US6117658A
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Methods of making polyhydroxyalkanoates comprising 4-hydroxybutyrate monomer units
Assignee: James Madison Innovations Inc
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Filing: 1998-02-12
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Granted: 2000-09-12
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Expires: 2018-02-12
Abstract
Abstract The PHA biosynthetic pathway is combined with a succinic semialdehyde metabolic pathway that metabolizes succinic semialdehyde via a 4-hydroxybutyryl-CoA intermediate in order to produce high levels of PHA comprising 4HB monomer units. This combination is particularly useful, in part, because the PHA biosynthetic pathway is well known and has been expressed to produce P(3HB) to levels as high as 70-80% of the cell dry weight, while the succinic semialdehyde metabolic pathway is also well known, and the combination of the two pathways means that production of PHAs comprising 4HB monomer units does not depend on immediate precursors of 4-hydroxybutyrate. Rather, the production of the PHAs comprising 4HB monomer units can occur simply from the extraction of succinate or succinyl-CoA from the citric acid cycle when the host cell is grown on an inexpensive carbon source such as glucose.
Inventors
- 👤 Douglas E. Dennis
- 👤 Henry E. Valentin
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 12:25
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0006; C12N15/00; C12N15/09; C12N15/11; C12N15/52; C12N15/63; C12N15/79; C12N15/82; C12N15/8241; C12N15/8242; C12N15/8243; C12N9/10; C12N9/13; C12P; C12P7/00; C12P7/62; C12P7/625
Claims Analysis
Total Claims: 72
Claim 1
Independent Claim
83 words
A process for producing a polyhydroxyalkanoate comprising 4-hydroxybutyrate monomer units, the method comprising: providing a host cell containing at least one recombinant nucleotide sequence that encodes a polyhydroxyalkanoate biosynthetic pathway, and at least one recombinant nucleotide sequence that encodes a succinic semialdehyde metabolic pathway that metabolizes succinic semialdehyde via a 4-hydroxybutyryl-CoA intermediate; and, cultivating the host cell under suitable conditions to obtain expression of both the polyhydroxyalkanoate biosynthetic pathway and the succinic semialdehyde metabolic pathway within the host cell, to produce the polyhydroxyalkanoate.
Claim 2
Dependent Claim
83 words
A process for producing a polyhydroxyalkanoate comprising 4-hydroxybutyrate monomer units, the method comprising: providing a host cell containing at least one recombinant nucleotide sequence that encodes a polyhydroxyalkanoate biosynthetic pathway, and at least one recombinant nucleotide sequence that encodes a succinic semialdehyde metabolic pathway that metabolizes succinic semialdehyde via a 4-hydroxybutyryl-CoA intermediate; and, cultivating the host cell under suitable conditions to obtain expression of both the polyhydroxyalkanoate biosynthetic pathway and the succinic semialdehyde metabolic pathway within the host cell, to produce the polyhydroxyalkanoate.
Claim 3
Dependent Claim
18 words
The process of claim 1 wherein the recombinant nucleotide sequences are contained on one or more vector constructs.
Claim 4
Dependent Claim
20 words
The process of claim 1 wherein the method further comprises the step of isolating the polyhydroxyalkanoate from the host cell.
Claim 5
Dependent Claim
30 words
The process of claim 1 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinic semialdehyde dehydrogenase.
Claim 6
Dependent Claim
18 words
The process of claim 4 wherein the succinic semialdehyde dehydrogenase is able to convert succinyl-CoA to succinic semialdehyde.
Claim 7
Dependent Claim
29 words
The process of claim 1 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinyl-CoA:CoA transferase.
Claim 8
Dependent Claim
47 words
The process of any one of claims 1-6 wherein the polyhydroxyalkanoate biosynthetic pathway is encoded by a single sequence recombinant nucleotide contained in a first vector construct, and the succinic semialdehyde metabolic pathway is encoded by a single sequence recombinant nucleotide contained in a second vector construct.
Claim 9
Dependent Claim
31 words
The process of any one of claims 1-6 wherein the polyhydroxyalkanoate biosynthetic pathway and the succinic semialdehyde metabolic pathway are encoded by sequences recombinant nucleotide contained in a single vector construct.
Claim 10
Dependent Claim
18 words
The process of any one of claims 1-6 wherein the succinic semialdehyde metabolic pathway is from Clostridium kluyveri.
Claim 11
Dependent Claim
17 words
The process of any one of claims 1-6 wherein the polyhydroxyalkanoate biosynthetic pathway is from Ralstonia eutropha.
Claim 12
Dependent Claim
20 words
The process of any one of claims 1-6 wherein the polyhydroxyalkanoate biosynthetic pathway comprises a polyhydroxyalkanoate synthase from Nocardia corallina.
Claim 13
Dependent Claim
18 words
The process of any one of claims 1-6 wherein the host cell is an Escherichia coli host cell.
Claim 14
Dependent Claim
17 words
The process of any one of claims 1-6 wherein the host cell is a Klebsiella host cell.
Claim 15
Dependent Claim
16 words
The process of claim 13 wherein the host cell is a Klebsiella aerogenes or Klebsiella oxytoca.
Claim 16
Dependent Claim
17 words
The process of any one of claims 1-6 wherein the host cell is a plant host cell.
Claim 17
Dependent Claim
17 words
The process of any one of claims 1-6 wherein the host cell is an insect host cell.
Claim 18
Dependent Claim
17 words
The process of any one of claims 1-6 wherein the host cell is a spider host cell.
Claim 19
Dependent Claim
24 words
The process of any one of claims 1-6 wherein the host cell has an inhibiting mutation in its CoA-independent NAD-dependent succinic independent semialdehyde dehydrogenase.
Claim 20
Dependent Claim
23 words
The process of any one of claims 1-6 wherein the host cell has an inhibiting mutation in its CoA-independent NADP-dependent succinic semialdehyde dehydrogenase.
Claim 21
Dependent Claim
31 words
The process of any one of claims 1-6 wherein the host cell has an inhibiting mutation in its CoA-independent NAD-dependent succinic independent semialdehyde dehydrogenase and its CoA-independent NADP-dependent succinic semialdehyde dehydrogenase.
Claim 22
Dependent Claim
34 words
The process of any one of claims 1-6 wherein the step of cultivating comprises cultivating the host cell on a carbon source that does not comprise 4-hydroxybutyrate or a structurally related precursor of 4-hydroxybutyrate.
Claim 23
Dependent Claim
14 words
The process of claim 21 wherein the structurally related precursor is 1,4-butanediol or 4-butyrolactone.
Claim 24
Dependent Claim
31 words
The process of any one of claims 1-6 wherein the step of cultivating comprises cultivating the host cell on a carbon source comprising a sugar having a 5-carbon or 6-carbon ring.
Claim 25
Dependent Claim
26 words
The process of any one of claims 1-6 wherein the step of cultivating comprises cultivating the host cell on a carbon source consisting essentially of glucose.
Claim 26
Dependent Claim
26 words
The process of any one of claims 1-6 wherein the step of cultivating comprises cultivating the host cell on a carbon source consisting essentially of sucrose.
Claim 27
Dependent Claim
79 words
A process for producing polyhydroxyalkanoate comprising 4-hydroxybutyrate monomer units, the method comprising: providing a host cell able to synthesize poly-β-hydroxyalkanoate via a polyhydroxyalkanoate biosynthetic pathway, the host cell containing at least one recombinant nucleotide sequence that encodes a succinic semialdehyde metabolic pathway that metabolizes succinic semialdehyde via a 4-hydroxybutyryl-CoA intermediate; and, cultivating the host cell under suitable conditions to obtain expression of the poly-β-hydroxyalkanoate pathway and the succinic semialdehyde metabolic pathway in the host cell, to produce the polyhydroxalkanoate.
Claim 28
Dependent Claim
79 words
A process for producing polyhydroxyalkanoate comprising 4-hydroxybutyrate monomer units, the method comprising: providing a host cell able to synthesize poly-β-hydroxyalkanoate via a polyhydroxyalkanoate biosynthetic pathway, the host cell containing at least one recombinant nucleotide sequence that encodes a succinic semialdehyde metabolic pathway that metabolizes succinic semialdehyde via a 4-hydroxybutyryl-CoA intermediate; and, cultivating the host cell under suitable conditions to obtain expression of the poly-β-hydroxyalkanoate pathway and the succinic semialdehyde metabolic pathway in the host cell, to produce the polyhydroxalkanoate.
Claim 29
Dependent Claim
20 words
The process of claim 26 wherein the method further comprises the step of isolating the polyhydroxalkanoate from the host cell.
Claim 30
Dependent Claim
29 words
The process of claim 26 or 27 wherein the at least one recombinant nucleotide sequence encoding the succinic semialdehyde metabolic pathway is contained on one or more vector constructs.
Claim 31
Dependent Claim
32 words
The process of claim 26 or 27 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinic semialdehyde dehydrogenase.
Claim 32
Dependent Claim
18 words
The process of claim 29 wherein the succinic semialdehyde dehydrogenase is able to convert succinyl-CoA to succinic semialdehyde.
Claim 33
Dependent Claim
31 words
The process of claim 26 or 27 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinyl-CoA:CoA transferase.
Claim 34
Dependent Claim
33 words
The process of claim 26 or 27 wherein the step of cultivating comprises cultivating the host cell on a carbon source that does not comprise 4-hydroxybutyrate or a structurally related precursor of 4-hydroxybutyrate.
Claim 35
Dependent Claim
14 words
The process of claim 32 wherein the structurally related precursor is 1,4-butanediol or 4-butyrolactone.
Claim 36
Dependent Claim
29 words
The process of claim 26 or 27 wherein the step of cultivating comprises cultivating the host cell on a carbon source consisting essentially of glucose, sucrose, fructose or lactose.
Claim 37
Dependent Claim
17 words
The process of claim 26 or 27 wherein the host cell is an Ralstonia eutropha host cell.
Claim 38
Dependent Claim
17 words
The process of claim 26 or 27 wherein the host cell is a Nocardia corallina host cell.
Claim 39
Dependent Claim
77 words
A process for producing a polyhydroxyalkanoate comprising 4-hydroxybutyrate monomer units, the method comprising: providing a host cell that metabolizes succinic semialdehyde via a succinic semialdehyde metabolic pathway that uses a 4-hydroxybutyryl-CoA intermediate, the host cell containing at least one recombinant nucleotide sequence that encodes a polyhydroxyalkanoate biosynthetic pathway; cultivating the host cell under suitable conditions to obtain expression of the succinic semialdehyde metabolic pathway and the polyhydroxyalkanoate biosynthetic pathway in the host cell, to produce the polyhydroxyalkanoate.
Claim 40
Dependent Claim
77 words
A process for producing a polyhydroxyalkanoate comprising 4-hydroxybutyrate monomer units, the method comprising: providing a host cell that metabolizes succinic semialdehyde via a succinic semialdehyde metabolic pathway that uses a 4-hydroxybutyryl-CoA intermediate, the host cell containing at least one recombinant nucleotide sequence that encodes a polyhydroxyalkanoate biosynthetic pathway; cultivating the host cell under suitable conditions to obtain expression of the succinic semialdehyde metabolic pathway and the polyhydroxyalkanoate biosynthetic pathway in the host cell, to produce the polyhydroxyalkanoate.
Claim 41
Dependent Claim
20 words
The process of claim 37 wherein the method further comprises the step of isolating the polyhydroxyalkanoate from the host cell.
Claim 42
Dependent Claim
28 words
The process of claim 37 or 38 wherein the at least one recombinant nucleotide sequence encoding the polyhydroxyalkanoate biosynthetic pathway is contained on one or more vector constructs.
Claim 43
Dependent Claim
32 words
The process of claim 37 or 38 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinic semialdehyde dehydrogenase.
Claim 44
Dependent Claim
21 words
The process of claim 26, 27 or 40 wherein the succinic semialdehyde dehydrogenase is able to convert succinyl-CoA to succinic semialdehyde.
Claim 45
Dependent Claim
31 words
The process of claim 37 or 38 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinyl-CoA:CoA transferase.
Claim 46
Dependent Claim
33 words
The process of claim 37 or 38 wherein the step of cultivating comprises cultivating the host cell on a carbon source that does not comprise 4-hydroxybutyrate or a structurally related precursor of 4-hydroxybutyrate.
Claim 47
Dependent Claim
14 words
The process of claim 43 wherein the structurally related precursor is 1,4-butanediol or 4-butyrolactone.
Claim 48
Dependent Claim
29 words
The process of claim 37 or 38 wherein the step of cultivating comprises cultivating the host cell on a carbon source consisting essentially of glucose, sucrose, fructose or lactose.
Claim 49
Dependent Claim
17 words
The process of claim 37 or 38 wherein the host cell is a Clostridium kluyveri host cell.
Claim 50
Dependent Claim
27 words
The process of any one of claims 1, 26 or 37 wherein the polyhydroxyalkanoate is poly(4-hydroxybutyrate) and wherein the polyhydroxyalkanoate biosynthetic pathway consists of a polyhydroxyalkanoate synthase.
Claim 51
Dependent Claim
38 words
A host cell containing at least one recombinant nucleotide sequence that encodes a polyhydroxyalkanoate biosynthetic pathway, and at least one recombinant nucleotide sequence that encodes a succinic semialdehyde metabolic pathway that metabolizes succinic semialdehyde via a 4-hydroxybutyryl-CoA intermediate.
Claim 52
Dependent Claim
38 words
A host cell containing at least one recombinant nucleotide sequence that encodes a polyhydroxyalkanoate biosynthetic pathway, and at least one recombinant nucleotide sequence that encodes a succinic semialdehyde metabolic pathway that metabolizes succinic semialdehyde via a 4-hydroxybutyryl-CoA intermediate.
Claim 53
Dependent Claim
25 words
The host cell of claim 48 wherein at least one of the recombinant nucleotide sequences is contained in a vector construct that expresses the sequence.
Claim 54
Dependent Claim
47 words
The host cell of claim 48 or 49 wherein the polyhydroxyalkanoate biosynthetic pathway is encoded by a single sequence recombinant nucleotide contained in a first vector construct, and the succinic semialdehyde metabolic pathway is encoded by a single sequence recombinant nucleotide contained in a second vector construct.
Claim 55
Dependent Claim
31 words
The host cell of claim 48 or 49 wherein the polyhydroxyalkanoate biosynthetic pathway and the succinic semialdehyde metabolic pathway are encoded by sequences recombinant nucleotide contained on a single vector construct.
Claim 56
Dependent Claim
33 words
The host cell of claim 48 or 49 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinic semialdehyde dehydrogenase.
Claim 57
Dependent Claim
21 words
The host cell of claim 48 or 49 wherein the succinic semialdehyde dehydrogenase is able to convert succinyl-CoA to succinic semialdehyde.
Claim 58
Dependent Claim
30 words
The host cell of claim 53 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinyl-CoA:CoA transferase.
Claim 59
Dependent Claim
18 words
The host cell of claim 48 or 49 wherein the host cell is an Escherichia coli host cell.
Claim 60
Dependent Claim
17 words
The host cell of claim 48 or 49 wherein the host cell is a Klebsiella host cell.
Claim 61
Dependent Claim
17 words
The host cell of claim 56 wherein the host cell is a Klebsiella aerogenes or Klebsiella oxytoca.
Claim 62
Dependent Claim
17 words
The host cell of claim 48 or 49 wherein the host cell is a plant host cell.
Claim 63
Dependent Claim
17 words
The host cell of claim 48 or 49 wherein the host cell is an insect host cell.
Claim 64
Dependent Claim
17 words
The host cell of claim 48 or 49 wherein the host cell is a spider host cell.
Claim 65
Dependent Claim
24 words
The host cell of claim 48 or 49 wherein the host cell has an inhibiting mutation in its CoA-independent NAD-dependent succinic independent semialdehyde dehydrogenase.
Claim 66
Dependent Claim
23 words
The host cell of claim 48 or 49 wherein the host cell has an inhibiting mutation in its CoA-independent NADP-dependent succinic semialdehyde dehydrogenase.
Claim 67
Dependent Claim
31 words
The host cell of claim 48 or 49 wherein the host cell has an inhibiting mutation in its CoA-independent NAD-dependent succinic independent semialdehyde dehydrogenase and its CoA-independent NADP-dependent succinic semialdehyde dehydrogenase.
Claim 68
Dependent Claim
31 words
A vector construct that expresses at least one sequence recombinant nucleotide that encodes a polyhydroxyalkanoate biosynthetic pathway, and at least one sequence recombinant nucleotide that encodes a succinic semialdehyde metabolic pathway.
Claim 69
Dependent Claim
31 words
A vector construct that expresses at least one sequence recombinant nucleotide that encodes a polyhydroxyalkanoate biosynthetic pathway, and at least one sequence recombinant nucleotide that encodes a succinic semialdehyde metabolic pathway.
Claim 70
Dependent Claim
31 words
The vector construct of claim 64 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinic semialdehyde dehydrogenase.
Claim 71
Dependent Claim
19 words
The vector construct of claim 65 wherein the succinic semialdehyde dehydrogenase is able to convert succinyl-CoA to succinic semialdehyde.
Claim 72
Dependent Claim
30 words
The vector construct of claim 64 wherein the at least one sequence recombinant nucleotide encoding the succinic semialdehyde metabolic pathway further comprises a sequence recombinant nucleotide encoding a succinyl-CoA:CoA transferase.
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