Zymomonas pentose-sugar fermenting strains and uses thereof

• US 20030162271A1
• Filed: 04/27/2002
• Published: 08/28/2003
• Est. Priority Date: 05/01/2000
• Status: Active Grant

• Current Assignee: Alliance For Sustainable Energy LLC
• Original Assignee: Midwest Research Institute
• Inventors: Chou, Yat-Chen, Zhang, Min, Evans, Kent, Howe, William, Eddy, Christine, Mohagheghi, Ali
• Granted Patent: US 7,223,575 B2
• Time in Patent Office: Days
• US Class Current: 435/161
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/92 Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/065 with microorganisms other than yeasts
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Disclosed in the present invention is a Zymomonas integrant and derivatives of these integrants that posses the ability to ferment pentose into ethanol. The genetic sequences encoding for the pentose-fermenting enzymes are integrated into the Zymomonas in a two-integration event of homologous recombination and transposition. Each operon includes more than one pentose-reducing enzyme encoding sequence. The integrant in some embodiments includes enzyme sequences encoding xylose isomerase, xylulokinase, transketolase and transketolase. The Zymomonas integrants are highly stable, and retain activity for producing the pentose-fermenting enzyme for between 80 to 160 generations. The integrants are also resistant to acetate inhibition, as the integrants demonstrate efficient ethanol production even in the presence of 8 up to 16 grams acetate per liter media. These stably integrated sequences provide a unique Zymomonas that may then be used for the efficient conversion of pentose sugars (xylose, arabinose) to ethanol. Method of using the Zymomonas integrants and derivatives thereof in production of ethanol from cellulosic feedstock is also disclosed. The invention also provides a method for preparing a Zymomonas integrant as part of the present invention. The host Zymomonas strain found particularly useful in the creation of these compositions and methods is Zymomonas mobilis 31821.

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.

20. A biocatalyst for pentose fermentation comprising the Zymomonas integrant of claim 16.

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.

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.