MICROORGANISMS WITH INACTIVATED LACTATE DEHYDROGENASE GENE (LDH) FOR CHEMICAL PRODUCTION

• US 20110230682A1
• Filed: 03/22/2010
• Published: 09/22/2011
• Est. Priority Date: 03/19/2010
• Status: Abandoned Application

• Current Assignee: Qteros, Inc.
• Original Assignee: Qteros, Inc.
• Inventors: Bowman, Tim, Schmalisch, Matthias, Huang, Kexue, Ali, Muhammad Abdul
• Application Number:
  US12/729,037
• Publication Number:
  US 20110230682A1
• Time in Patent Office: Days
• US Class Current: 568/840
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12R 2001/145 Clostridium
• CPC Class Codes: C12Y 101/01027 L-Lactate dehydrogenase (1.1.1.27)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

This invention provides systems and methods for the increased production of ethanol and other chemical compounds by recombinant Clostridium species whereby the recombinant species are genetically-engineered to disrupt lactate dehydrogenase activity and to hydrolyze and ferment carbonaceous biomass and synthesize compounds of commercial value without production of lactic acid.

Claims

1. An isolated microorganism genetically modified to lack expression of a wild-type lactate dehydrogenase gene, wherein the microorganism can hydrolyze both C5 and C6 polysaccharides.

2. The microorganism of claim 1, wherein said microorganism further does not express pyruvate decarboxylase.

3. The microorganism according to claim 1, wherein said microorganism does not comprise an integration element in a lactate dehydrogenase gene.

4. The microorganism according to claim 1, wherein said microorganism is a mesophilic microorganism.

5. The microorganism according to claim 1, wherein said lactate dehydrogenase gene is Cphy_—

1232 or Cphy_—

1117.

6. The microorganism according to claim 1, wherein said microorganism is gram negative.

7. The microorganism according to claims 1, wherein said microorganism is gram positive.

8. The microorganism of claim 1, wherein said microorganism is a Clostridium species.

9. The microorganism of claim 8, wherein said Clostridium species is C. phytofermentans.

10. An isolated microorganism selected from the group consisting of:

NRRL Accession No. NRRL B 50351, NRRL B-50352, NRRL B-50353, NRRL B 50354, NRRL B-50355, NRRL B-50356, NRRL B-50357, NRRL B-50358, and NRRL B-50359.

11. An isolated mesophilic microorganism that lacks lactate dehydrogenase activity, wherein said mesophilic microorganism ferments polysaccharides.

13. The microorganism according to claim 11 or 12, wherein said microorganism is an anaerobic microorganism that can saccharify both C5 and C6 polysaccharides.

14. The microorganism according to claim 11 or 12, wherein said microorganism is gram negative.

15. The microorganism according to claim 11 or 12, wherein said microorganism is gram positive.

12. An isolated mesophilic microorganism, genetically modified to produce higher amounts of ethanol, wherein said modification comprises inactivation of lactic acid synthesis as it occurs in the wild-type mesophilic microorganism.

16. A product for production of a chemical product comprising:

a. a fermentation vessel comprising a carbonaceous biomass; and

b. a microorganism genetically modified to inactivate a lactate dehydrogenase gene of the wild-type organism, wherein the microorganism hydrolyzes both C5 and C6 polysaccharides and produces a chemical product.

17. The product of claim 16, wherein said microorganism is capable of direct fermentation of C5 and C6 carbohydrates.

18. The product according to claim 16, wherein said microorganism is a mesophilic microorganism.

19. The product according to claims claim 16, wherein said microorganism is a Clostridium species.

20. The product according to claim 16, wherein said lactate dehydrogenase gene is Cphy_—

1232 or Cphy_—

1117.

21. The product according to claim 16, wherein said biomass comprises cellulosic or lignocellulosic materials.

22. The product according to claim 16, wherein said biomass comprises one or more of corn steep solids, corn steep liquor, malt syrup, xylan, cellulose, hemicellulose, fructose, glucose, mannose, rhamnose, or xylose.

23. The product according to claims claim 16, wherein said biomass comprises municipal waste, wood, plant material, plant material extract, bagasse, switch grass, algae, corn stover, poplar, a natural or synthetic polymer, or a combination thereof.

24. The product according to claim 16, wherein said biomass is pre-conditioned with acid treatment.

25. The product according to claim 16, wherein said chemical product is an alcohol, a gas, an acid, a fatty acid, an isoprenoid, or a polyisoprene.

26. The product according to claim 16, wherein said microorganism is capable of hydrolyzing xylose.

27. The product according to claim 16, wherein said chemical product is ethanol, butanol, methanol, methane, or hydrogen.

28. A process for producing a chemical product comprising:

a. contacting a carbonaceous biomass with a microorganism genetically modified to inactivate a lactate dehydrogenase gene of the wild-type organism, wherein the microorganism hydrolyzes both C5 and C6 polysaccharides;

b. allowing sufficient time for said hydrolysis and fermentation to produce the chemical product.

29. The process of claim 28, wherein said microorganism is modified to enhance production of a fermentive end-product is selected from the group consisting of Clostridium phytofermentans, Thermoanaerobacter ethanolicus, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium. cellovorans, Clostridium tyrobutyricum, Clostridium thermobutyricum, Thermoanaerobacterium saccharolyticum, Thermoanaerobacter thermohydrosulfuricus, and Saccharomyces cerevisiae, Clostridium acetobutylicum, Moorella ssp., Carboxydocella ssp., Zymomonas mobilis, E. coli, and Klebsiella oxytoca.

30. The process of claim 28, wherein said microorganism is capable of direct fermentation of C5 and C6 carbohydrates.

31. The process according to claim 28, wherein said microorganism is a Clostridium.

32. The process according to claim 28, wherein said microorganism is a mesophilic microorganism.

33. The process according to claim 28, wherein said microorganism is C. phytofermentans.

34. The process according to claim 28, wherein said lactate dehydrogenase gene is Cphy_—

1232 or Cphy_—

1117.

35. The process according to claim 28, wherein said biomass comprises cellulosic or lignocellulosic materials.

36. The process according to claim 28, wherein said biomass comprises one or more of corn steep solids, corn steep liquor, malt syrup, xylan, cellulose, hemicellulose, fructose, glucose, mannose, rhamnose, or xylose.

37. The process according to claim 28, wherein said biomass comprises municipal waste, wood, plant material, plant material extract, bagasse, switch grass, algae, corn stover, poplar, a natural or synthetic polymer, or a combination thereof.

38. The process according to claim 28, wherein said biomass is pre-conditioned with acid treatment.

39. The process according to claim 28, wherein said process occurs at a temperature between 10°

C. and 35°

C.

40. A chemical product produced by the process according to claim 28.

41. The chemical product of claim 40, wherein said product is an alcohol.

42. The chemical product of claim 41, wherein said alcohol is ethanol.

43. An isolated microorganism that ferments cellulosic or lignocellulosic materials to produce ethanol in a concentration that is at least 90% of a theoretical yield, wherein the microorganism is genetically modified to inactivate a lactate dehydrogenase gene of the wild-type organism.

44. The isolated microorganism of claim 43, wherein the microorganism saccharifies C5 and C6 polysaccharides.

45. The isolated microorganism of claim 43, wherein the microorganism is gram negative.

46. The isolated microorganism of claim 43, wherein the microorganism is gram positive.

47. The isolated microorganism of claim 43, wherein said lactate dehydrogenase gene is Cphy_—

1232 or Cphy_—

1117.

48. A plasmid comprising SEQ ID NO:

1.

49. A primer selected from the group comprising SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, and SEQ ID NO;

5.

50. A plasmid comprising a nucleic acid sequence with about 70% similarity to SEQ ID NO:

1.

51. A primer comprising a nucleic acid sequence with about 70% similarity to SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, or SEQ ID NO;

5.