Claims
- 1. A process for producing poly-.beta.-hydroxybutyrate comprising:
- providing a culture of Escherichia coli bacterial host cells transformed by (1) a first vector containing a DNA sequence encoding the biosynthetic pathway of poly-.beta.-hydroxybutyrate from Alcaligenes eutrophus, wherein the DNA sequence coding for poly-.beta.-hydroxybutyrate comprises thee p4A plasmid, and (2) a second vector containing a DNA sequence of a T7 bacteriophage lysozyme gene;
- growing the culture in a suitable medium and obtaining expression of the poly-.beta.--hydroxybutyrate biosynthetic pathway and the lysozyme gene in the Escherichia coli bacterial host cells to provide poly-.beta.-hydroxybutyrate and lysozyme;
- lysing the Escherichia coli bacterial host cells;
- isolating the poly-.beta.-hydroxybutyrate; and,
- collecting the poly-.beta.-hydroxybutyrate.
- 2. A process for producing poly-.beta.-hydroxybutyrate comprising:
- providing a culture of Escherichia coli bacterial host cells transformed by (1) a first vector containing a DNA sequence encoding the biosynthetic pathway of poly-.beta.-hydroxybutyrate from Alcaligenes eutrophus, wherein the first vector is a plasmid selected from the group consisting of pJM9101, pJM9113, pJM9114, pJM9115, pJM9116, pJM9117, pJM9118, pJM9119, pJM9120, pJM9125, and pJM9126, and (2) a second vector containing a DNA sequence of a T7 bacteriophage lysozyme gene;
- growing the culture in a suitable medium and obtaining expression of the poly-.beta.-hydroxybutyrate biosynthetic pathway and the lysozyme gene in the Escherichia coli bacterial host cells to provide poly-.beta.-hydroxybutyrate and lysozyme;
- lysing the Escherichia coli bacterial host cells;
- isolating the poly-.beta.-hydroxybutyrate; and,
- collecting the poly-.beta.-hydroxybutyrate.
- 3. A process for producting poly-.beta.-hydroxybutyrate comprising:
- providing a culture of Escherichia coli bacterial host cells, in which the Escherichia coli bacterial host cells comprise a strain (S4) designated as E. coli HMS174(p4A (BstB-),pLysS), deposited with the American Type Culture Collection under ATCC Accession No. 69001;
- growing the culture in a suitable medium and obtaining expression of the poly-.beta.-hydroxybutyrate biosynthetic pathway and the lysozyme gene in the Escharichia coli bacterial host cells to provide poly-.beta.-hydroxybutyrate and lysozyme;
- lysing the Escherichia coli bacterial host cells;
- isolating the poly-.beta.-hydroxybutyrate; and,
- collecting the poly-.beta.-hydroxybutyrate.
- 4. A process for producing poly-.beta.-hydroxybutyrate comprising:
- providing a culture of Escherichia coli bacterial host cells transformed by (1) a first vector containing a DNA sequence encoding the biosynthetic pathway of poly-.beta.-hydroxybutyrate from Alcaligenes eutrophus, wherein the DNA sequence of the biosynthetic pathway of poly-.beta.-hydroxybutyrate is contained on a high copy number plasmid or on a runaway replication vector, and (2) a second vector containing a DNA sequence of a T7 bacteriophage lysozyme gene;
- growing the culture in a suitable medium and obtaining expression of the poly-.beta.-hydroxybutyrate biosynthetic pathway and the lysozyme gene in the Escherichia coli bacterial host cells to provide poly-.beta.-hydroxybutyrate and lysozyme;
- lysing the Escherichia coli bacterial host cells:
- isolating the poly-.beta.-hydroxybutyrate; and,
- collecting the poly-.beta.-hydroxybutyrate at levels of about 90 to about 95%, by weight, of dry cell weight.
- 5. A process for producing poly-.beta.-hydroxybutyrate comprising:
- providing a culture of Escherichia coli bacterial host cells transformed by (1) a first vector containing a DNA sequence encoding the biosynthetic pathway of poly-.beta.-hydroxybutyrate from Alcaligenes eutrophus, in which the DNA sequence coding for poly-.beta.-hydroxybutyrate is contained on a high copy number plasmid, and (2) a second vector containing a DNA sequence of a T7 bacteriophage lysozyme gene;
- growing the culture in a suitable medium and obtaining expression of the poly-.beta.-hydroxybutyrate biosynthetic pathway and the lysozyme gene in the Escherichia coli bacterial host cells to provide poly-.beta.-hydroxybutyrate and lysozyme;
- lysing the Escherichia coli bacterial host cells;
- isolating the poly-.beta.-hydroxybutyrate; and, collecting the poly-.beta.-hydroxybutyrate.
- 6. A process for production of poly-.beta.-hydroxybutyrate, the method comprising transforming an Escherichia coli having a dna.beta.am mutation with a vector containing (1) Alcaligenes eutrophus genes coding the biosynthetic pathway of poly-.beta.-hydroxybutyrate and (2) a stabilization gene selected from the group consisting of parB and supF, wherein the genes are expressed by the transformed Escherichia coli at sufficient levels to result in the production of poly-.beta.-hydroxybutyrate as a fermentation product when the Escherichia coli is grown in an appropriate medium, and wherein said vector is a plasmid selected from the group consisting of, pJM9113, pJM9114, pJM9117, pJM9118, pJM9125 and pJM9126.
- 7. The method according to claim 6, wherein the Escherichia coli has been transformed with pJM9126.
- 8. The process according to claim 6, wherein the stabilization gene comprises a supF gene.
- 9. A process for producing poly-.beta.-hydroxybutyrate comprising:
- providing a culture of Escherichia coli bacterial host cells transformed by (1) a first vector containing a DNA sequence encoding the biosynthetic pathway of poly-.beta.-hydroxybutyrate from Alcaligenes eutrophus, wherein the isolated DNA sequence coding for poly-.beta.-hydroxybutyrate is contained on a runway replication vector, and (2) a second vector containing a DNA sequence of a T7 bacteriophage lysozyme gene;
- growing the culture in a suitable medium and obtaining expression of the poly-.beta.-hydroxybutyrate biosynthetic pathway and the lysozyme gene in the Escherichia coli bacterial host cells to provide poly-.beta.-hydroxybutyrate and lysozyme;
- lysing the Escherichia coli bacterial host cells;
- isolating the poly-.beta.-hydroxybutyrate; and
- collecting the poly-.beta.-hydroxybutyrate.
- 10. A process for production of poly-.beta.-hydroxybutyrate, the method comprising transforming an Escherichia coli having a dnaBam mutation with a vector containing (1) Alcaligenes eutrophus genes coding for the biosynthethic pathway of poly-.beta.-hydroxybutyrate and (2) a stabilization gene selected from the group consisting of parB and supF, wherein the genes are expressed by the transformed Escherichia coli at sufficient levels to result in the production of poly-.beta.-hydroxybutyrate as a fermentation product when the Escherichia coli is grown in an appropriate medium, and wherein the DNA sequence of the biosynthetic pathway of poly-.beta.-hydroxybutyrate is contained on a runaway replication vector.
- 11. A process for producing poly-.beta.-hydroxybutyrate comprising:
- providing a culture of Escherichia coli bacterial host cells tranformed by (1) an isolated DNA sequence coding for the Alcaligenes eutrophus biosynthetic pathway of poly-.beta.-hydroxybutyrate, and (2) an isolated DNA sequence of a T7 bacteriophage lysozyme gene;
- growing the culture in a suitable medium and obtaining expression of the poly-.beta.-hydroxybutyrate biosynthetic pathway and the lysozyme gene in each Escherichia coli bacterial host cell;
- isolating the poly-.beta.-hydroxybutyrate by lysing the Escherichia coli bacterial host cells; and
- collecting the poly-.beta.-hydroxybutyrate.
- 12. The process of claim 11 in which the Escherichia coli bacterial host cells are lysed by exposing the cells to a permeabilizing agent of sufficient strength to permeabilize the host cell's inner membrane.
- 13. The process of claim 12, wherein the host cells are further exposed to a suitable surfactant.
- 14. The process of claim 11 in which the Escherichia coli bacterial host cells are lysed by pelleting the cells and resuspending the cells in a permeabilizing agent.
- 15. The process of claim 11 in which the Escherichia coli bacterial host cells are lysed by pelleting the cells, resuspending the cells in a permeabilizing agent, and thereafter, freezing the cells.
- 16. The process of claim 11 in which the Escherichia coli bacterial host cells comprise Escherichia coli HMS 174.
- 17. The process of claim 11 in which the plasmid is selected from the group consisting of pJM9101, pJM9113, pJM9114, plM9115, plM9116, pJM9117, pMJ9118, pJM9119, pJM9120, pJM9125, and pJM9126.
- 18. The process of claim 11 in which the Escherichia coli bacterial host is transformed by a vector having a DNA sequence coding for poly-.beta.-hydroxybutyrate and a DNA sequence coding for T7 lysozyme.
- 19. The process of claim 11 in which the isolated DNA sequence of the biosynthetic pathway of poly-.beta.-hydroxybutyrate is contained on a high copy number plasmid.
- 20. The process of claim 19 in which poly-.beta.-hydroxybutyrate is collected at levels of about 90 to about 95%, by weight, of dry cell weight.
Parent Case Info
This is a continuation-in-part of U.S. Ser. No. 07/705,806, filed May 24, 1991 which is a continuation-in-part of U.S. Ser. No. 528,549, filed May 25, 1990, both of which are expressly incorporated herein by reference, both now abandoned.
Non-Patent Literature Citations (2)
Entry |
Slater et al., "Cloning and Expression in E. coli of the A. eutrophus H16 PHB Biosynth. Pathway", J. Bact. 170(10):4431-4436 (Oct. 1988). |
Studier et al., "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes", Meth. Enz. 185:60-89 (1990). |
Continuation in Parts (2)
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Number |
Date |
Country |
Parent |
705806 |
May 1991 |
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Parent |
528549 |
May 1990 |
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