US5750848A
Expired / Lapsed
US
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DNA sequence useful for the production of polyhydroxyalkanoates
Assignee: Yield10 Bioscience Inc
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Filing: 1996-08-13
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Granted: 1998-05-12
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Expires: 2016-08-13
Abstract
Abstract A genomic fragment harboring the gene phaG was cloned by phenotypic complementation of Pseudomonas putida KT2440 mutants, defective in the polyhydroxyalkanoic acid (PHA) synthesis via de novo fatty acid biosynthesis but not affected in PHA biosynthesis via fatty acid beta -oxidation. The 885-bp phaG gene encodes a protein of 295 amino acids with a molecular weight of 33,876 Da. The transcriptional induction of phaG could be observed under conditions of PHA synthesis via de novo fatty acid biosynthesis whereas no induction was detected under conditions which favor PHA synthesis via fatty acid degradation by beta -oxidation. The phaG gene is useful for the production of PHAs in bacteria and plants.
Inventors
- 👤 Niels Kruger
- 👤 Alexander Steinbuchel
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 03:21
CPC Classifications
C; C12; C12P; C12P7/00; C12P7/62; C12P7/625; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/82; C12N15/8241; C12N15/8242; C12N15/8243; C12N9/00; C12N9/10; C12N9/1025; C12N9/1029
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
128 words
An isolated DNA molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of the coding strand shown in SEQ ID NO: 1, positions 911 through 1795, or the complement thereof; (b) a nucleotide sequence that hybridizes to said nucleotide sequence of (a) under a wash stringency equivalent to 0.5X SSC to 2X SSC, 0.1% SDS, at 55°-65° C., and which encodes a PHA synthase protein; (c) a Pseudomonas putida nucleotide sequence having at least 40% identity with the nucleotide sequence of (a) and which encodes a PHA synthase protein; (d) a nucleotide sequence encoding the same amino acid sequence as said nucleotide sequence of (a); and (e) a nucleotide sequence encoding the same amino acid sequence as said nucleotide sequence of (b).
Claim 2
Dependent Claim
128 words
An isolated DNA molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence of the coding strand shown in SEQ ID NO: 1, positions 911 through 1795, or the complement thereof; (b) a nucleotide sequence that hybridizes to said nucleotide sequence of (a) under a wash stringency equivalent to 0.5X SSC to 2X SSC, 0.1% SDS, at 55°-65° C., and which encodes a PHA synthase protein; (c) a Pseudomonas putida nucleotide sequence having at least 40% identity with the nucleotide sequence of (a) and which encodes a PHA synthase protein; (d) a nucleotide sequence encoding the same amino acid sequence as said nucleotide sequence of (a); and (e) a nucleotide sequence encoding the same amino acid sequence as said nucleotide sequence of (b).
Claim 3
Dependent Claim
19 words
The DNA molecule of claim 1 having the DNA sequence of SEQ ID NO: 1, positions 911 through 1795.
Claim 4
Dependent Claim
30 words
A recombinant DNA comprising a DNA molecule of claim 1 and a promoter region, operatively linked such that the promoter enhances transcription of said DNA molecule in a host cell.
Claim 5
Dependent Claim
23 words
The recombinant DNA of claim 3 wherein said DNA molecule has the DNA sequence of SEQ ID NO: 1, positions 911 through 1795.
Claim 6
Dependent Claim
12 words
A recombinant vector comprising a DNA of any one of claims 1-4.
Claim 7
Dependent Claim
22 words
A recombinant host cell comprising a DNA of claims 3 or 4, wherein said DNA molecule is heterologous to said promoter region.
Claim 8
Dependent Claim
13 words
The recombinant cell of claim 6 which is a microbe or plant cell.
Claim 9
Dependent Claim
16 words
The recombinant cell of claim 6 which is a Pseudomonas fluorescens rRNA homology group I bacterium.
Claim 10
Dependent Claim
72 words
A method of transforming a host cell capable of producing polyhydroxyalkanoates comprising: providing a host cell having a functional PHA synthase gene; preparing a recombinant vector comprising a DNA molecule of claim 1 and a promoter region, operatively linked such that the promoter causes transcription of said DNA molecule in said host cell, and wherein said DNA molecule is heterologous to said promoter region; and transforming the host cell with said vector.
Claim 11
Dependent Claim
25 words
The method of claim 9 wherein the recombinant vector comprises a DNA molecule having the nucleotide sequence of SEQ ID NO:1, positions 911 through 1795.
Claim 12
Dependent Claim
16 words
The method of claim 9 wherein the host cell is a microbe or a plant cell.
Claim 13
Dependent Claim
21 words
The method of claim 9 wherein the host cell is selected from bacteria of the Pseudomonas fluorescens rRNA homology group I.
Claim 14
Independent Claim
40 words
A method for preparing polyhydroxyalkanoates composed of repeating units having 3-14 carbon atoms (PHA) comprising: culturing transformed cells produced according to any one of claims 9-12 to allow expression of the pha genes; and recovering the PHA from the cells.
Claim 15
Dependent Claim
19 words
The method of claim 13 wherein the transformed cells are cultured in a media containing a simple carbohydrate substrate.
Claim 16
Dependent Claim
15 words
The method of claim 14 wherein the media comprises gluconate, glucose, sucrose, fructose or lactose.
Claim 17
Dependent Claim
19 words
The method of claim 13 wherein the transformed cells are cultured in a media comprising corn syrup or molasses.
Claim 18
Dependent Claim
13 words
A substantially purified protein having the amino acid sequence of SEQ ID NO:2.
Claim 19
Dependent Claim
13 words
A substantially purified protein having the amino acid sequence of SEQ ID NO:2.
Claim 20
Dependent Claim
70 words
A method of producing a genetically transformed plant which expresses PhaG comprising the steps of: inserting into the genome of a host plant cell a recombinant, double-stranded DNA molecule comprising: (i) a promoter which functions in plant cells to enhance transcription of an adjacent DNA coding sequence; (ii) a DNA molecule of claim 1 operatively linked to the promoter; and regenerating a genetically transformed plant from said host plant cell.
Claim 21
Dependent Claim
17 words
The method of claim 18 wherein the DNA molecule is SEQ ID NO:1, positions 911 through 1795.
Claim 22
Dependent Claim
20 words
The method of claim 18 wherein the genome of the host plant cell also includes a functional PHA synthase gene.
Claim 23
Dependent Claim
14 words
The method of claim 18 further comprising harvesting the transformed plant or plant parts.
Claim 24
Dependent Claim
15 words
The method of claim 21 wherein the plant parts comprise leaves, roots, seeds or tubers.
Claim 25
Dependent Claim
37 words
The method of claim 18 wherein the plant is maize, potato, sugar beet, tobacco, wheat, or Arabidonsis; or a high oil seed plant selected from the group consisting of soybean, canola, rape seed, sunflower, flax and peanut.
Claim 26
Dependent Claim
14 words
A plant produced by the method of any one of claims 18-20 or 23.
Claim 27
Dependent Claim
19 words
A mutant Pseudomonas putida microorganism unable to synthesize PHA from gluconate but able to synthesize PHA from octanoic acid.
Claim 28
Dependent Claim
19 words
A mutant Pseudomonas putida microorganism unable to synthesize PHA from gluconate but able to synthesize PHA from octanoic acid.
Claim 29
Dependent Claim
27 words
The mutant of claim 25 wherein the phenotypic ability to synthesize PHA from gluconate is restored by complementation with a DNA sequence of SEQ ID NO: 1.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-05-11 | 2001-11-10 | 2002-05-12 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-05-11 | 2005-11-10 | 2006-05-12 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-05-11 | 2009-11-10 | 2010-05-12 | $7700.00 | Window Closed / Lapsed |