding the IRES of the turnip mosaic potyvirus ((SEQ ID NO:8); Basso, et al., J Gen. Virol. 75:3157-65 (1994)) can be isolated from plasmid pTUMIA. (American Type Culture Collection 107) using primers TMV.c and TMV.r.

TMV.c 5 'cggatccaaaatataaaaactcaacacaacata (SEQ ID NQ:3) TMV.r 5'catg~oggtgttggtgattgctttgataacgacaa (SEQ ID NO:4) The sequence encoding the IRES of the cowpea mosaic virus ((SEQ ID NO:9); van Wezenbeek, et al., EMBO J 2:941-46 (1983)) can be isolated from plasmid pTMB 120 (American Type Culture Collection #45 054) using primers CMV.c and CMV.r.

CMV.c 5'cgggatccatgttttctttcactgaagcgaaatca (SEQ ID NO: CMV.r 5'catgccataugcaaatttgggcagaatatacaga (SEQ ID NO:6) Poly Adenviation Signals At the extreme 3' end of the transcript, a polyadenylation signal can be engineered. A polyadenylation signal refers to any sequence that can result in polyadenylation of the mRNA in the nucleus prior to export of the ruRNA to the cytosol, such as the 3' region of nopaline synthase (Bevan, et al., Nucleic Acids Res. 11:369-85 (1983)).

WO 00/78985 PCTIUSOO/17197 Protein Encoding Sequences The protein encoding sequences (also commonly referred to as "genes", although in this context not typically including a promoter or other regulatory sequences) can encode any protein which is to be expressed.

In a preferred embodiment, the protein coding sequences encode enzymes required for the production of polyhydroxyalkanoate biopolymers.

In another embodiment, the protein coding sequences encode different subunits of a single enzyme or multienzyme complex. Preferred two subunit enzymes include the two subunit PHA synthases. Preferred multi-enzyme complexes include the fatty acid oxidation complexes. In another embodiment, the protein coding sequences encode proteins which impart insect and/or pest resistance to the plant. In the case of a protein coding for insect resistance, a Bacillus thuringenesis toxin is preferred, in the case of a herbicide resistance gene, the coding sequence imparts resistance to glyphosate, sulphosate or Liberty herbicides.

Marker Genes Selectable marker genes useful in practicing the methods described herein include proteins conferring antibiotic resistance, herbicide resistances, and detectable proteins such as the green fluorescent protein. Examples of antibiotic resistance genes include the neomycin phosphotransferase gene nptlI Patent No. 5,034,322 and No. 5,530,196), hygromycin resistance gene 5,668,298), and the bar gene encoding resistance to phosphinothricin Patent No. 5,276,268). EP 0 530 129 Al describes a positive selection system which enables the transformed plants to outgrow the non-transformed lines by expressing a transgene encoding an enzyme that activates an inactive compound added to the growth media. U.S. Patent No. 5,767,378 describes the use of mannose or xylose for the positive selection of transgenic plants.

Representative screenable marker genes useful for practicing the methods described herein include the P-glucuronidase gene (Jefferson et al., EMBO J.

6:3901-07 (1987); U.S. Patent No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., Trends Biochem Sci. 20:448-55 WO 00/78985 PCT/US00/17197 (1995); Pang et al., Plant Physiol. 112:893-900 (1996)). Some of these markers have the added advantage of introducing a trait, herbicide resistance, into the plant of interest, thereby providing an additional agronomic value on the input side.

Enzymes required for Production of Polvhydroxvalkanoates Polyhydroxyalkanoates Several types of PHAs are known. It is useful to broadly divide the PHAs into two groups according to the length of their side chains and according to their pathways for biosynthesis. Those with short side chains, such as polyhydroxybutyrate (PHB), a homopolymer of R-3-hydroxybutyric acid units, are crystalline thermoplastics; PHAs with long side chains are more elastomeric. The former polymers have been known for about seventy years (Lemoigne Roukhelman 1925), while the latter polymers are a relatively recent discovery (deSmet, et al., J Bacteriol., 154:870-78 (1983)).

Before this designation, however, PHAs of microbial origin containing both R-3-hydroxybutyric acid units and longer side chain units from C5 to C16 were identified (Wallen Rowheder, Environ. Sci. Technol., 8:576-79 (1974)). A number of bacteria which produce copolymers of D-3hydroxybutyric acid and one or more long side chain hydroxyacid units containing from five to sixteen carbon atoms have been identified more recently (Steinbuchel Wiese, Appl. Microbiol. Biotechnol., 37:691-97 (1992); Valentin et al., Appl. Microbiol. Biotechnol., 36: 507-14 (1992); Valentin et al., Appl. Microbiol. Biotechnol., 40:710-16 (1994); Abe et al., Int. J. Biol. Macromol., 16:115-19 (1994); Lee et al., Appl. Microbiol.

Biotechnol., 42:901-09 (1995); Kato et al., Appl. Microbiol. Biotechnol., 45:363-70 (1996); Valentin et al., Appl. Microbiol. Biotechnol., 46:261-67 (1996); U.S. Patent No. 4,876,331 to Doi). Useful examples of specific twocomponent copolymers include PHB-co-3-hydroxyhexanoate (Brandl et al., Int. J. Biol. Macromol., 11:49-55 (1989); Amos Mclnerey, Arch.

Microbiol., 155:103-06 (1991); U.S. Patent No. 5,292,860 to Shiotani et al.).

Other representative PHAs are described in Steinbichel Valentin, FEMS Microbiol. Lett., 128:219-28 (1995). Chemical synthetic methods have also WO 00/78985 PCT/US00/17197 been applied to prepare racemic PHB copolymers of this type for -applications testing(PCTW--952Q614,_PCT WO 95/20615, and PCTWO 96/20621).

Useful molecular weights of the polymers are between about 10,000 and 4 million Daltons, and preferably between about 50,000 and 1.5 million Daltons. The PHAs preferably contain one or more units of the following formula: -OCR'R2CRR 4 )nCOwherein n is 0 or an integer; and wherein R 2

R

3 and R 4 are independently selected from saturated and unsaturated hydrocarbon radicals, halo- and hydroxy- substituted radicals, hydroxy radicals, halogen radicals, nitrogen-substituted radicals, oxygen-substituted radicals, and hydrogen atoms.

Monomeric units generally include hydroxybutyrate, hydroxyvalerate, hydroxyhexanoate, hydroxyheptanoate, hydroxyoctanoate, hydroxynonanoate, hydroxydecanoate, hydroxyundecanoate, and hydroxydodecanoate units. PHAs can include monomers and polymers and derivatives of 3-hydroxyacids, 4-hydroxyacids and Enzymes for Polymer Production The following is a general description of enzymes useful for polymer production in a plant.

ACP-CoA transacylase refers to an enzyme capable of converting phydroxy-acyl ACPs to p-hydroxy-acyl CoAs, such as phaG encoded protein from Pseudomonasputida (Rehm, et al., J. Biol. Chem. 273:24044-51 (1998)). PHA synthase refers to a gene encoding an enzyme that polymerizes hydroxyacyl CoA monomer units to form polymer. Examples of PHA synthases include a synthase with medium chain length substrate specificity, such as phaCI from Pseudomonas oleovorans (Peoples Sinskey, PCT WO 91/00917; Huisman, et al.,J. Biol. Chem. 266:2191-98 (1991)) or Pseudomonas aeruginosa (Timm Steinbuchel, Eur. J. Biochem.

209:15-30 (1992)) or the PHB synthase from Alcaligenes eutrophus with short chain length specificity (Peoples Sinskey J. Biol. Chem. 264:15298- ~W i fr 'u Y M&i~ y ,iI lvWr ,:AIm' WO 00/78985 PCT/US00/17197 303 (1989)). A range of PHA synthase genes and genes encoding enzymes involved in additional steps in PHA biosynthesis are described in Madison Huisman, Microbiology and Molecular Biology Reviews 63:21-53 (1999)).

An a subunit of P-oxidation enzyme complex refers to a multifunctional enzyme that minimally possesses hydratase and dehydrogenase activities (Figure The subunit may also possess epimerase and A3-cis, A2-trans isomerase activities. Examples of a subunits of Poxidation are FadB from E. coli (DiRusso, J. Bacteriol. 172:6459-68 (1990)), FaoA from Pseudomonasfragi (Sato, et al., J. Biochem. 111:8-15 (1992)), and the E. coli open reading frame f714 that contains homology to multifunctional a subunits of P-oxidation (Genbank Accession 1788682).

A p subunit of P-oxidation enzyme complex refers to a polypeptide capable of forming a multifunctional enzyme complex with its partner a subunit. The P subunit possesses thiolase activity (Figure Examples of P subunits are FadA from E. coli (DiRusso, J. Bacteriol. 172:6459-68 (1990)), FaoB from Pseudomonasfragi (Sato, et al., J. Biochem. 111:8-15 (1992)), and the E. coli open reading frame f436 that contains homology to a subunits of P-oxidation (Genbank Accession AE000322; gene b2342).

A reductase refers to an enzyme that can reduce p-ketoacyl CoAs to R-3-OH-acyl CoAs, such as the NADH dependent reductase from Chromatium vinosum (Liebergesell Steinbuchel, Eur. J. Biochem.

209:135-50 (1992)), the NADPH dependent reductase from Alcaligenes eutropus (Peoples Sinskey J. Biol. Chem. 264:15293-97 (1989)), or the NADPH reductase from Zoogleola ramigera (Peoples, et al., J. Biol. Chem.

262:97-102 (1987); Peoples Sinskey J Molecular Microbiol. 3:349-57 (1989)).

A p-ketothiolase refers to an enzyme that can catalyze the conversion of acetyl CoA and an acyl CoA to a p-ketoacyl CoA, a reaction that is reversible (Figure An example of such a thiolase is PhaA from Alcaligenes eutropus (Peoples Sinskey J. Biol. Chem. 1989, 264:15293- 97).

L V-119014 IW&WA%' MOAMM <rn"i k"I. ,Uv F WO 00/78985 PCTIUS00/17197 An acyl CoA oxidase refers to an enzyme capable of converting saturated acyl CoAs to A2 unsaturated acyl CoAs (Figure Examples of acyl CoA oxidases are POXI from Saccharomyces cerevisiae (Dmochowska, et al., Gene 88:247-52 (1990)) and ACX1 from Arabidopsis thaliana (Genbank Accession AF057044).

A catalase refers to an enzyme capable of converting hydrogen peroxide to hydrogen and oxygen. Examples of catalases are KatB from Pseudomonas aeruginosa (Brown, et al., J. Bacteriol. 177:6536-44 (1995)) and KatG from E. coli (Triggs-Raine Loewen, Gene 52:121-28 (1987)).

Multi-step enzyme pathways are known for the biosynthesis of PHA copolymers from normal cellular metabolites and are particularly suited to the methods and constructs described herein. For example, pathways for incorporation of 3-hydroxyvalerate are described in PCT WO 98/00557.

Pathways for incorporation of 4-hydroxybutyrate are described in PCT WO 98/36078 and PCT WO 99/14313.

Plants The transformation of suitable agronomic plant hosts using these vectors can be accomplished by a range of methods and plant tissues.

Representative plants suitable for carrying out the methods include the Brassica family including napus, rappa, sp. carinata andjuncea; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards including Sinapis alba; and flax. Representative tissues which are suitable for transformation using these vectors include protoplasts, cells, callus tissue, leaf discs, pollen, and meristems.

II. Methods Transformation Processes Examples of suitable transformation procedures include Agrobacterium-mediated transformation, biolistics, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome-mediated transformation, and silicon fiber-mediated transformation Patent No. 5,464,765; Gene Transfer to Plants, Potrykus Spangenberg, eds., Springer-Verlag Berlin Heidelberg New York (1995); WO 00/78985 PCT/US00/17197 Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins. Owen Pen, eds., John Wiley Sons Ltd. England (1996); and Methods in Plant Molecular Biology-a laboratory course manual, Maliga, et al., eds., Cold Spring Laboratory Press, New York (1995).

In order to generate transgenic plants using the constructs of the present invention, following transformation by any one of the methods described above, the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene at such that the level of desired polypeptides is obtained in the desired tissue and cellular location.

For the specific crops useful for practicing the described invention, transformation procedures have been established (Gene Transfer to Plants, Potrykus Spangenberg, eds., Springer-Verlag Berlin Heidelberg New York (1995); Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins, Owen Pen, eds., John Wiley Sons Ltd.

England (1996); and Methods in Plant Molecular Biology-a laboratory course manual, Maliga, et al., eds., Cold Spring Laboratory Press, New York (1995)).

Brassica napus can be transformed as described, for example, in U.S.

Patents No. 5,188,958 and No. 5,463,174. Other Brassica such as rappa, carinata andjuncea, as well as Sinapis alba, can be transformed as described in Moloney et al., Plant Cell Reports 8:238-42 (1989)). Soybean can be transformed by a number of procedures, such as those described in U.S.

Patents No. 5,015,580; No. 5,015,944; No. 5,024,944; No. 5,322,783; No.

5,416,011; and No. 5,169,770).

A number of transformation procedures have been reported for the production of transgenic maize plants including pollen transformation (U.S.

Patent No. 5,629,183), silicon fiber-mediated transformation Patent No. 5,464,765), electroporation of protoplasts Patents No. 5,231,019; No. 5,472,869; and No. 5,384,253), gene gun Patents No. 5,538,877 WO 00/78985 PCT/US00/17197 and No. 5,538,880), and Agrobacterium-mediated transformation (EP 0 604 662Al; PCT WO 94/00977). The Agrobacterium-mediated procedure is particularly preferred, as single integration events of the transgene constructs are more readily obtained using this procedure which greatly facilitates subsequent plant breeding.

Cotton can be transformed by particle bombardment Patents No. 5,004,863 and No. 5,159,135). Sunflower can be transformed using a combination of particle bombardment and Agrobacterium infection (EP 0 486 233 A2; U.S. Patent No. 5,030,572). Flax can be transformed by either particle bombardment or Agrobacterium-mediated transformation.

Recombinase technologies which are useful in practicing the methods described herein include the cre-lox FLP/FRT and Gin systems. Methods by which these technologies can be used for the purposes described herein are described, for example, in U.S. Patent No. 5,527,695; Dale Ow, Proc.

Natl. Acad. Sci. USA 88:10558-62 (1991); and Medberry et al., Nucleic Acids Res. 23:485-90 (1995).

Producing Plants Containing Multiple Stacked Input Traits The production of a plant that is tolerant to the herbicide glyphosate and that produces the Bacillus thuringiensis (BT) toxin can be used to illustrate the usefulness of poly-cistronic vectors for the creation of plants with stacked input traits. Glyphosate is a herbicide that prevents the production of aromatic amino acids in plants by inhibiting the enzyme enolpyruvylshikimate-3-phosphate synthase (EPSP synthase). The overexpression of EPSP synthase in a crop of interest allows the application of glyphosate as a weed killer without killing the genetically engineered plant (Suh, et al., Plant Mol. Biol. 22:195-205 (1993)). BT toxin is a protein that is lethal to many insects, providing the plant that produces it protection against pests (Barton, et al., Plant Physiol. 85:1103-09 (1987)). Combining the two traits into one plant using a polycistronic IRES containing vector enables the production of stacked traits in a single transformation event, a major improvement over current art.

IVIV41"AWTOW "100"W"WA 09VIVIVW-KIIII WO 00/78985 PCT/US00/17197 The compositions and methods described herein will be further understood with reference to the following non-limiting examples.

Example 1: IRES Containing Bicistronic Constructs The in vivo expression of two proteins from an IRES containing bicistronic construct with only one promoter and one polyadenylation signal can be demonstrated in protoplast transient expression assays.

A. Preparation of Constructs A suitable construct contains the following genetic elements (see Figure a promoter active in leaves such as the 35S-C4PPDK light inducible plant promoter (Sheen, J. EMBO, 12:3497-505 (1993)); a gene encoding P-glucuronidase (GUS) (Jefferson, et al., EMBO J 6:3901-07 (1987)); an IRES; a gene encoding an enhanced green fluorescent protein (EGFP; Clontech, Palo Alto, CA); and a polyadenylation signal.

To demonstrate the functionality of various IRES sequences in promoting translation of the second coding region in bicistronic constructs, plasmid pC4PPDK-GUS-TEV-EGFP was constructed. This construct allows the easy replacement of the tobacco etch virus (TEV) enhancer (Carrington Freed, J. Virology 1990, 64:1590-97), cloned between genes encoding GUS and EGFP, with any IRES sequence flanked by a 5' BamHI and a 3' Nco I restriction site. Plasmid pC4PPDK-GUS-TEV-EGFP is unable to express GFP since the TEV enhancer is not able to promote cap-independent translation of the second gene in the bicistronic construct. Replacement of the TEV enhancer with a functional IRES upstream of the EGFP gene allows GFP expression.

Plasmid pC4PPDK-GUS-TEV-EGFP was constructed using the following multi-step procedure. A fragment containing the 35S-C4PPDK promoter was excised from plasmid 35S-C4PPDK-GFP (Sheen et al., Plant Journal, 8:777-84 (1995)) as an XhoI/BamHI fragment and inserted into the equivalent sites of plasmid pIRES2.EGFP (Clontech, Palo Alto, California) forming plasmid pIRES2/C4PPDK. Plasmid plRES2/C4PPDK contains the 35S-C4PPDK promoter inserted in front of the IRES from the ~W t~4~i, W4~fl ~f~f WO 00/78985 PCT/US00/17197 encephalomyocarditis virus (EMCV), the EGFP protein, and the polyadenylation signal of the bovine growth hormone.

A fragment containing the 35S-C4PPDK promoter, the EMCV IRES, and the EGFP gene was released from plasmid pIRES2/C4PPDK by digestion with Xho I/Not I and cloned into the Xho I/Not I sites of C4PPDK-GFP forming plasmid pUC18/C4PPDK-EMCVIRES-EGFP-nos.

Plasmid pUC18/C4PPDK-EMCVIRES-EGFP-nos contains the C4PPDK promoter, the EMCV IRES, and the EGFP gene in front of the plant polyadenylation signal of plasmid 35S-C4PPDK-GFP.

The EMCV-IRES of plasmid pUC 18/C4PPDK-EMCVIRES-EGFPnos was replaced with a fragment encoding the TEV enhancer (Carrington Freed) by digesting plasmid pUC 18/C4PPDK-EMCVIRES-EGFP-nos with BamHI and Nco I. The TEV enhancer was ligated into the previously prepared vector DNA forming pC4PPDK-TEV-EGFP. Plasmid pC4PPDK- TEV-EGFP contains the C4PPDK promoter, the TEV enhancer, the EGFP gene, and a plant polyadenylation signal.

A fragment encoding GUS was ligated into the BamHI site of pC4PPDK-TEV-EGFP as follows. Plasmid pBI 101.2 (Clontech, Palo Alto, CA), containing GUS flanked by SacI and BamHI sites, was digested with SacI and the protruding ends were filled in with Klenow. BamHI linkers were attached to the blunt-ended fragments with T4 ligase. The sample was digested with BamHI resulting in the formation of a GUS fragment flanked by BamHI restriction sites. The GUS gene was cloned into the BamHI site of plasmid pC4PPDK-TEV-EGFP forming plasmid pC4PPDK-GUS-TEV-

EGFP.

IRES sequences can be tested for functionality in vivo in plants by removing the TEV enhancer with BamHI and Nco I and inserting the sequence to be tested as a BamHI/Nco I fragment. EGFP, the second coding region in the bicistronic construct, requires the insertion of a functional IRES immediately upstream in order for expression of EGFP to occur.

WO 00/78985 PCT/US00/17197 B. Expression of the GUS and EGFP Genes SExpression of the GUS and EGFP genes in IRES containing bicistronic constructs were tested using the following protoplast transient expression procedure. Two well-expanded leaves from 4-6 week old plants of Arabidopsis thaliana were harvested and the leaves were cut perpendicularly, with respect to the length of the leaf, into the smallest strips possible. The cut leaves were transferred to 20 mL of a solution containing 0.4 M mannitol and 10 mM MES, pH 5.7, in a 250 mL side armed flask.

Additional leaves were cut such that the total number of leaves processed was between 50 and 60. After all leaves were cut, the solution in the flask was removed with a pipette and 20 mL of a cellulase/macerozyme solution was added. The enzyme solution was prepared as follows: 8.6 mL of H20, mL of 0.8 M mannitol, and 400 uL 0.5M MES, pH 5.7, were mixed and heated to 55 0 C. R-10 cellulase (0.3 g, Serva) and R-10 macerozyme (0.08 g, Serva) was added and the solution was mixed by inversion. The enzyme solution was incubated at room temperature for 10-15 min. A 400 pL aliquot of 1M KCI and 600 uL of 1M CaC12 were added to the enzyme solution, mixed, and the resulting solution was sterile filtered through a 0.2 pM filter.

After addition of the enzyme solution, the flask was swirled gently to mix the leaf pieces and a house vacuum was applied for 5 minutes. Prior to releasing the vacuum, the flask was swirled gently to release air bubbles from the leaf cuts. The leaves were digested for 2-3 hours at room temperature.

Protoplasts were released from the leaves by gently swirling the flask for 1 min and filtering the protoplast containing solution was filtered through nylon mesh (62 uM mesh). The eluent was transferred to a sterile, screw top, 40 mL conical glass centrifuge tube and centrifuged at 115 g for 2 min.

The supernatant was removed with a Pasteur pipette and 10 mL ice cold solution was added (W5 solution containing 154 mM NaC1, 125 mM CaC1 2 mM KC1, 5 mM glucose, and 1.5 mM MES, pH The sample was mixed by rocking the tube end over end until all of the pellet was in solution.

The sample was centrifuged as described above and the supematant was removed with a Pasteur pipette. The protoplast pellet was resuspended in 'A WINAX(MM'", IffiarMyNt WO 00/78985 PCT/US00/17197 mL of ice cold W5. The sample was incubated for 30 minutes on ice so that _the protoplasts became competent for transformation. Intact protoplasts were quantitated using a hemacytometer. The protoplasts were isolated by centrifugation and resuspended in an ice cold solution containing 0.4 M mannitol, 15 mM MgCI 2 and 5 mM MES, pH 5.7, to approximately 2 x 10 6 protoplasts/mL.

Plasmid DNA samples (160 Vg, 1 pg/utL stock) for transformation were placed in 40 mL glass conical centrifuge tubes. An aliquot of protoplasts (800 ViL) was added to an individual tube followed immediately by 800 1 L of a solution containing 40% PEG 3350 0.4 M mannitol, and 100 mM Ca (NO 3 2 The sample was mixed by gentle inversion and the procedure repeated for remaining samples.

All transformation tubes were incubated at room temperature for minutes. Protoplasts samples were diluted sequentially with 1.6 mL, 4 mL, 8 mL, and 12 mL of W5 solution. Between each dilution step, the sample was gently mixed by inversion and incubated at room temperature for 5 minutes.

Protoplasts were harvested by centrifugation (115 g) and the supernatant was carefully removed with a Pasteur pipette. Protoplasts were resuspended in 4 mL of a solution containing 0.5 M mannitol, 5 mM MES, pH 5.7, 20 mM KC1, and 5 mM CaC12. For transient expression of the transformed DNA, the samples were incubated at room temperature for 16 hours under a Watt table top plant light.

GUS activity in protoplasts can be measured as described, for example, in Jefferson, Plant Mol. Biol. Rep. 5:387-405 (1987). For detection of GFP expression, Arabidopsis protoplast samples can be analyzed by fluorescent microscopy Sheen, et al., Plant J, 8:777-84 (1995)).

Protoplasts expressing GFP fluoresce under green light such that transformed cells appear green. Untransformed cells are red due to the autofluorescence of the chloroplasts. Alternatively, GFP expression can be analyzed by Western detection of the protein.

Samples from transient expression experiments were prepared for Western analysis as follows. Protoplasts were harvested by centrifugation WO 00/78985 PCTIUS00/17197 (115 g) and the supernatant removed. An aliquot (14 jiL) of 7X stock of protease inhibitor stock was added to the sample and the sample brought to a final volume of 100 jgL with a solution containing 0.5 M mannitol, 5 mM MES, pH- 5.7, 20 mM KC1, 5 mM CaC12. The 7X stock of protease inhibitors was prepared by dissolving one "Complete Mini Protease Inhibitor Tablet" (Boehringer Manneheim) in 1.5 mL 0.5 M mannitol, 5 mM MES, pH 5.7, 20 mM KCI, 5 mM CaCI 2 The protoplasts were disrupted in a 1.5 mL centrifuge tube using a pellet pestle mixer (Kontes) for 30 s. Soluble proteins were separated from insoluble proteins by centrifugation at maximum speed in a microcentrifuge (10 min, 4 The protein concentration of the soluble fraction was quantitated using the Bradford dyebinding procedure with bovine serum albumin as a standard (Bradford, Anal.

Biochem. 1976, 72:248-54). The insoluble protein was resuspended in 100 pL IX gel loading buffer (New England Biolabs, Beverly, MA) and a volume equal to that loaded for the soluble fraction was prepared for analysis. Samples from the soluble and insoluble fractions of the protoplast transient expression experiment, as well as standards of green fluorescent protein (Clontech, Palo Alto, CA), were resolved by SDS-PAGE and proteins were blotted onto PVDF. Detection of transiently expressed proteins was performed by Western analysis using Living Colors Peptide Antibody to GFP (Clontech, Palo Alto, CA) and the Immun-Star Chemiluminescent Protein Detection System (BioRad, Hercules, CA).

The results demonstrated that the proteins were expressed.

Example 2: Transformation of Tobacco Plants In Pseudomonads of the RNA I homology group, PHAs consisting of saturated medium-chain-length 3-hydroxyalkanoic acids are synthesized from monomer units that are thought to be derived from acyl carrier protein (ACP) intermediates of de novo fatty acid biosynthesis (Eggink, et al., FEMS Microbiol. Rev. 1992, 103:159-64; Saito Doi, Int. J. Biol. Macromol.

1993, 15:287-92; Huijberts, et al., J. Bacteriol. 1994, 176:1661-66). A transacylase that is capable of converting acyl ACPs to acyl CoAs has recently been cloned (Rehm, et al., J. Biol. Chem. 1998, 273:24044-51).

WATIMUNO ANWOVOW"4101A WO 00/78985 PCT/US00/17197 Targeting of both the transacylase and a medium chain length synthase to the chloroplasts of a plant, the site of fatty acid biosynthesis, should divert 3hydroxyacyl ACPs from fatty acid biosynthesis to polymer (Figure 7).

Expression of all constructs can be targeted to the leaves, using a leaf specific promoter, or the seeds, using a seed specific promoter.

The genes encoding PhaC and PhaG can be inserted into a plant in a single transformation event using a multi-gene construct where each gene is flanked by its own promoter or polyadenylation signal. A suitable construct for this purpose would include the following genetic elements (Figure a leaf specific promoter, a chloroplast targeting signal, the coding sequence of PhaG from Pseudomonas putida, a polyadenylation sequence, a leaf specific promoter, a chloroplast targeting signal, the coding sequence of a medium chain length PhaC, and a polyadenylation sequence. The construct can be transformed into a plant, such as tobacco, for PHA production in leaves.

Alternatively, a construct containing only one promoter, one polyadenylation signal, and an IRES can be constructed. A suitable construct for this purpose would include the following genetic elements (Figure 9a): a leaf specific promoter, a chloroplast targeting signal fused to a gene encoding a ACP-CoA transacylase, an IRES sequence, a chloroplast targeting signal fused to a gene encoding a PHA synthase that accepts medium chain length substrates, and a polyadenylation signal at the extreme 3' end of the cassette. The construct can be transformed into a plant, such as tobacco, for PHA production in leaves.

Constructs were transformed into tobacco using the following procedure. In a laminar flowhood under aseptic conditions, leaves from a tobacco plant were sterilized for 15 minutes in a one liter beaker containing a solution of 10% bleach and 0.1% Tween 20. The sterilized leaves were washed in one liter of water for 10 minutes, the water decanted, and the washing step repeated two additional times. The intact part of the leaves were cut in small pieces with a scalpel, avoiding any injured areas of the leaves. An aliquot (20 mL) of MS-suspension was mixed with 5 mL of an overnight culture of Agrobacterium, carrying the construct to be transformed 23 ;mamlld!*Ml t igw l lle4rimritAlt "10A "M AT'* P111"WIN- WO 00/78985 PCT/USOO/17197 [MS-suspension contains (per L) 4.3 g MS salts, 1 mL of B5 vitamins __Sigma, St. Louis, MO), 30_g sucrose, 2.mgp-chlorophenoxyacetic acid, and 0.05 mg kinetin, pH The tobacco leaf pieces were introduced into the solution and vortexed for a few seconds. The leaves were removed, wiped on sterile filter paper, and placed in a petri dish to remove the excess Agrobacterium solution. An aliquot (1 mL) of tobacco cell culture was added on top of solidified MS-104 medium in a petri dish and a sterile piece of filter paper was placed directly on the top of the culture [MS-104 medium contains (per L) 4.3 g MS salts, pH 5.8, 1 mL B5 vitamins, 30 g sucrose, 1 mg benzylaminopurine, 0.1 mg napthalene acetic acid, and 8 g of phytagar].

The tobacco leaf pieces were placed on top of the filter and incubated for two days at 25 The leaf pieces were transferred, face-up, to a petri dish containing MS-selection medium and gently pressed into the medium [MSselection medium contains (per L) 4.3 g MS salts, pH 5.8, 1 mL B5 vitamins, 30 g sucrose, 1 mg benzylaminopurine, 0.1 mg napthalene acetic acid, 500 mg of carbenicillin, appropriate drug for selection of resistance of transformation vector, and 6.5 g ofphytagar]. The dishes were wrapped with parafilm and incubated at 25 OC for 3 weeks. The leaves were transferred to fresh MS-selection medium and incubation at 25 °C is continued until plantlets appear. Plantlets were separated from the callus and placed in testtubes (24 x 3 cm) containing 10 mL of MS-rooting medium [MS-rooting medium contains (per L) 4.3 g MS salts, pH 5.8, 1 mL B5 vitamins, 30 g sucrose, and 6.5 g ofphytagar]. When roots were 1 cm in length, the transformed plants were transferred to soil and covered with an inverted, transparent, plastic cup in which a hole had been pierced in the bottom.

After 4 or 5 days, the cup was removed and transformed tobacco plants were grown under standard conditions.

The results demonstrated expression in the chloroplasts of the leaves.

Example 3: PHA Formation in the Peroxisomes of Plant Leaves Pseudomonads, such as Pseudomonas oleovorans, are also capable of producing medium chain length polymers when grown in the presence of fatty acids (Lageveen et al., Appl. Environ. Microbiol. 54:2924-32 (1988); fflWftI#VM# WINIW#Mq Rjfflj##W MWWW AR-1 OW WAr lip At RIP" Ramsay, et al., Appl. Environ. Microbiol. 57: 625-29 (1991)). The 3-hydroxyacyl CoA monomer units are derived from intermediates formed during degradation of the fatty acid to acetyl CoA using P-oxidation pathways (Figure 2).

Medium chain length PHAs can be formed from fatty acids in the peroxisomes of leaves. A suitable construct for this purpose contains a leaf specific promoter, a gene encoding a PHA synthase that accepts medium chain length substrates fused to a peroxisomal targeting signal, an IRES, a multifunctional a subunit of P-oxidation fused to a peroxisomal targeting signal, and a polyadenylation signal. Plants naturally contain a and p subunits of P-oxidation in the peroxisomes that convert fatty acyl CoAs to acetyl CoA. It has been demonstrated that targeting of a medium chain length synthase to the peroxisomes ofArabidopsis thaliana (Mittendorf, et al., Proc. Natl. Acad. Sci.

USA 95: 13397-402 (1998)) or tobacco plants (Hahn, J. Ph. D. Thesis, University of Minnesota, Feb. 1998) produce a small amount of polymer. The expression of a transgene encoding an additional a subunit of P-oxidation increases the ratio of a subunits, containing activities capable of monomer formation, to P-subunits, containing S a thiolase activity that will channel the substrate down the degradative pathway (Figure This increased ratio may help to divert more carbon from fatty acid degradation to i PHA formation in the peroxisomes. The construct described above can be transformed into crops for the production of medium chain length polymer in the peroxisomes of leaves. For example, tobacco can be transformed as previously described.

Example 4: Constructs Transformed into Brassica napus The yields of polymer previously observed in Arabidopsis peroxisomes upon targeting a medium chain length synthase to the peroxisomes (Mittendorf, et al., Proc.

Natl. Acad. Sci. USA 1998,95: 13397402) may be low due to the rapid degradation of o 25 fatty acids to acetyl CoA, preventing diversion of 3-hydroxyacyl CoAs to PHAs (Figure Creation of an artificial p-oxidation pathway in the cytosol would allow the optimal .enzymes to be chosen for both the degradation of fatty acids to the appropriate chain length and for the diversion of carbon from the fatty acid degradation pathway to polymer formation. A suitable construct for this purpose includes a seed specific promoter, a gene encoding acyl CoA oxidase in which the peroxisomal targeting signal has been removed, an IRES, a gene encoding a medium chain length synthase, an IRES, a gene encoding an oc subunit of P-oxidation, an IRES, a gene encoding a P subunit of P-oxidation, and a polyadenylation signal.

The construct can be transformed into oil seed crops for PHA production within the seeds. For example, the constructs can be transformed into Brassica napus using the following procedure (Moloney et al., Plant Cell, 8: 238-42 (1989)). Seeds of Brassica napus cv. Westar were surfaced sterilized in 10% commercial bleach (Javex, Colgate Palmolive Canada Inc.) for 30 min. with gentle shaking. The seeds were washed three times in sterile distilled water. Seeds were placed on germination medium comprising Murashige-Skoog (MS) salts and vitamins, 3% sucrose and 0.7% phytagar, pH 5.8 at a density of 20 per plate and maintained at 24 0 C in a 16 h light/8 h dark photoperiod at a light intensity of 60-80 Em- 2 s-1 for 4-5 days. Constructs were introduced into Agrobacterium tumefaciens strain EHA101 (Hood et al, J. Bacteriol.

1986, 168: 1291-301) by electroporation. Prior to transformation of cotyledonary petioles, single colonies of strain EHA101 harboring each construct were grown in 15 mL of minimal medium, supplemented with the appropriate selection antibiotics for the transformation vector, for 48 h at 28 0 C. One mL of bacterial suspension was pelleted by centrifugation for 1 min in a microfuge. The pellet was resuspended in 1 mL minimal medium.

For transformation, cotyledons were excised from 4 to 5 day old seedlings so that they included approximately2 mm of petiole at the base. Individual cotyledons with the cut surface of their petioles were immersed in diluted bacterial suspension for 1s and immediately embedded to a depth of approximately 2 mm in co-cultivation medium, MS medium with 3% sucrose and 0.7% phytagar, enriched with benzyladenine. The inoculated cotyledons were plated at a density of 10 per plate and 25 incubated under the same growth conditions for 48 h. After co-cultivation, the cotyledons were transferred to regeneration medium comprising MS medium supplemented with 3% sucrose, 20 pM benzyladenine, 0.7% phytagar, pH 5.8, 300 mg/L timentinin and the appropriate antibiotics for selection of the plant transformation vector.

After 2-3 weeks, regenerant shoots were obtained, cut, and maintained on 'shoot elongation' medium (MS medium containing 3% sucrose, 300 mg/L timentin, 0.7% 26 ~f Ti i~j. ~WT k~f~0I~ phytagar and the appropriate antibiotic) in Magenta jars. The elongated shoots were transferred to 'rooting' medium comprising MS medium, 3% sucrose, 2 mg/L indole butyric acid, 0.7% phytagar and 500 mg/L carbenicillin. After the emergence of roots, plantlets were transferred to potting mix (Redi Earth, W. R. Grace Co. Canada Ltd.).

The plants were maintained in a misting chamber (75% relative humidity) under the same growth conditions.

The results demonstrate expression.

Example 5: Cytosolic Production of Short Chain PHAs in Leaves Short chain PHAs can be produced by co-expressing enzymes that divert acetyl CoA from a plant's metabolism with a synthase that accepts short chain length substrates. p-ketothiolase and acetoacetyl CoA reductase will convert acetyl CoA to R- 3-hydroxybutyryl CoA, the substrate for PHA synthase. Since acetyl CoA is found in multiple tissues and organelles in the plant, a multitude of strategies can be developed for short chain production by using the appropriate organelle targeting signals and/or tissue specific promoters. For the cytosolic production of short chain PHAs in leaves, an appropriate expression cassette includes the following: a leaf specific promoter, a reductase, an IRES, a synthase that accepts short chain substrates, and a polyadenylation signal at the extreme 3' end of the cassette. PHB production has been demonstrated in the leaves ofArabidopsis thaliana by inserting transgenes expressing synthase and reductase while depending on the endogenous thiolase in the cytoplasm for conversion of acetyl CoA to acetoacetyl CoA (Poirier, et al., Science 1992,256: 520-23). Insertion of a transgene expressing thiolase into the construct may increase the yield of PHB in the cytoplasm of the leaves. For this purpose, an additional IRES element as well as a nucleotide sequence encoding p-ketothiolase, can be inserted in the construct. Both the 25 expression cassette and the construct containing the additional IRES element and nucleotide sequence encoding P-ketothiolase, can be transformed into tobacco plants, as previously described, for production of short chain PHAs in tobacco leaves.

Example 6: Cytosolic Production of Short Chain PHAs in Oilseeds Short chain PHAs can also be produced in the cytosol of oilseeds. A seed specific promoter construct would be an appropriate expression cassette, and could include the following: a seed specific promoter, a reductase, an IRES, a synthase that .A ;~uu sMUMTh w4msa 541 wN accepts short chain substrates, and a polyadenylation signal at the extreme 3' end of the cassette. Insertion of a transgene expressing thiolase may increase the yield of PHB in the cytoplasm of seeds. For this purpose, an additional IRES element, as well as a gene expressing P-ketothiolase, can be inserted in the seed specific promoter construct described above.

Example 7: Short Chain PHA Production in the Chloroplasts of Leaves For short chain PHA production in the chloroplasts of leaves, an appropriate expression cassette includes the following: a leaf specific promoter, a chloroplast targeting signal fused to a reductase, an IRES, a chloroplast targeting signal fused to a synthase that accepts short chain substrates, and a polyadenylation signal at the extreme 3' end of the cassette. Insertion of a transgene expressing thiolase may increase the yield of PHB in the chloroplasts of the leaves. For this purpose, an additional IRES element as well as a gene expressing P-ketothiolase fused to a chloroplast targeting signal, can be inserted into the construct. Both the expression cassette and the construct containing the additional IRES element and nucleotide sequence encoding 3ketothiolase, can be transformed into plants, such as tobacco, for production of short chain PHAs in the chloroplasts of leaves.

Example 8: Short Chain PHA Production in the Plastids of Oilseeds For short chain PHA production in the plastids of oilseeds, an appropriate expression cassette includes the following: a seed specific promoter, a chloroplast targeting signal fused to a reductase, an IRES, a chloroplast targeting signal fused to a synthase that accepts short chain substrates, and a polyadenylation signal at the extreme 3' end of the cassette. Insertion of a transgene expressing thiolase may increase the yield of PHB in the plastids of seeds. For this purpose, an additional IRES element as 25 well as a gene expressing P-ketothiolase fused to a chloroplast targeting signal, can be inserted in the construct. The above constructs can be transformed into an oil seed crop, such Brassica napus whose transformation has been described above, for production of short chained PHAs in the plastids of oil seeds.

Example 9: Short Chain PHA Production in the Peroxisomes of Leaves Short chain length PHAs can be formed in the peroxisomes of leaves by engineering enzymes that will capture the acetyl CoA as fatty acids are degraded by p- 28 "YOM~~vwridI ~uY*u I~~ga oxidation enzymes. An appropriate construct for targeting reductase and synthase to the peroxisomes of leaves would contain the following: a leaf specific promoter, a gene encoding reductase fused to a peroxisomal targeting signal, an IRES sequence, a gene encoding a short chain synthase fused to a peroxisomal targeting signal, and a polyadenylation signal. The expression of an additional thiolase in the peroxisomes of seeds may serve to increase PHA yield. For this purpose, an additional IRES sequence, as well as a gene encoding a thiolase fused to a peroxisomal targeting signal, can be inserted in the construct.

Example 10: Short Chain PHA Production from Fatty Acids Short chain PHA production from fatty acids can be achieved in the cytosol by engineering an artificial pathway for fatty acid P-oxidation (Figure A suitable construct for this purpose contains the following: a seed specific promoter, a gene encoding an acyl CoA oxidase, an IRES, a gene encoding a short chain length synthase, an IRES, a gene encoding an a-subunit of P-oxidation, an IRES, a gene encoding a atsubunit of P-oxidation, an IRES, a gene encoding a reductase, and a polyadenylation signal. The addition of a p-ketothiolase may allow more PHA to be formed. For this purpose, the above described construct can be modified to contain an additional IRES and a gene encoding a P-ketothiolase.

I: Acyl CoA oxidases form hydrogen peroxide as a byproduct when converting acyl CoAs to A2 unsaturated acyl CoAs. If hydrogen peroxide proves harmful to the plants, a gene encoding catalase can be inserted to convert the hydrogen peroxide to water and oxygen. For this purpose, the initial construct described in this example can be modified to contain an additional IRES sequence and a gene encoding a catalase.

The above constructs can be transformed into oil seed crops, such as Brassica S 25 napus, as previously described.

Example 11: Glyphosate-Resistant Plant A construct in accordance with the invention can be used to create a plant that is resistant to glyphosate and produces BT toxin using a single transformation event. The construct may contain a constitutive promoter that is active in all plant tissues, a gene encoding either wild-type enolpyruvylshikimate-3-phosphate synthase or a glyphosate resistant mutant of the enzyme (Stalker, et al., J. Biol. Chem. 1985,260: 4724-28), an M''Wiro AMMIMMO PXM 6WIM 0% IRES, a gene encoding the Bacillus thuringiensis endotoxin (Schnepf, et al., J Biol.

Chem. 1985, 260: 6264-72 Schnepf, et al., J Biol. Chem. 1985,260: 6273-80), and a polyadenylation signal. This construct can be transformed into any agronomically important crop to provide a plant that is herbicide resistant and that produces its own insecticide.

S

*5*5 h IhZUl~r ~IY*Lil I Ir i~iibjYVI~II~V~~. Y~4~i~iWU~ah~)l l~i n~ili~UihUil)l*I*I liU;lli~li~illl EDITORIAL NOTE APPLICATION NUMBER 54991/00 The following Sequence Listing pages 1 to 9 are part of the description.

The claims pages follow on pages "31" to "34".

WO 00/78985 WO 0078985PCT/JSOO/17197 SEQUENCE LISTING <110> Metabolix, Inc.

<120> Plant Multi-gene Expression Constructs <130> MBX 037 PCT <140> Not Yet Assigned <141> 2000-06-23 <150> 60/140,768 <151> 1999-06-24 <160> 13 <170> Patentln Ver. 2.1 <210> 1 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 1 cgggatccac aacataagaa aaacaacgca aaaac <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 2 catgccatgg agtatgctag taaatgaagg aaat 34 <210> 3 <211> 34 <212> DNA <2 13> Artificial Sequence <220> M~ 2 WO OOn8985 WO 0078985PCTUSOO/17197 <223> Description of Artificial Sequence: Primer <400> 3 cgggatccaa aatataaaaa ctcaacacaa cata 34 .<210> 4 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 4 catgccatgg tgttggtgat tgctttgata acgacaa 37 <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> cgggatccat gttttctttc actgaagcga aatca <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 6 catgccatgg caaatttggg cagaatatac aga 33 <210> 7 <211> 169 <212> DNA <213> Potato virus Y <220>

NO

WO 00/78985 WO 0078985PCT/USOO/17197 <221> misc feature <222> (166)..(168) <223> Nucleotides encoding N-terminal methiolifle <400> 7 acaacataag aaaaacaacg caaaaacact acttgtaaat ttagetttgg tctttttctt cagtttaaca aactatttca tttccttcat tacaaacgct gtgatctttt ttactagcat cattttctct caacgaagca taacgatatt gaaaatcgtc 120 actccatgg 169 <210> 8 <211> 127 <212> DNA <213> Turnip Mosaic Virus <400> 8 aaaatataaa aactcaacac aacatacaca aaacgattaa agcaaacaca atctttcaaa gcattcaaag cattcaagca atcaaagatt ttcaaatctt ttgtcgttat caaagcaatc 120 accaaca 127 <210> 9 <211> 354 <212> DNA <213> Cowpea Mosaic virus <220> <221> misc feature <222> (3) <223> Nucleotides encoding N-terminal methionine <22D> <221> misc feature <222> (352)..(354) <223> Nucleotides encoding N-terminal Methiolifle <400> 9 atgttttctt ttaaataacg ggaggctgct aaatatctc t gctgattggt cgaacttgga <210> <211> 1885 <212> DN~A tcactgaagc tgtacttgtc gttcagcccc acttctgctt tctataagaa gaaagattgt gaaatcaaag ctattcttgt atacattact gacgaggtat atctagtatt taagc ttctg atatctttgt cggtgtggte tgttacgatt tgttgcctgt ttctttgaaa tatattctgc ggacacgtag ttgggaaaag ctgCtgactt acttctttct cagagttttc ccaaatttga tgcggcgcca aaagcttgct tcggcgggtg tcttCttctt ccgtggtttt aatg V 1 WO 00/78985 WO 0078985PCT/USOO/17197 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chloroplast targeting signal form the alfalfa rubisco protein fused to the coding sequence of PhaC from P.

aerugino sa <220> <221> misc feature <222> <223> Nco I restriction site <220> <221> misc feature <222> <223> N-terminal ATG of targeting sequence <220> <221> misc feature <222> (175)..(180) <223> Sph I restriction site <220> <221> misc feature <222> (192) (194) <223> N-terminal ATG of protein <220> <221> <222> <223> misc feature (1878) (1885) Not I restriction site <400> ccatggettc tacaatctgg caaaggtcaa gaaggat ttc cgctgaacct tggtcctgct gcctggagc t gacgcttttc acctggcctg acatcagtcg gcctgagcaa acggcctcgg acatggacgc ctctatgatg cgtggtggct caaagacatt tatgagtcag gaatccggtg ccaggcggtg gaagaacgtc cgatccggcc gcgcaaggag tggccagttc cccggcggcg ccacctggcc cttcgaggtg tcctcttcag ccattcgttg acttccattg aagaacaata atcggcatcc cgccagccgc ctgctcggcc tggagccaga ctgcacagct gtcatcaacc gtcaagcgct aaggacctgg ggcaagaacc ctgtgactac gac taaagtc caagcaatgg aegagcttcc ggggcaagga tgcacagcgc agtcggagc t atccactgta ggatcagcca tgctgaccga tcttcgagac.

tgaacaacgg tggccaccac ggccgatcac.

agttaaccgt aatggctggc tggaagagta caagcaagcc cctgcteac caggcacgtg acgcccaggc caagcgctac cagcgaectg ggcgatgtcg cggcggcaag cgggatgccg cgagggcgc cgagtcggtg gcctc ttcgg ttcccagtta aactgeatgc gcggaaa&a tccgcgcgca gcgcatttca gatgacgacc atgcagacct tcgccgcagg ccgaccaaca agcctgctgg agccaggtgg gtggtgttcc cacgaacgcc gcaacggcgt gctggaactg atccagtacc YOWA' 4"W'R'-A"Tr- WO 00/78985 PTUO/79 PCT/USOO/17197 cgctgctggt agagcctggc acccgaccaa ccatcgaggt ccggcgggat tcaacgcc tt tgttcgccga tggagggcaa actactgggt ggaacaacga agagcaaccc agcaggtgac agtcgtgcta gcggtcacat atccggaac t cgtggtggtt ccgccagcc t atgaacgatg ggtgccgccg gcgcttctgc gtcgcagcgc agtcctgtcg caccaccgcg cacccaactg cgagaagact ggacatggcc caacaactac caccacgcgc gc tgaaccgc ttgcgacttc c aagtcggcc ccagagcatc gcccgccgag gcactggcag gggcaacaag agaattcgcg cagatcaaca c tgc gc aac g gaatggggcc atcaccggca accctggtcg gtcagcgtgc c tggaggccg aaggtgttcg c tgctcggca ctgcccgccg cccggcgccc tac tgtg tcg aggctgctgg ctcaacccac cccaaggcct caatggc tgg acctatccgg gccgc agttctacgt gcgtgcagac tgaccaccta gcaaggacct gccactacgt tcgacttcga ccaagcgtcg cctggatgcg ac cagccgcc cgctgcacgg tggaggtc tc ccggtctgaa gtggcaagtg cgggcaaccc ggctggaaca ccgaacgc tc ccggcgaagc cttcgacctg cttcatagtc tatcgaggcg caacctcctc ggccagcggc actgaatacc ttcctaccag ccccaacgac ggcgttcgac cgagttcgtc cggcacgccc cgaccacatc cgagttcatc caaggcacgc ggccggcaag cggcaagacc cgcgcccgga tcgccggaca agttggcgca ctcaaggagg ggcgcc tgc t gagaagaagg caggtcgcgc tccggcgtgc ctgatctgga atcctctact gaactgttca atcgacctga accccctggg ctctccaaca ttcatgacca cacgccgac t cgcaaggcgc acctacgtgc 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1885 <210> 11 <211> 1232 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Pea Rubisco Chloroplast Targeting Signal <220> <221> misc feature <222> (6) <223> Barn HI restriction site <220> <221> misc feature <222> (9) <223> N-terminal ATG which encodes methionine for targeting signal <220> <221> misc feature <222> (327) .(332) <223> Xba I restriction site <220> <221> misc feature <222> (335) WO 2WWM 4 Wer"11 UNA WYMMMU" im $Jlhm'j wo oon8985 WO 0078985PCT[USOO/17 197 <223> N-terminal ATG which encodes methionine for PhaG <220> <221> misc feature <222> (1227)..(1232) <223> Barn HI restriction site <400> 11 ggatccatgg gggcaatccg agaaggtcaa ggtgacagaa taacgtt tat cctatttgcc tccaaggtca tcatcctgat tgcacccaca acaaccgtca tcgagcactt tggcgctggc.

tgatcaacga accgttatca tcaaacgc tt acttccacat atatcaacat atgcgcggca gccacttcct gcttcctcaa agcatgcatt cttctatgat ccgcagtggc cactgacatt acatatacat gttgaatatt accattgacg gtatcgggtt caaeggctcg gttcaacgtg ggaacggctg ccaggcagac gcaccagccg gccgatgcgc ggtcggcaac caactaccgc caaccaggtg cccggtgctg gttcagcaag ggacatggag gcc aaccgtg tgccatctga atcctcttcc tcattcggcg acttccatta atatatatat taggtgtggc agagattcta tacacggagt c tggccacca gttctgttcg atcagcaagg cacgtgatgt cggtacgtga gactatctgg ctggtcaatg catgtgagca ctggagcacg ttcatcaacg catgtgggca aacaagaccg cgtgaacccc gctgtgacaa gcctcaaatc.

caagcaatgg agttgaatat ctccaattgg gaatgaggc tctatcgcgc cggcctcgtt accagccgta agaccgaggc ctttttcgtg agaaggcagt accgtggctg acaccategg gcctggacag acctggaacg gcgagcgcga gaagccagtt cctgcgagaa gccaacgtta cagtcagccg catgactgga tgaagagtaa cagtaatgat aaagaagttt agaaatcgc t ggatgcggcc cgcccagacg ttcaggcaag gcatatcctc gggtggcgca ggtgagttcg ccagtacctg caagcacttg ccacgagtac tgcgctgcaa cgagtacacc cagcgtgatc cacccgcaat ccaacccgtg tgcctctagg ttcccagtga agtgcatgca tcaagtttgt gagactcttt gtac ttgata gaaaacacga gtacgtaacc tccaagccgc cttgagctga agcacgctga ttctcgccag gccgcc tgcg ccgtcgctgt gcacagatgc ggcgcgcgca acagtcgagg cgcgatgcgg gtcatgetgg cagcaggggc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1.140 1200 3.232 ggtaccggat cc <210> 12 <211> 940 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Chioroplast targeting signal from the alfalfa rubisco protein fused to the coding sequence of reductase from A.

eutrophus <220> <221> misc feature <222> <223> Nco I restriction site <220> wo oon8985 WO 0078985PCTIUSOO/17197 <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> misc-feature (15) (17) N-terminal ATG of the targeting signal misc feature (175) (180) Sph I restriction site misc feature (192) (194) N-terminal ATG of PhaC <220> <221> misc feature <222> (933) .(940) <223> Not I restriction site <400> 12 ccatggcttc tacaatctgg caaaggtcaa gaaggatttc ccgccatttg actcgccgcg ectcggaagg ccgaggtcgg tccgcaagat tcaacgtcac acatctcgtc aggccggcct ccgtcaacac acgtgc tcga tcgcctcgat tctcgctcaa ctctatgatg cgtggtggct caaagacatt tatgactcag ccagcggctg ccgcgaaaag caatgtggct cgaggttgat gacccgcgec caagcaggtg ggtgaacggg gcatggcttc ggtctctccg caagatcgte ctgcgcc tgg cggcggcctg tcctcttcag ccattcgttg acttccattg cgcattgcgt gccaaggatg tggctggagc gactgggact gtgctgatca gac tgggatg atcgacggca cagaagggcc accatggcac ggc tatatcg gcgacgatcc ttgtcgtcgg catatgggct ctgtgactac gactaaagtc caagcaatgg atgtgaccgg gc tttcgtgt agcagaaggc cgaccaagac acaacgccgg cggtgatcga tggccgaccg agttcggcca tggcgcagga ccaccgacat cggtcaagcg aggagtccgg gagcggccgc agttaaccgt aatggc tggc tggaagagta cggcatgggt ggtggccggt Cctgggcttc cgcattcgac tatcacccgc caccaacctg tggctggggc gaccaactac agtggcgacc ggtcaaggcg Cctgggcctg tttctcgacc gcctcttcgg ttcccagtta aactgcatgc ggtatcggaa tgcggcccca gatttcattg aaggtcaagt gacgtggtgt acctcgctgt cgcatcgtca tccaccgcca aagggcgtga atccgccagg ccggaagaga ggcgccgact 4210> 13 <211> 2108 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Pea Rubisco Chloroplast Targeting Signal <220> I .it~i~ A AiA~f WO OOn8985 WO 0078985PCT/USOO/17197 <221> misc-feature <222> (6) <223> BaMHI restriction site <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> misc-feature N-terminal ATG of targeting signal misc feature (327) (332) Xba I restriction site misc feature (333) (335) N terminal ATG of PhaC <220> <221> misc feature <222> (2103)..(2106) <223> BamHI restriction site <400> 13 ggatccatgg gggcaatccg agaaggtcaa ggtgacagaa taacgtttat cctatttgcc ccacgcagga catggctgga ccggcattcc atatccagca aggccgaggc acctcccata tggccgatgc aatgggtcga tgctgatcga tgacacgcgg cggtgaccga cgctgaccga actacatcct atacggtgtt acgactacat aggacaagat cggtgctgc cttctatgat ccgcagtggc cactgacatt acatatacat gttgaatatt accattgacg aggcaagtcc atggtcccgc.

gggcc tggat gcgctacatg caccggtccg tcgcttcgct cgtcgaggcc tgcgatgtcg gtcgggcggc caagatctcg aggcgccgtg caaggtgcac ggacctgcag tctggtgtcg cgagcacgcg caacgtgctc cgcgcgcggc atcctcttcc tc attcggcg acttccatta atatatatat taggtgtggc agagattcta caaccattca cagtggcagg gcgctggcag aaggacttct c tgcacgacc gccgcgttct gatgccaaga c ccgc caac t gaatcgctgc cagaccgacg gtcttcgaga gcgcgcccgc ccggagagct tggcgcaatc gccatcCgCg ggcttctgcg gagcacccgg gc tgtgacaa gcctcaaatc caagcaatgg agttgaatat ctccaattgg gaatggc tac aggtcacgcc gc actgaagg gcgtcaagat cagcgctgtg ggcgcttcgC acctgctcaa cccgccagcg tccttgacac gtgccggcgt agagcgcgtt acgagtactt tgctgatggt cgc tggtgcg cggacgccag ccatcgaagt tgggcggcac ccgccagcgt cagtcagccg catgactgga tgaagagtaa cagtaatgat aaagaagt tt cggcaaaggc ggggccattc caacggccac cgcgccggcg gcaggccatg cggcgacgca tgcgcgCc c atccgc ttc caatcccgag gcgcaacatg tgaggtcggc ccagctgttg gccgccgtgc ccatgtggtg catggccggC cgcgcgegac cattgtctcg cacgctgctg tgcctctagg ttcccagtga agtgcatgca tcaagtttgt gagactcttt gcggcagctt gatccagcca gcggccgcgt cagc tgggtg gccgagggca tggcgcacca t tgaccgagc gcgatctcgc gcgcagcgca atggaagacc cgcaatgtcg cagtacaage atcaacaagt gagcagggac agcacc tggg atcagcggcc accgcgctgg accacgctga 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 Lt"~5h~~ VI WO 00/78985 PCTUSOO/17 197 tggactttgc gcgaggccac tggccaatac actacctgaa ccaacctgcc tcaaggtacc tgccgactcta cctcgaccgc ccggtgtgat cggagtcgcc actggaccgc gcaatgcgcg gaggatcc cgacacgggc gctgggcggc cttctcgttc gggcaacacg ggggccgtgg gggcaagc tg tatctacggc gc tgc tggcg caacccgccg gcagcaatgg atggctggcc ctatcgcgca atcctcgacg ggcgccggcg ttgcgcccga ccggtgccgt tactgctggt accgtgtgcg tcgcgcgaag aacaagc tgc gccaagaaca ctggccggcg gggcaggccg atcgaacceg tctttgtcga cgccgtgcgc acgacctggt tcgacc tgct acctgcgcca gcgtgccggt accatatcgt gcttcgtgct agcgcagcca ccatcgagca gcgcgaaacg cgcc tgggcg cgagggccat gctgctgcgc gtggaactac gttctggaac cacctacctg ggacctggcc gccgtggacc gggtgcgtCg c tggac taac tcacggcagc cgccgcgccc atacgtcaaa gtgcagttgc ggcc ttgagc gtggtcgaca ggcgacgcca cagaacgagc agcatcgacg gcggcc tatg ggccatatcg gatgcgc tgc tggtggccgg gccaactatg gccaaggcat 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2108

Claims (25)

1. An expression cassette comprising a single plant promoter at the 5'-end of the construct, a first protein encoding sequence 3' to the promoter, an internal ribosome entry site sequence 3' to the first protein encoding sequence, a second protein encoding sequence immediately 3' to the internal ribosome entry site, and a polyadenylation sequence 3' to the second protein encoding sequence. wherein the encoded proteins function in the production of polyhydroxyalkanoates.

2. The cassette of claim 1 wherein the promoter is selected from the group consisting of inducible, constitutive, and tissue specific plant promoters.

3 The cassette of claim 2 wherein the tissue specific plant promoter is selected from the group consisting of carboxylase promoter, 2S albumin promoter, seed storage protein promoter, phaseolin promoter, oleosin promoter, zein promoter, glutelin promoter, starch synthase promoter, starch branching enzyme promoter, and leaf expression specific promoters.

4. The cassette of any one of claims 1 to 3 further comprising at least one nucleotide sequence 5' to at least one protein encoding sequence, the sequence encoding a peptide signal that targets the protein to a particular compartment of the cell. 25

5. The cassette of claim 4 wherein the compartment is selected from the group consisting of chloroplasts and plastids and peroxisomes.

S6. The cassette of any one of claims 1 to 5 wherein the protein encoding sequences encode at least one marker protein selected from the group consisting of proteins conferring 30 antibiotic resistance, herbicide resistances, and detectable proteins. 31

7. The cassette of any one of claims 1 to 6 further comprising a second internal ribosome entry site sequence and a third protein encoding sequence.

8. The cassette of claim 7 further comprising a third internal ribosome entry site sequence and a fourth protein encoding sequence.

9. The cassette of claim 7 or claim 8, wherein the internal ribosome entry sites are from different plant viruses.

10. The cassette of any one of claims 1 to 5 or 7 or 8 wherein the protein encoding sequences encode proteins selected from the group consisting of ACP-CoA transacylase, PHA synthase, an a subunit of p-oxidation enzyme complex, a P subunit of p-oxidation enzyme complex, a reductase, a P-ketothiolase, an acyl CoA oxidase, and a catalase.

11. The cassette of claim 1 comprising a leaf specific promoter, a chloroplast targeting signal fused to a gene encoding a ACP-CoA transacylase, an internal ribosome entry site sequence, a chloroplast targeting signal fused to a gene encoding a PHA synthase that accepts medium chain length substrates, and a polyadenylation signal at the extreme 3'end of the cassette.

12. The cassette of claim 1 comprising a leaf specific promoter, a gene encoding a PHA synthase that accepts medium chain length substrates fused to a peroxisomal targeting signal, an internal ribosome entry site sequence, a multifunctional a subunit of p-oxidation complex fused to a peroxisomal targeting signal, and a polyadenylation signal.

13. The cassette of claim 8 comprising a seed specific promoter, a gene encoding acyl CoA oxidase in which the peroxisomal targeting signal has been removed, an internal ribosome entry site sequence, a gene encoding a medium chain length synthase, an IRES, a gene encoding an a subunit of P-oxidation, an IRES, a gene encoding a P subunit of p- 30 oxidation, and a polyadenylation signal. 32 i" 1 A'C~W Iflr ~l#*iI

14. Plant material transformed with an expression cassette comprising a single plant promoter at the 5'-end of the construct, a first protein encoding sequence 3' to the promoter, an internal ribosome entry site 3' to the first protein encoding sequence, a second protein encoding sequence immediately 3' to the internal ribosome entry site, and a polyadenylation sequence 3' to the second protein encoding sequence, wherein the encoded proteins function in the production of polyhydroxyalkanoates.

15. The plant material of claim 14 wherein the plants are selected from the group consisting of Brassica; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards; and flax.

16. The plant material of either of claim 14 or claim 15 wherein the material is tissue selected from the group consisting of protoplasts, cells, callus tissue, leaf discs, pollen and meristems.

17. The plant material of any one of claims 14 to 16 wherein the expression cassette is as defined by any of claims 2-13.

18. A method for expression of heterologous genes in plant material comprising transforming the material with an expression cassette comprising a single plant promoter at the 5'-end of the construct, a first protein encoding sequence 3' to the promoter, 25 an internal ribosome entry site 3' to the first protein encoding sequence, a second protein encoding sequence immediately 3' to the internal ribosome entry site, and a polyadenylation sequence 3' to the second protein encoding sequence, wherein the encoded proteins function in the production of polyhydroxyalkanoates. 0 •i. hlu~iui~rur**ll*wn~vIl~?v_~uvpi~wl~v;~lr u~wh~.;lriira/ii~~~~~

19. The method of claim 18 wherein the plant material is transformed using a method selected from the group consisting of Agrobacterium mediated transformation, biolistics, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome-mediated transformation, and silicon fiber-mediated transformation.

The method of either of claim 18 or claim 19 wherein the cassette is as defined by any of claims 2-13.

21. A multi-gene expression cassette substantially as hereinbefore described with reference to the examples and the drawings.

22. Plant material transformed with a multi-gene expression cassette according to claim 21.

23. A method for expression of heterologous genes in plant material comprising transforming the material with a multi-gene expression cassette according to claim 22.

24. Plant material transformed with a multi-gene expression cassette substantially as hereinbefore described with reference to the examples and the drawings.

:25. A method for expression of heterologous genes in plant material comprising t "':'.transforming the material with an expression cassette substantially as hereinbefore described with reference to the examples and the drawings. o lo •DATED THIS NINTH DAY OF DECEMBER 2003 METABOLIX, INC. PIZZEYS PATENT AND TRADE MARK ATTORNEYS 34 MAY UN &NOWNWWWAN"I'VIN