AU2003200941B2 - Polyhydroxyalkanoate biopolymer compositions - Google Patents

Description

n 1 O POLYHYDROXYALKANOATE BIOPOLYMER COMPOSITIONS Background to the Invention Numerous microorganisms have the ability to accumulate intracellular reserves of PHA polymers. Poly [(R)-3-hydroxyalkanoates] (PHAs) are biodegradable and biocompatible thermoplastic materials, produced from Srenewable resources, with a broad range of industrial and biomedical Sapplications (Williams and Peoples, 1996, CHEMTECH 26,38-44). Around 100 different monomers have been incorporated into PHA polymers, as reported in the literature (Steinbuchel and Valentin, 1995, FEMS Microbiol.

Lett. 128; 219-228) and the biology and genetics of their metabolism has recently been reviewed (Huisman and Madison, 1998, Microbiology and Molecular Biology Reviews, 63: 21-53).

To date, PHAs have seen limited commercial availability, with only the copolymer poly(3-hyd roxybutyrate-co-3-hyd roxyvalerate) (PHBV) being available in development quantities. This copolymer has been produced by fermentation of the bacterium Ralstonia eutropha. Fermentation and recovery processes for other PHA types have also been developed using a range of bacteria including Azotobacter, Alcaligenes latus, Comamonas testosterone and genetically engineered E. coli and Klebsiella and have recently been reviewed (Braunegg et al., 1998, Journal of Biotechnology 65:127-161; Choi and Lee, 1999, Appl. Microbiol. Biotechnol. 51:13-21). More traditional polymer synthesis approaches have also been examined, including direct condensation and ring-opening polymerization of the corresponding lactones (Jesudason and Marchessault, 1994, Macromolecules 27: 2595-2602).

Synthesis of PHA polymers containing the monomer 4-hydroxybutyrate (PHB4HB, Doi, Y. 1995, Macromol. Symp. 98, 585-599) or 4-hydroxyvalerate and 4-hydroxyhexanoate containing PHA polyesters have been described (Valentin et al., 1992, Appl.Microbiol. Biotechnol. 36,507 WO 99/61624 PCT/US99/I1417 514 and Valentin et al., 1994, AppI. Microbiol. Biotechnol. 40, 710-716).

These polyestern have been manufactured using methods similar to that originally described for PHBV in which the microorganisms are fed a relatively expensive non-carbohydrate feedstock in order to force the incorporation of the monomer into the PHA polyester. The PHB4HB copolymers can be produced with a range of monomer compositions which again provides a range of polymer (Saito, Y, Nakamura, Hiramitsu, M.

and Doi, 1996, Polym. Int. 39: 169).

PHA copolymers of 3-hydroxybutyrate-co-3-hydroxypropionate have also been described (Shimamura et. al., 1994, Macromolecules 27: 4429-4435; Cao et. al., 1997, Macromol. Chem. Phys. 198: 3539-3557). The highest level of 3hydroxypropionate incorporated into these copolymers 88 mol (Shimamura et. al., 1994, Macromolecules 27: 4429-4435).

PHA terpolymers containing 4-hydroxyvalerate have been produced by feeding a genetically engineered Pseudomonasputida strain on 4-hydroxyvalerate or levulinic acid which resulted in a three component PHA, Poly(3-hydroxybutyrate-co- 3-hydroxyvalerate-4-hydroxyvalerate) (Valentin et. al., 1992, Appl. Microbiol.

Biotechnol. 36: 507-514; Steinbfichel and Gorenflo, 1997, Macromol. Symp. 123: 61-66). It is desirable to develop biological systems to produce two component polymers comprising 4-hydroxyvalerate or poly(4-hydroxyvalerate) homopolymer.

The results of Steinbichel and Gorenflo (1997, Macromol. Symp. 123: 61-66) indicate that Pseudomonasputida has the ability to convert levulinic acid to 4hydroxyvalerate.

Hein et al. (1997) attempted to synthesize poly-4HV using transgenic Escherichia coli strain XL1-Blue but were unsuccessful. These cells carried a plasmid which permitted expression of the A. eutrophus PHA synthase and the Clostridium kluyveri 4-hydroxybutyryl-CoA transferase genes. When the transgenic E. coli were fed 4HV, O-valerolactone, or levulinic acid, they produced only a small amount of PHB homopolymer.

It is clearly desirable for industrial reasons to be able to produce a range of defined PHA homopolymer, copolyer and terpolymer compositions. To accomplish this, it is desirable to be able to control the availability of the individual enzymes in the corresponding PHA biosynthetic pathways.

WO 99/61624 PCT/US99/11417 It is therefore an object of the present invention to provide a range of defined PHA homopolymer, copolyer and terpolymer compositions.

It is another object of the present invention to provide a method and matierlas to control the availability of the individual enzymes in the corresponding PHA biosynthetic pathways.

Summary of the Invention Several novel PHA polymer compositions produced using biological systems include monomers such as 3-hydroxybutyrate, 3-hydroxypropionate, 2hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate, 4-hydroxyvalerate and hydroxyvalerate. These PHA compositions can readily be extended to incorporate additional monomers including, for example, 3-hydroxyhexanoate, 4hydroxyhexanoate, 6-hydroxyhexanoate or other longer chain 3-hydroxyacids containing seven or more carbons. This can be accomplished by taking natural PHA producers and mutating through chemical or transposon mutagenesis to delete or inactivate genes encoding undesirable activities. Alternatively, the strains can be genetically engineered to express only those enzymes required for the production of the desired polymer composition. Methods for genetically engineering PHA producing microbes are widely known in the art (Huisman and Madison, 1998, Microbiology and Molecular Biology Reviews, 63: 21-53). These polymers have a variety of uses in medical, industrial and other commercial areas.

Brief Description of the Drawings Figure 1 is a schematic of the pathway from levulinic acid to poly-4hydroxyvalerate.

Figure 2 is a schematic of a construct of plasmid pFS16, which includes the lad (inducer) gene, ampicillin resistance gene, and hbcT gene.

Figure 3 is a schematic of a construct of plasmid pFS30, which includes the lad (inducer) gene, ampicillin resistance gene, polyhydroxyalkanoate polymerase (phaC) gene, and hbcT gene.

WO 99/61624 PCT/IUS99/11417 Detailed Description of the Invention Several hovel PHA polymer compositions have been produced using biological systems to incorporate monomers such as 3-hydroxybutyrate, 3hydroxypropionate, 2-hydroxybutyrate, 3-hydroxyvalerate, 4hydroxybutyrate, 4-hydroxyvalerate and 5-hydroxyvalerate. These PHA compositions can readily be extended to incorporate additional monomers including, for example, 3-hydroxyhexanoate, 4-hydroxyhexanoate, 6hydroxyhexanoate or other longer chain 3-hydroxyacids containing seven or more carbons. Techniques and procedures to engineer transgenic organisms that synthesize PHAs containing one or more of these monomers either as sole constituent or as co-monomer have been developed. In these systems the transgenic organism is either a bacterium eg. Escherichia coli, K pneumoniae, Ralstonia eutropha (formerly Alcaligenes eutrophus), Alcaligenes latus or other microorganisms able to synthesize PHAs, or a higher plant or plant component, such as the seed of an oil crop (Brassica, sunflower, soybean, corn, safflower, flax, palm or coconut or starch accumulating plants (potato, tapioca, cassava).

It is crucial for efficient PHA synthesis in recombinant E. coli strains that the expression of all the genes involved in the pathway be adequate. To this end, the genes of interest can be expressed from extrachromosomal

DNA

molecules such as plasmids, which intrinsically results in a copy number effect and consequently high expression levels, or, more preferably, they can be expressed from the chromosome. For large scale fermentations of commodity type products it is generally known that plasmid-based systems are unsatisfactory due to the extra burden of maintaining the plasmids and the problems of stable expression. These drawbacks can be overcome using chromosomally encoded enzymes by improving the transcriptional and translational signals preceding the gene of interest such that expression is sufficient and stable.

The biological systems must express one or more enzymes as required to convert the monomers into polymers. Suitable substrates include 3hydroxybutyrate, 3 -hydroxypropionate, 2-hydroxybutyrate, 3hydroxyvalerate, 4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate, 3- 4 WO 99/61624 PCT/US99/11417 hydroxyhexanoate, 4-hydroxyhexanoate, 6-hydroxyhexanoate and other longer chain 3-hydroxyacids containing seven or more carbons. These enzymes include polyhydroxyalkanoate synthase, acyl-CoA transferase and hydroxyacyl CoA transferase, and hydroxyacyl CoA synthetase. These enzymes can be used with these substrates to produce in a biological system such as bacteria, yeast, fungi, or plants, polymer such as poly(3hydroxybutyrate-co-4-hydroxyvalerate), poly(4-hydroxyvalerate), poly(3poly(2-hydroxybutyrate), poly(2hydroxybutyrate-co-3-hydroxybutyrate), and poly(3-hydroxypropionate).

Genes encoding the required enzymes can be acquired from multiple sources. U.S. Patent Nos. 5,798,235 and 5,534,432 to Peoples, et al., describe polyhydroxyalkanoate synthetase, reductase and thiolase. A 4hydroxybutyryl CoA transferase gene from C. aminobutyricum is described by Willadsen and Buckel, FEMS Microbiol. Lett. (1990) 70: 187-192) or from C. kluyveri is described by Sohling and Gottschalk, 1996, J. Bacteriol.

178, 871-880). An acyl coenzyme A synthetase from Neurospora crassa is described by Hii and Courtright, J. Bacteriol. 1982.150(2), 981-983. A hydroxyacyl transferase from Clostridium is described by Hofmeister and Bucker, Eur. J. Biochem. 1992, 206(2), 547-552.

It is important for efficient PHA production that strains do not lose the capability to synthesize the biopolymer for the duration of the inoculum train and the production run. Loss of any of thepha genes results in loss of product. Both are undesirable and stable propagation of the strain is therefore required. Merely integrating the gene encoding the transferase or synthase may not result in significant polymer production. Enzyme expression can be enhanced through alteration of the promoter region or mutagenesis or other known techniques, followed by screening for polymer production. Growth and morphology of these recombinant PHA producers is not compromised by the presence ofpha genes on the chromosome.

The present invention will be further understood by reference to the following non-limiting examples.

WO 99/61624 PCT/US99/11417 Example 1. Poly(3HB-co-4HV) from 4-hydroxyvalerate and glucose in E. coli.

Construction of pFS 16.

The plasmid pTrcN is a derivative ofpTrc99a (Pharmacia; Uppsala, Sweden); the modification that distinguishes pTrcN is the removal of the Ncol restriction site by digestion with NcoI, treatment with T4 DNA polymerase, and self-ligation. The orjZ gene encoding the 4-hydroxybutyryl-CoA transferase from Clostridium kluyveri was amplified using the polymerase chain reaction (PCR) and a kit from Perkin Elmer (Foster City, CA) using plasmid pCK3 (Sohling and Gottschalk, 1996, J. Bacteriol.

178: 871-880) as the target DNA and the following oligonucleotide primers:

TCCCCTAGGATTCAGGAGGTTTTTATGGAGTGGGAAGAGATATATAAAG

-3' (orJZ 5' AvrI) CCTTAAGTCGACAAATTCTAAAATCTCTTTTTAAATTC 3' (orZ 3' Sall) The resulting PCR product was digested withAvrII and Sall and ligated to pTrcN that had been digested with Xbal (which is compatible with Avrll) and Sall to form plasmid pFS 16 such that the 4-hydroxybutyryl-CoA transferase can be expressed from the IPTG (isopropyl-B-D-glucopyranoside) inducible trcpromoter.

Construction The plasmid pFS30 was derived from pFS 16 by adding the Ralstonia eutropha PHA synthase (phaC) gene (Peoples and Sinskey, 1989. J. Biol.

Chem. 264:15298-15303) which had been modified by the addition of a strong E. coli ribosome binding site as described by (Gerngross et. al., 1994.

Biochemistry 33: 9311-9320). The plasmid pAeT414 was digested with Xmal and Stul so that the R. eutropha promoter and the structural phaC gene were present on one fragment. pFS16 was cut with BamH, treated with T4 DNA polymerase to create blunt ends, then digested with Xmal. The two DNA fragments thus obtained were ligated together to form pFS30. In this 6 WO 99/61624 PCT/US99/11417 construct the PHB synthase and 4-hydroxybutyryl-CoA transferase are expressed from the A. eutrophus phbC promoter (Peoples and Sinskey, 1989.

J. Biol. Chem. 264:15298-15303). Other suitable plasmids expressing PHB synthase and 4-hydroxybutyryl-CoA transferase have been described (Hein et.

al., 1997, FEMS Microbiol. Lett. 153: 411-418; Valentin and Dennis, 1997, J.

Biotechnol. 58 :33-38).

E coli MBX769 has a PHA synthase integrated into its chromosome. This strain is capable of synthesizing poly(3-hydroxybutyrate) (PHB) from glucose with no extrachromosomal genes present. MBX769 is also deficient infadR, the repressor of the fatty-acid-degradation pathway and effector of many other cellular functions, it is deficient in rpoS, a regulator of stationary-phase gene expression, and it is deficient in atoA, one subunit of the acetoacetyl-CoA transferase. MBX769 also expresses atoC, a positive regulator of the acetoacetate system, constitutively.

E. coli MBX769 carrying the plasmid pFS16 (Figure which permitted the expression of the Clostridium kluyveri 4-hydroxybutyryl-CoA transferase, was precultured at 37 OC in 100 mL of LB medium containing 100 gg/mL sodium ampicillin in a 250-mL Erlenmeyer flask with shaking at 200 rpm. The cells were centrifuged at 5000g for 10 minutes to remove them from the LB medium after 16 hours, and they were resuspended in 100 mL of a medium containing, per liter: 4.1 or 12.4 g sodium 4-hydroxyvalerate (4HV); 5 g/L sodium 4-hydroxybutyrate (4HB); 2 g glucose; 2.5 g LB broth powder (Difco; Detroit, Mich.); 50 mmol potassium phosphate, pH 7; 100 jg/mL sodium ampicillin; and 0.1 nunol isopropyl-P-Dthiogalactopyranoside (WPTG). The sodium 4-hydroxyvalerate was obtained by saponification of y-valerolactbne in a solution of sodium hydroxide. The cells were incubated in this medium for 3 days with shaking at 200 rpm at 32 °C in the same flask in which they had been precultured. When 4.1 g/L sodium 4-hydroxyvalerate was present initially, the cells accumulated a polymer to 52.6% of the dry cell weight that consisted of 63.4% 3HB units and 36.6% 4HB units but no 4HV units.

When 12.4 g/L sodium 4HV was present initially, the cells accumulated a polymer to 45.9% of the dry cell weight that consisted of 95.5% 3HB units and 4HV units but no detectable 4HB units. The identity of the PHB-co-4HV polymer was verified by nuclear magnetic resonance (NMR) analysis of the solid product obtained by chloroform extraction of whole cells followed by filtration, ethanol WO 99i61624 PCT/US99/11417 precipitation of the polymer from the filtrate, and washing of the polymer with water.

It was also verified by gas chromatographic (GC) analysis, which was carried out as follows. Extracted polymer (1-20 mg) or lyophilized whole cells (15-50 mg) were incubated in 3 mL of a propanolysis solution consisting of 50% 1,2-dichloroethane, 40% 1-propanol, and 10% concentrated hydrochloric acid at 100 OC for 5 hours.

The water-soluble components of the resulting mixture were removed by extraction with 3 mL water. The organic phase (1 tL at a split ratio of 1:50 at an overall flow rate of 2 mL/min) was analyzed on an SPB-1 fused silica capillary GC column (30 m; 0.32 mm ID; 0.25 rnm film; Supelco; Bellefonte, Pa.) with the following temperature profile: 80 2 min; 10 C' per min to 250 250 2 min. The standard used to test for the presence of 4HV units in the polymer was y-valerolactone, which, like 4hydroxyvaleric acid, forms propyl 4-hydroxyvalerate upon propanolysis. The standard used to test for 3H-B units in the polymer was PHB.

Example 2. Poly(4HV) from 4-hydroxyvalerate in E. coli.

Escherichia coli MBX1177 is not capable of synthesizing poly(3hydroxybutyrate) (PHB) from glucose. MBXI 177 is a spontaneous mutant of strain 0 that is able to use 4-hydroxybutyric acid as a carbon source. MBX1177 carrying the plasmid pFS30 (Figure which permitted the expression of the Clostridium kluyveri 4HB-CoA transferase and the Ralstonia eutropha PHA synthase, was precultured at 37 °C in 100 mL ofLB medium containing 100 pg/mL sodium ampicillin.

The cells were centrifuged at 5000g for 10 minutes to remove them from the LB medium after 16 hours, and they were resuspended in 100 mL of a medium containing, per liter: 5 g sodium 4-hydroxyvalerate (4HV); 2 g glucose; 2.5 g LB broth powder; 100 mmol potassium phosphate, pH 7; 100 ig/mL sodium ampicillin; and 0.1 mmol IPTG. The cells were incubated in this medium for 3 days with shaking at 200 rpm at 30 oC in the same flask in which they had been precultured.

The cells accumulated a polymer to 0.25% of the dry cell weight that consisted of 100% 4HV units. The identity of the poly(4HV) polymer was verified by GC analysis of whole cells that had been washed with water and propanolyzed in a mixture of 50% 1,2-dichloroethane, 40% 1-propanol, and 10% concentrated hydrochloric acid at 100 OC for 5 hours, with y-valerolactone as the standard.

WO 99/61624 PCT/US99/11417 Example 3. Poly(3HB-co-2HB) from 2-hydroxybutyrate and glucose in E. colil.

E. coli MBX769 carrying the plasmid pFS16 was precultured at 37 °C in 100 mL of LB medium containing 100 ig/mL sodium ampicillin in a 250-mL Erlenmeyer flask with shaking at 200 rpm. The cells were centrifuged at 5000g for 10 minutes to remove them from the LB medium after 16 hours, and they were resuspended in 100 mL of a medium containing, per liter: 5 g sodium 2-hydroxybutyrate (2HB); 2 g glucose; 2.5 g LB broth powder; 50 mmol potassium phosphate, pH 7; 100 ig/mL sodium ampicillin; and 0.1 mmol IPTG. The cells were incubated in this medium for 3 days with shaking at 150 rpm at 33 °C in the same flask in which they had been precultured. The cells accumulated a polymer to 19.0% of the dry cell weight that consisted of 99.7% 3HB units and 0.3% 2HB units. The identity of the poly(3HBco-2HB) polymer was verified by GC analysis of the solid product obtained by chloroform extraction of whole cells followed by filtration, ethanol precipitation of the polymer from the filtrate, and washing of the polymer with water. It was also verified by GC analysis of whole cells that had been washed with water and propanolyzed in a mixture of 50% 1,2-dichloroethane, 40% 1-propanol, and concentrated hydrochloric acid at 100 OC for 5 hours, with PHB and sodium 2hydroxybutyrate as the standards.

Example 4. Poly(2HB) from 2-hydroxybutyrate in E. coli.

Escherichia coli MBX184 is not capable of synthesizing poly(3hydroxybutyrate) (PHB) from glucose. MBX184 is deficient infadR and expresses atoC constitutively.

MBX184 carrying theplasmid pFS30 was precultured at 37 °C in 100 mL of LB medium containing 100 pg/mL sodium ampicillin. The cells were centrifuged at 5000g for 10 minutes to remove them from the LB medium after 16 hours, and they were resuspended in 100 mL of a medium containing, per liter: 5 g sodium 2hydroxybutyrate (2HB); 2 g glucose; 2.5 g LB broth powder; 50 mmol potassium phosphate, pH 7; 100 gg/mL sodium ampicillin; and 0.1 mmol IPTG. The cells were incubated in this medium for 3 days with shaking at 150 rpm at 33 OC in the same flask in which they had been precultured.

The cells accumulated a polymer to 1.0% of the dry cell weight that consisted of 100% 2HB units. The identity of the poly(2HB) polymer was verified by GC 9 WO 99/61624 PCT/US99/ 1417 analysis of whole cells that had been washed with water and propanolyzed in a mixture of 50% 1,2-dichloroethane, 40% 1-propanol, and 10% concentrated hydrochloric acid at 100 °C for 5 hours, with sodium 2-hydroxybutyrate as the standard.

Example 5. Poly-3HP and poly-3HP-co-5HV from 1,3-propanediol and from Escherichia coli MBX184 carrying the plasmid pFS30 was precultured at 37 °C in 100 mL of LB medium containing 100 gg/mL sodium ampicillin. The cells were centrifuged at 5 000g for 10 minutes to remove them from the LB medium after 16 hours, and they were resuspended in 100 mL of a medium containing, per liter: 10 g 1,3-propanediol (1,3-PD) or 1,5-pentanediol 2 g glucose; 2.5 g LB broth powder; 50 mmol potassium phosphate, pH 7; 100 pg/mL sodium ampicillin; and 0.1 mmol IPTG. The cells were incubated in this medium for 3 days with shaking at 200 rpm at 30 °C in the same flask in which they had been precultured. When the diol substrate was 1,3-PD, the cells accumulated a polymer to 7.0% of the dry cell weight that consisted entirely of 3HP units. When the substrate was 1,5-PD, the cells accumulated a polymer to 22.1% of the dry cell weight that consisted of greater than 3-hydroxypropionate units and less than 10% 5-hydroxyvalerate units. The identity of the poly(3-hydroxypropionate) polymer was verified by NMR analysis of the solid product obtained by sodium hypochlorite extraction of whole cells followed by centrifugation and washing of the polymer with water. The identity of both polymers was verified by GC analysis of sodium hypochlorite-extracted polymer that was propanolyzed in a mixture of 50% 1,2-diclloroethane, 40% 1-propanol, and concentrated hydrochloric acid at 100 °C for 5 hours, with P-propiolactone and 8-valerolactone as the standards.

Example 6. Poly-5HV from 5-hydroxyvaleric acid.

Escherichia coli MBX1177 carrying the plasmid pFS30 was precultured at 37 °C in 50 mL of LB medium containing 100 pg/mL sodium ampicillin. The cells were centrifuged at 5000g for 10 minutes to remove them from the LB medium after 8 hours, and they were resuspended in 100 mL of a medium containing, per liter: g sodium 5-hydroxyvalerate (5HV); 5 g glucose; 2.5 g LB broth powder; 50 mmol potassium phosphate, pH 7; 100 itg/mL sodium ampicillin; and 0.1 mmol IPTG. The sodium 5HV was obtained by saponification ofd-valerolactone. The cells were WO 99/61624 PCT/US99/11417 incubated in this medium for 3 days with shaking at 200 rpm at 30 °C in the same flask in which they had been precultured. GC analysis was conducted with lyophilized whole cells that were butanolyzed in a mixture of 90% 1-butanol and concentrated hydrochloric acid at 110 oC for 5 hours; the standard was sodium hydroxyvalerate. This analysis showed that the cells had accumulated poly(5HV) to 13.9% of the dry cell weight. The identity of the poly(5-hydroxyvalerate) polymer was verified by NMR analysis of the solid product obtained by 1,2-dichloroethane extraction of whole cells followed by centrifugation and washing of the polymer with water.

Claims (17)

1. 2. The polymer of claim 1 wherein the 2-hydroxyacid is 2- hydroxybutyrate.
2. 3. The polymer of claim 1 selected from the group consisting of poly(2-hydroxybutyrate) and poly(2- hydroxybutyrate-co-3-hydroxybutyrate).
3. 4. The polymer of claim 1 produced by providing 2-hydroxybutyrate and one or more substrates selected from the group consisting of 3- hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate and 6- hydroxyhexaboate. O o 5. The polymer of claim 1 which is poly(2-hydroxybutyrate-co-4- Shydroxyvalerate). C)
4. 6. The polymer of any one of claims 1 to 5, wherein the biological system is genetically engineered to express one or more genes encoding the enzymes. 0 7. A biological system selected from the group consisting of bacteria, cc, fungi and yeast genetically engineered to express one or more enzymes selected from the group consisting of polyhydroxyalkanoate synthase, acyl-CoA transferase, hydroxyacyl CoA tranferase, and hydroxyacyl CoA synthetase wherein the genes encoding the enzymes are integrated into the chromosome of the biological system and stably expressed.
5. 8. The biological system of claim 7 which is a bacterium.
6. 9. The biological system of claim 8 which is E. coli. A biological system genetically engineered to express one or more enzymes selected from the group consisting of acyl-CoA transferase, hydroxyacyl CoA transferase, and hydroxyacyl CoA synthetase, wherein the biological system is selected from the group consisting of a plant, fungi, and yeast.
7. 11. A method for making polymers in a biological system comprising providing one or more substrates selected from the group consisting of: 2-hydroxyacids; 3-hydroxypropionate; 4-hydroxyvalerate; the combination of 3-hydroxybutyrate and 4-hydroxyvalerate; and in a biological system selected from the group consisting of bacteria, yeast, fungi and plants; inI 't 0 o wherein the biological system expresses one or more genes enzymes selected Sfrom the group consisting of polyhydroxyalkanoate synthase, acyl-CoA Cr) transferase, hydroxyacyl CoA transferase, and hydroxyacyl CoA synthetase such that the polymers accumulate; and wherein the polymer is selected form the group consisting of poly (3- hydroxypropionate), poly (3-hyd roxypropionate -co-5-hyd roxyvalerate), poly (3- hydroxybutyrate-co-4-hydroxyvalerate), poly(4-hydroxyvalerate), poly 0 0 hydroxyvalerate), and polymers comprising 2-hydroxyacid monomers. Cc 0 0 10 12. The method of claim 11 wherein the biological system is a bacterium.
8. 13. The method of claim 11 wherein the biological system is a plant.
9. 14. The method of any one of claims 10 to 12 wherein the biological system expresses at least one heterologous gene encoding the enzymes. The method of claim 11 comprising providing with the 2- hydroxybutyrate one or more substrates selected from the group consisting of 3- hydroxypropionate, 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxyhexanoate, 4-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate and 6- hydroxyhexanoate.
10. 16. The method of claim 11 wherein the one or more substrates are 2- hydroxybutyrate and 4-hydroxyvalerate.
11. 17. A method for making polymers in a biological system comprising providing a substrate that forms 3-hydroxyproprionyl-CoA in the biological system, wherein the biological system expresses one or more enzymes selected from the group consisting of polyhydroxyalkanoate synthase, acyl-CoA transferase, hydroxyacyl CoA tranferase, and hydroxyacyl CoA synthetase such that the polymers accumulate; and O o wherein the polymer is selected from poly (3-hydroxypropionate) and poly Q^ (3-hydroxypropionate C)
12. 018. The method of claim 17 wherein the biological system is a bacterium.
13. 19. The method of claim 17 wherein the biological system is a plant. 0 0 c 20. A method for making polymers in a biological system comprising 0 10 providing a substrate that forms 4-hydroxyvaleryl-CoA in the biological system, wherein the biological system expresses enzymes selected from the group consisting of polyhydroxyalkanoate synthase, acyl-CoA transferase, hydroxyacyl CoA tranferase, and hydroxyacyl CoA synthetase such that the polymers accumulate; and wherein the polymer is selected from poly (3-hydroxybutyrate -co-4- hydroxyvalerate)and poly (4-hydroxyvalerate).
14. 21. The method of claim 18 wherein the biological system is a bacterium.
15. 22. The method of claim 18 wherein the biological system is a plant..
16. 23. The method of any one of claims 11 to 22, wherein the biological system is genetically engineered to express one or more of the enzymes.
17. 24. A method for making polymers in a biological system, substantially as hereinbefore described with reference to any one of examples 1-6. METABOLIX, INC. DATED THIS NINETEENTH DAY OF SEPTEMBER 2005 BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS