US5229279A
Expired / Lapsed
US
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Method for producing novel polyester biopolymers
Assignee: Massachusetts Institute of Technology
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Filing: 1990-08-13
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Granted: 1993-07-20
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Expires: 2010-08-13
Abstract
Abstract The present invention invention is a method for controlling biopolymer synthesis by determining the genetics and enzymology of polyhydroxybutyrate (PHB) biosynthesis at the molecular level. The purified enzymes and genes provide the means for developing new PHB-like biopolymers having polyester backbones. Specific aims are to 1) control the chain length of the polymers produced in fermentation processes through genetic manipulation, 2) incorporate different monomers into the polymers to produce copolymers with different physical properties, and 3) examine the physical/ rheological properties of these new biopolymers in order to develop further design criteria at the molecular level. The method for engineering biopolymer synthesis includes: isolation and characterization of the genes for the enzymes in the synthetic pathway (beta-ketothiolase, acetoacetyl-CoA reductase and PHB synthetase); cloning of the genes in a vector(s); placement of the vector(s) under the control of regulated promoters; expression of the genes; determination of the function and use of other factors such as substrate specificity in polymer production and composition; and isolation and physical and chemical analysis of the resulting polymers.
Inventors
- 👤 Oliver P. Peoples
- 👤 Anthony J. Sinskey
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 21:38
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0006; C12N15/00; C12N15/09; C12N15/11; C12N15/52; C12N9/10; C12N9/1025; C12N9/1029; C12P; C12P7/00; C12P7/62; C12P7/625
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
106 words
A method for constructing polyhydroxybutyrate-like polyester polymers in a bacterial host comprising: selecting a bacterial host for expression of isolated structural genes encoding enzymes required for synthesis of polyhydroxybutyrate polymers, wherein the host does not produce polyhydroxybutyrate polymerase, providing isolated structural genes encoding a beta-ketothiolase, an acetoacetyl-CoA reductase, and a polyhydroxybutyrate polymerase in one or more vectors for expression of the isolated structural genes in the bacterial host, as required to produce polyhydroxybutyrate polymers, introducing said expression vector, or vectors, into the bacterial host, expressing the enzymes encoded by the introduced isolated structural genes, and providing appropriate substrates for the expressed enzymes to synthesize polyhydroxybutyrate-like polymers.
Claim 2
Independent Claim
106 words
A method for constructing polyhydroxybutyrate-like polyester polymers in a bacterial host comprising: selecting a bacterial host for expression of isolated structural genes encoding enzymes required for synthesis of polyhydroxybutyrate polymers, wherein the host does not produce polyhydroxybutyrate polymerase, providing isolated structural genes encoding a beta-ketothiolase, an acetoacetyl-CoA reductase, and a polyhydroxybutyrate polymerase in one or more vectors for expression of the isolated structural genes in the bacterial host, as required to produce polyhydroxybutyrate polymers, introducing said expression vector, or vectors, into the bacterial host, expressing the enzymes encoded by the introduced isolated structural genes, and providing appropriate substrates for the expressed enzymes to synthesize polyhydroxybutyrate-like polymers.
Claim 3
Dependent Claim
16 words
The method of claim 1 further comprising providing sequences regulating the expression of said structural genes.
Claim 4
Dependent Claim
17 words
The method of claim 1 further comprising selecting said enzymes on the basis of their substrate specificity.
Claim 5
Dependent Claim
21 words
The method of claim 3 further comprising modifying the side chains of said polymer by providing substrates for said selected enzymes.
Claim 6
Dependent Claim
46 words
The method of claim 1 further comprising providing a temperature sensitive regulatory sequence in the expression vector, wherein expression of the genes encoding the enzymes required for polymer synthesis and synthesis of the resulting polymer is regulated by the temperature at which the host is grown.
Claim 7
Dependent Claim
38 words
The method of claim 1 further comprising providing regulatory sequences in the expression vector wherein the genes encoding the enzymes required for polymer synthesis can be expressed at different times to alter the polymer produced by said enzymes.
Claim 8
Dependent Claim
27 words
The method of claim 1 wherein said structural genes are from bacteria selected from the group consisting of Zoogloea, Azotobacter, Alcaligenes, Bacillus, Nocardia, Clostridium, Halobacterium, and Pseudomonas.
Claim 9
Dependent Claim
34 words
The method of claim 1 further comprising providing structural genes encoding enzymes selected from the group consisting of beta-ketothiolases, acetoacetyl-CoA reductases, and polyhydroxybutyrate polymerases that are stereospecific for the D isomer of the substrate.
Claim 10
Dependent Claim
14 words
The method of claim 1 wherein said structural genes are of different bacterial origin.
Claim 11
Dependent Claim
14 words
The method of claim 1 wherein said expression host is a Gram negative bacteria.
Claim 12
Dependent Claim
56 words
A bacterial host synthesizing a beta-ketothiolase and acetoacetyl-CoA reductase but not a polyhydroxybutyrate polymerase, having inserted therein an isolated structural gene encoding a polyhydroxybutyrate polymerase in a vector for expression of the gene in a bacterial host, wherein the host is cultured in the presence of appropriate substrates for the expressed enzyme to synthesize polyhydroxybutyrate-like polymers.
Claim 13
Dependent Claim
56 words
A bacterial host synthesizing a beta-ketothiolase and acetoacetyl-CoA reductase but not a polyhydroxybutyrate polymerase, having inserted therein an isolated structural gene encoding a polyhydroxybutyrate polymerase in a vector for expression of the gene in a bacterial host, wherein the host is cultured in the presence of appropriate substrates for the expressed enzyme to synthesize polyhydroxybutyrate-like polymers.
Claim 14
Dependent Claim
36 words
The host of claim 11 wherein the structural gene encoding a polyhydroxybutyrate polymerase is in plasmid pAeT29, deposited Feb. 24, 1992 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, ATCC designation 75207.
References Cited (Backward Citations)
US4477654A
Cited
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