Claims
- 1. A process for using a genetically modified Zymomonas integrant to produce ethanol from a pentose sugar, wherein said process comprises:
preparing a Zymomonas integrant comprising a sequence encoding more than one pentose-sugar fermenting enzymes, wherein said pentose fermenting enzymes comprise xylose isomerase, xylulokinase, transketolase, and transaldolase; adding the integrant Zymomonas to a feedstock comprising pentose under appropriate fermentation conditions; and fermenting the pentose sugar to provide a composition comprising ethanol, wherein said sequence encoding the pentose fermenting enzymes is incorporated into said integrant Zymomonas by a first integration event and a second integration event.
- 2. The process of claim 1 wherein the first integration event is a transposition and wherein said second integration event is a homologous recombination.
- 3. The process of claim 2 wherein the first integration event comprises a transposition integration of a first operon comprising a sequence encoding more than one pentose-fermenting enzyme.
- 4. The process of claim 3 wherein the first operon includes a sequence encoding transaldolase and transketolase.
- 5. The process of claim 3 wherein the first operon is further defined as comprising Penotaltkt or Pgaptaltkt.
- 6. The process of claim 2 wherein the second integration event comprises a homologous recombination with a second operon comprising a sequence encoding more than one pentose-fermenting enzyme.
- 7. The process of claim 6 wherein the second operon is further defined as comprising PgapxylAB.
- 8. The process of claim 6 wherein the second operon includes a sequence encoding xylose isomerase and xylulokinase.
- 9. The process of claim 1 wherein the Zymomonas integrant is ATCC31821-5C Penotaltkt/PgapxylAB.
- 10. The process of claim 1 wherein the Zymomonas integrant is ATCC31821-5C Pgaptaltkt/PgapxylAB.
- 11. The process of claim 1 wherein the first integration event comprises a homologous recombination and the second integration event comprises a transposition event.
- 12. A method for preparing a Zymomonas integrant capable of fermenting a pentose-containing feedstock to ethanol, said method comprising:
conducting a first integration event on a native Zymomonas strain to incorporate a first operon comprising a sequence encoding a pentose-fermenting enzyme to provide a partial Zymomonas integrant; conducting a second integration event on the partial Zymomonas integrant to incorporate a second operon comprising a sequence encoding more than one pentose—fermenting enzyme other than the enzyme of the first operon to provide a Zymomonas integrant capable of fermenting a pentose-containing feedstock to ethanol.
- 13. The method of claim 12 wherein the Zymomonas integrant comprises a sequence encoding at least four pentose-sugar fermenting enzymes, wherein said pentose fermenting enzymes comprise xylose isomerase, xylulokinase, transketolase, and transaldolase.
- 14. The method of claim 12 wherein the first operon is further defined as comprising PgapxylAB.
- 15. The method of claim 12 wherein the second operon is further defined as comprising Penotaltkt or Pgaptaltkt.
- 16. A Zymomonas integrant capable of fermenting pentose prepared by the process of claim 12.
- 17. The method of claim 12 wherein the Zymomonas integrant is ATCC31821-5C Penotaltkt/PgapxylAB.
- 18. The method of claim 12 wherein the Zymomonas integrant is ATCC31821-5C Pgaptaltkt/PgapxylAB.
- 19. A Zymomonas integrant capable of growth in the presence of 8 grams acetate/acetic acid per liter media and capable of fermenting pentose sugar to ethanol.
- 20. A biocatalyst for pentose fermentation comprising the Zymomonas integrant of claim 16.
- 21. A Zymomonas integrant capable of fermenting pentose to ethanol comprising chromosomally or native plasmid integrated sequences encoding more than one pentose-fermenting enzyme, wherein said integrant is stable for at least 80 generations.
- 22. The Zymomonas integrant of claim 21 further defined as capable of fermenting a pentose sugar to ethanol in the presence of acetate.
- 23. The Zymomonas integrant of claim 22 further defined as capable of fermenting a pentose sugar in the presence of 8 grams acetate/acetic acid gram per liter media.
- 24. The Zymomonas integrant of claim 21 further comprising a sequence encoding a mannose-fermenting enzyme.
Parent Case Info
[0001] The present application is a continuation-in-part of U.S. patent application Ser. No. 09/565,233, filed May 1, 2000. The present application is related to PCT application PCT/US01/11334, filed Apr. 6, 2001, claiming priority to U.S. Ser. No.09/565,233.
CONTRACTUAL ORIGIN OF THE INVENTION
[0002] The United States Government has rights in this invention pursuant to contract No. DE-AC36-99G010337 between the United States Department of Energy and the Midwest Research Institute.
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
09565233 |
May 2000 |
US |
Child |
10134655 |
Apr 2002 |
US |