Fermentive production of four carbon alcohols

• US 20080182308A1
• Filed: 09/27/2006
• Published: 07/31/2008
• Est. Priority Date: 09/29/2005
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Suh, Wonchul, Donaldson, Gail K., Maggio-Hall, Lori Ann, Nagarajan, Vasantha, Huang, Lixuan Lisa, Nakamura, Charles E.
• Granted Patent: US 9,297,028 B2
• Time in Patent Office: Days
• US Class Current: 435/160
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12Y 101/01157 3-Hydroxybutyryl-CoA dehydrogenase (1.1.1.157)
• CPC Class Codes: C12Y 103/99002 Butyryl-CoA dehydrogenase (1.3.99.2), i.e. short chain acyl-CoA dehydrogenase
• CPC Class Codes: C12Y 203/01009 Acetyl-CoA C-acetyltransferase (2.3.1.9)
• CPC Class Codes: C12Y 402/01055 3-Hydroxybutyryl-CoA dehydratase (4.2.1.55)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Methods for the fermentive production of four carbon alcohols is provided. Specifically, butanol, preferably 1-butanol is produced by the fermentive growth of a recombinant bacterium expressing a 1-butanol biosynthetic pathway.

Claims

1. A recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:

a) acetyl-CoA to acetoacetyl-CoA

b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA

c) 3-hydroxybutyryl-CoA to crotonyl-CoA

d) crotonyl-CoA to butyryl-CoA

e) butyryl-CoA to butyraldehyde and

f) butyraldehyde to 1-butanol;

wherein the at least one DNA molecule is heterologous to said microbial host cell and wherein said microbial host cell produces 1-butanol.

2. A host cell according to claim 1 wherein the polypeptide that catalyzes a substrate to product conversion of acetyl-CoA to acetoacetyl-CoA is acetyl-CoA acetyltransferase.

3. A host cell according to claim 1 wherein the polypeptide that catalyzes a substrate to product conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA is 3-hydroxybutyryl-CoA dehydrogenase.

4. A host cell according to claim 1 wherein the polypeptide that catalyzes a substrate to product conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA is crotonase.

5. A host cell according to claim 1 wherein the polypeptide that catalyzes a substrate to product conversion of crotonyl-CoA to butyryl-CoA is butyryl-CoA dehydrogenase.

6. A host cell according to claim 1 wherein the polypeptide that catalyzes a substrate to product conversion of butyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase.

7. A host cell according to claim 1 wherein the polypeptide that catalyzes a substrate to product conversion of butyraldehyde to 1-butanol is butanol dehydrogenase.

8. A host cell according to claim 1 wherein the cell is selected from the group consisting of:

a bacterium, a cyanobacterium, a filamentous fungus and a yeast.

9. A host cell according to claim 8 wherein the cell is a member of a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

10. A host cell according to claim 9 wherein the cell is Escherichia coli.

11. A host cell according to claim 9 wherein the cell is Alcaligenes eutrophus.

12. A host cell according to claim 9 wherein the cell is Bacillus licheniformis.

13. A host cell according to claim 9 wherein the cell is Paenibacillus macerans.

14. A host cell according to claim 9 wherein the cell is Rhodococcus erythropolis.

15. A host cell according to claim 9 wherein the cell is Pseudomonas putida.

16. A host cell according to claim 9 wherein the cell is Bacillus subtilis.

17. A host cell according to claim 9 wherein the cell is Lactobacillus plantarum.

18. A host cell according to claim 9 wherein the cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium, and Enterococcus faecalis.

19. A host cell according to claim 9 wherein the cell is Saccharomyces cerevisiae.

20. A host cell according to claim 3 wherein the acetyl-CoA acetyltransferase has an amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

4, SEQ ID NO;

129, SEQ ID NO;

131, and SEQ ID NO;

133.

21. A host cell according to claim 4 wherein the 3-hydroxybutyryl-CoA dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

6, SEQ ID NO;

135, SEQ ID NO;

137, and SEQ ID NO;

139.

22. A host cell according to claim 5 wherein the crotonase has an amino acid sequence selected from the group consisting of SEQ ID NO:

8, SEQ ID NO;

141, SEQ ID NO;

143, and SEQ ID NO;

145.

23. A host cell according to claim 6 wherein the butyryl-CoA dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

10, SEQ ID NO;

147, SEQ ID NO;

149, SEQ ID NO;

151, and SEQ ID NO;

187.

24. A host cell according to claim 7 wherein the butyraldehyde dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

12, SEQ ID NO;

153, and SEQ ID NO;

189.

25. A host cell according to claim 8 wherein the butanol dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

14, SEQ ID NO;

16, SEQ ID NO;

153, SEQ ID NO;

155, and SEQ ID NO;

157.

26. A host cell according to claim 1 wherein the host cell is a facultative anaerobe.

27. A method for the production of 1-butanol comprising:

i) providing a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of;

a) acetyl-CoA to acetoacetyl-CoA

b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA

c) 3-hydroxybutyryl-CoA to crotonyl-CoA

d) crotonyl-CoA to butyryl-CoA

e) butyryl-CoA to butyraldehyde and

f) butyraldehyde to 1-butanol;

wherein the at least one DNA molecule is heterologous to said microbial host cell; and

ii) contacting the host cell of (i) with a fermentable carbon substrate under conditions whereby 1-butanol is produced.

28. A method according to claim 27 wherein the fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides.

29. A method according to claim 27 wherein the carbon substrate is selected from the group consisting of glucose, sucrose, and fructose.

30. A method according to claim 27 wherein the conditions whereby 1-butanol is produced are anaerobic.

31. A method according to claim 27 wherein the conditions whereby 1-butanol is produced are microaerobic.

32. A method according to claim 27 wherein the host cell is contacted with the carbon substrate in minimal media.

33. A method according to claim 27 wherein the polypeptide that catalyzes a substrate to product conversion of acetyl-CoA to acetoacetyl-CoA is acetyl-CoA acetyltransferase.

34. A method according to claim 27 wherein the polypeptide that catalyzes a substrate to product conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA is 3-hydroxybutyryl-CoA dehydrogenase.

35. A method according to claim 27 wherein the polypeptide that catalyzes a substrate to product conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA is crotonase.

36. A method according to claim 27 wherein the polypeptide that catalyzes a substrate to product conversion of crotonyl-CoA to butyryl-CoA is butyryl-CoA dehydrogenase.

37. A method according to claim 27 wherein the polypeptide that catalyzes a substrate to product conversion of butyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase.

38. A method according to claim 27 wherein the polypeptide that catalyzes a substrate to product conversion of butyraldehyde to 1-butanol is butanol dehydrogenase.

39. A method according to claim 27 wherein the host cell is selected from the group consisting of:

a bacterium, a cyanobacterium, a filamentous fungus and a yeast.

40. A method according to claim 39 wherein the host cell is a member of a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

41. A method according to claim 40 wherein the host cell is Escherichia coli.

42. A method according to claim 40 wherein the host cell is Alcaligenes eutrophus.

43. A method according to claim 40 wherein the host cell is Bacillus licheniformis.

44. A method according to claim 40 wherein the host cell is Paenibacillus macerans.

45. A method according to claim 40 wherein the host cell is Rhodococcus erythropolis.

46. A method according to claim 40 wherein the host cell is Pseudomonas putida.

47. A method according to claim 40 wherein the host cell is Bacillus subtilis.

48. A host cell according to claim 40 wherein the cell is Lactobacillus plantarum.

49. A host cell according to claim 40 wherein the cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium, and Enterococcus faecalis.

50. A host cell according to claim 40 wherein the cell is Saccharomyces cerevisiae.

51. A method according to claim 33 wherein the acetyl-CoA acetyltransferase has an amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

4, SEQ ID NO;

129, SEQ ID NO;

131, and SEQ ID NO;

133.

52. A method according to claim 34 wherein the 3-hydroxybutyryl-CoA dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

6, SEQ ID NO;

135, SEQ ID NO;

137, and SEQ ID NO;

139.

53. A method according to claim 35 wherein the crotonase has an amino acid sequence selected from the group consisting of SEQ ID NO:

8, SEQ ID NO;

141, SEQ ID NO;

143, and SEQ ID NO;

145.

54. A method according to claim 36 wherein the butyryl-CoA dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

10, SEQ ID NO;

147, SEQ ID NO;

149, SEQ ID NO;

151, and SEQ ID NO;

187.

55. A method according to claim 37 wherein the butyraldehyde dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

12, SEQ ID NO;

153, and SEQ ID NO;

189.

56. A method according to claim 38 wherein the butanol dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

14, SEQ ID NO;

16, SEQ ID NO;

153, SEQ ID NO;

155, and SEQ ID NO;

157.

57. A method according to claim 27 wherein the host cell is a facultative anaerobe.