Methods and Compositions for Improving the Production of Fuels in Microorganisms

• US 20090286294A1
• Filed: 04/06/2009
• Published: 11/19/2009
• Est. Priority Date: 04/04/2008
• Status: Abandoned Application

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: BLANCHARD, JEFFREY, FABEL, JOHN, LESCHINE, SUSAN, PETIT, ELSA
• Application Number:
  US12/419,211
• Publication Number:
  US 20090286294A1
• Time in Patent Office: Days
• US Class Current: 435/161
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/0095 acting on iron-sulfur proteins as donor (1.18)
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/08 produced as by-product or from waste or cellulosic material substrate
• CPC Class Codes: C12Y 102/07001 Pyruvate synthase (1.2.7.1), i.e. pyruvate ferredoxin oxidoreductase
• CPC Class Codes: C12Y 118/01002 Ferredoxin-NADP+ reductase (1.18.1.2)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The invention relates to compositions, systems, and methods for producing fuels, such as ethanol and hydrogen, and related compounds. More specifically, compositions and methods are provided for making recombinant microorganisms for the production of fuels using genes from the Clostridium phytofermentans ethanol and hydrogen pathways disclosed herein.

Claims

1. An isolated polynucleotide that encodes a polypeptide that modulates fuel production in C. phytofermentans.

2. The polynucleotide of claim 1, wherein the polynucleotide comprises a nicotinamide adenine dinucleotide (NADH) ferredoxin oxidoreductase (Nfo) subunit.

3. The polynucleotide of claim 1, wherein the polynucleotide comprises a C. phytofermentans rnf operon.

4. The polynucleotide of claim 1, wherein the polynucleotide comprises a nucleic acid sequence corresponding to a region of the C. phytofermentans chromosome extending from about position 259945 to about position 265175.

5. The polynucleotide of claim 1, wherein the polynucleotide comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, and SEQ ID NO;

6.

6. The polynucleotide of claim 2, wherein the Nfo subunit is selected from the group consisting of RnfC, RnfD, RnfG, RnfE, RnfA, and RnfB.

7. The polynucleotide of claim 1, wherein the polynucleotide comprises a nucleic acid sequence encoding RnfC, RnfD, RnfG, RnfE, RnfA, and RnfB.

8. The polynucleotide of claim 1, wherein the polynucleotide further comprises a nucleic acid sequence encoding an enzyme selected from the group consisting of pyruvate ferredoxin oxidoreductase (Pfo), acetaldehyde dehydrogenase, ethanol dehydrogenase, and hydrogenase.

9. An expression cassette that enables an organism to produce a fuel, the expression cassette comprising an isolated polynucleotide that encodes at least one polypeptide that modulates fuel production in C. phytofermentans.

10. The expression cassette of claim 9, wherein the polynucleotide comprises a nucleic acid sequence encoding a nicotinamide adenine dinucleotide (NADH) ferredoxin oxidoreductase (Nfo) subunit.

11. The expression cassette of claim 9, wherein the polynucleotide comprises a C. phy rnf operon.

12. The expression cassette of claim 10, wherein the Nfo subunit is selected from the group consisting of RnfC, RnfD, RnfG, RnfE, RnfA, and RnfB.

13. The expression cassette of claim 9, wherein the polynucleotide comprises a nucleic acid sequence encoding RnfC, RnfD, RnfG, RnfE, RnfA, and RnfB.

14. The expression cassette of claim 9, wherein the polynucleotide further comprises a nucleic acid sequence encoding an enzyme selected from the group consisting of pyruvate ferredoxin oxidoreductase (Pfo), acetaldehyde dehydrogenase, ethanol dehydrogenase, and hydrogenase.

15. The expression cassette of claim 9, wherein the polynucleotide further comprises a sequence encoding a selectable marker.

16. An isolated microorganism comprising a heterologous polynucleotide encoding at least one polypeptide that encodes a polypeptide that modulates fuel production in C. phytofermentans.

17. The microorganism of claim 16, wherein the microorganism ferments cellulose-containing biomass to produce at least one fuel.

18. The microorganism of claim 16, wherein the heterologous polynucleotide comprises a nucleic acid sequence corresponding to a gene from a C. phytofermentans metabolic pathway.

19. The microorganism of claim 16, wherein the heterologous polynucleotide comprises a nucleic acid sequence encoding a nicotinamide adenine dinucleotide (NADH) ferredoxin oxidoreductase (Nfo) subunit.

20. The microorganism of claim 16, wherein the heterologous polynucleotide comprises the C. phy rnf operon.

21. The microorganism of claim 16, wherein the heterologous polynucleotide comprises a nucleic acid sequence encoding RnfC, RnfD, RnfG, RnfE, RnfA, and RnfB.

22. The microorganism of claim 19, wherein the Nfo subunit is selected from the group consisting of RnfC, RnfD, RnfG, RnfE, RnfA, and RnfB.

23. The microorganism of claim 16, wherein the heterologous polynucleotide further comprises a nucleic acid sequence encoding an enzyme selected from the group consisting of pyruvate ferredoxin oxidoreductase (Pfo), acetaldehyde dehydrogenase, ethanol dehydrogenase, and hydrogenase.

24. The microorganism of claim 16, wherein the heterologous polynucleotide further comprises a sequence encoding a selectable marker.

25. The microorganism of claim 16, wherein the microorganism is a prokaryote or eukaryote.

26. The microorganism of claim 16, wherein the microorganism is selected from a group consisting of Escherichia, Zymomonas, Saccharomyces, Candida, Pichia, Streptomyces, Bacillus, Lactobacillus, and Clostridium.

27. The microorganism of claim 16, wherein the microorganisms is selected from the group consisting of Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium celerecrescens, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella cellulolytica, Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacterium saccharolyticum.

28. The microorganism of claim 16, wherein the microorganism produces a fuel in recoverable quantities greater than about 10 mM fuel after a 5 day fermentation.

29. The microorganism of claim 16, wherein said fuel is ethanol.

30. A method for producing fuel, the method comprising culturing a microorganism of claim 16 in a culture medium.

31. The method of claim 30, wherein the microorganism comprises a polynucleotide encoding a nicotinamide adenine dinucleotide (NADH) ferredoxin oxidoreductase (Nfo) subunit.

32. The method of claim 31, wherein the Nfo subunit is selected from the group consisting of RnfC, RnfD, RnfG, RnfE, RnfA, and RnfB.

33. The method of claim 30, wherein the polynucleotide comprises an rnf operon.

34. The method of claim 30, wherein the heterologous polynucleotide comprises a nucleic acid sequence encoding RnfC, RnfD, RnfG, RnfE, RnfA, and RnfB.

35. The method of claim 30, wherein the heterologous polynucleotide further comprises a nucleic acid sequence encoding an enzyme selected from the group consisting of pyruvate ferredoxin oxidoreductase (Pfo), acetaldehyde dehydrogenase, ethanol dehydrogenase, and hydrogenase.

36. The method of claim 30, wherein the microorganism is a prokaryote or eukaryote.

37. The method of claim 30, wherein the microorganism is selected from a group consisting of Escherichia, Zymomonas, Saccharomyces, Candida, Pichia, Streptomyces, Bacillus, Lactobacillus, and Clostridium.

38. The method of claim 30, wherein the microorganisms is selected from the group consisting of Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium celerecrescens, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella cellulolytica, Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacterium saccharolyticum.

39. The method of claim 30, wherein the microorganism produces a fuel in recoverable quantities greater than about 10 mM fuel after a 5 day fermentation.

40. The method of claim 30, wherein the fuel is hydrogen or ethanol.

41. The method of claim 30, wherein the culturing is performed in normal batch fermentation, fed-batch fermentation, or continuous fermentation.

42. The method of claims 30, where the culture medium comprises pretreated or non-pretreated feedstock.

43. The method of claim 42, wherein the feedstock comprises cellulosic, hemicellulosic, and/or lignocellulosic material.

44. The method of claim 43, wherein the culture medium comprises glucose, cellulose, xylan, or a combination thereof.

45. The method of claim 43, wherein the fuel is ethanol.