Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-05-21
2001-11-27
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S141000
Reexamination Certificate
active
06323010
ABSTRACT:
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 renewable resources, with a broad range of industrial and biomedical applications (Williams and Peoples, 1996, CHEMTECH 26, 38-44). Around 100 different monomers have been incorporated into PHA polymers, as reported in the literature (Steinbüchel 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-hydroxybutyrate-co-3-hydroxyvalerate) (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, 507-514 and Valentin et al., 1994, Appl. Microbiol. Biotechnol. 40, 710-716). These polyesters 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, S., Hiramitsu, M. and Doi, Y., 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 3-hydroxypropionate 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
Pseudomonas putida
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; Steinbüchel 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 Steinbüchel and Gorenflo (1997, Macromol. Symp. 123: 61-66) indicate that
Pseudomonas putida
has the ability to convert levulinic acid to 4-hydroxyvalerate.
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, □-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.
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 materials 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, 2-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate, 4-hydroxyvalerate and 5-hydroxyvalerate. These PHA compositions can readily be extended to incorporate additional monomers including, for example, 3-hydroxyhexanoate, 4-hydroxyhexanoate, 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.
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Choi & Lee, “Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation,”Appl. Microbiol. Biotechnol.51:13-21 (1999).
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Peoples, et al., “Biosynthetic Thiolase fromZoogloea ramigera,” J. Biol. Chem.262:97-102 (1987).
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Saito, et al.,
Peoples Oliver P.
Skraly Frank A.
Coe Susan D.
Holland & Knight LLP
Metabolix Inc.
Prats Francisco
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