Modified pseudomonas oleovorans phaC1 nucleic acids encoding bispecific polyhydroxyalkanoate polymerase
First Claim
1. A method for making a nucleic acid encoding a non-naturally occurring bispecific polyhydroxyalkanoate (PHA) polymerase which polymerizes a first carbon substrate and a second carbon substrate comprising:
- providing a Pseudomonas oleovorans phaC1 nucleic acid encoding a PHA polymerase which polymerizes the first carbon substrate; and
altering the nucleic acid to yield a modified phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase which copolymerizes the first carbon substrate and the second.
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Abstract
A genetically engineered Pseudomonas oleovorans phaC1 polyhydroxyalkanoate (PHA) polymerase having tailored substrate specificity is provided. The modified PHA polymerase is preferably a "bispecific" PHA polymerase capable of copolymerizing a short chain length monomer and a medium chain length monomer is provided. Methods for making the modified PHA polymerase and for making nucleic acids encoding the modified PHA polymerase are also disclosed, as are methods of producing PHA using the modified PHA polymerase. The invention further includes methods to assay for altered substrate specificity.
29 Citations
24 Claims
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1. A method for making a nucleic acid encoding a non-naturally occurring bispecific polyhydroxyalkanoate (PHA) polymerase which polymerizes a first carbon substrate and a second carbon substrate comprising:
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providing a Pseudomonas oleovorans phaC1 nucleic acid encoding a PHA polymerase which polymerizes the first carbon substrate; and altering the nucleic acid to yield a modified phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase which copolymerizes the first carbon substrate and the second. - View Dependent Claims (2)
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3. A modified P. oleovorans phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase.
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4. A vector comprising an inducible promoter operably linked to each of a nucleotide sequence encoding a β
- -ketothiolase and a nucleotide sequence encoding an acetoacetyl-CoA reductase, and a constitutive promoter operably linked to a modified P. oleovorans phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase.
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5. A vector comprising a promoter operably linked to a modified P. oleovorans phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase.
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6. A method for making a non-naturally occurring bispecific PHA polymerase comprising:
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providing a modified P. oleovorans phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase which copolymerizes a first carbon substrate and a second carbon substrate; and transforming a bacterial, yeast or plant host cell with the nucleic acid to yield a transformed host cell that produces the non-naturally occurring bispecific PHA polymerase. - View Dependent Claims (7, 8, 9)
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10. A method for making a polyhydroxyalkanoate (PHA) copolymer comprising:
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(a) transforming a bacterial, yeast or plant host cell with a modified P. oleovorans phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase to yield a transformed host cell that produces the non-naturally occurring bispecific PHA polymerase, resulting in the production of a PHA copolymer comprising at least one C3-C5 monomer and at least one C6-C14 monomer; and (b) isolating the PHA copolymer from the bacterial, yeast or plant host cell. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18)
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19. A method for making a polyhydroxyalkanoate (PHA) copolymer in a transgenic plant comprising:
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(a) providing a transgenic plant comprising a modified P. oleovorans phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase, wherein the transgenic plant produces the non-naturally occurring bispecific PHA polymerase resulting in production of a PHA copolymer comprising at least one C3-C5 monomer and at least one C6-C14 monomer; and (b) isolating the PHA copolymer from the plant. - View Dependent Claims (20, 21, 22)
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- 23. A transgenic organism comprising a modified P. oleovorans phaC1 nucleic acid encoding a non-naturally occurring bispecific PHA polymerase, wherein the transgenic organism is selected from the group consisting of a bacterium, a yeast and a plant.
Specification