Fermentive production of four carbon alcohols

• US 20070092957A1
• Filed: 10/25/2006
• Published: 04/26/2007
• Est. Priority Date: 10/26/2005
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Flint, Dennis, Nagarajan, Vasantha, Donaldson, Gail, Eliot, Andrew, Maggio-Hall, Lori
• Granted Patent: US 7,851,188 B2
• Time in Patent Office: Days
• US Class Current: 435/157
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• 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 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 15/75 for Bacillus
• CPC Class Codes: C12N 15/81 for yeasts
• 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 9/0016 with NAD or NADP as acceptor (1.4.1)
• CPC Class Codes: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• CPC Class Codes: C12N 9/1096 transferring nitrogenous groups (2.6)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12Y 101/01001 Alcohol dehydrogenase (1.1.1.1)
• CPC Class Codes: C12Y 101/01002 Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase
• CPC Class Codes: C12Y 101/01086 Ketol-acid reductoisomerase (1.1.1.86)
• CPC Class Codes: C12Y 102/00 Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12Y 102/01003 Aldehyde dehydrogenase (NAD+) (1.2.1.3)
• CPC Class Codes: C12Y 102/04004 3-Methyl-2-oxobutanoate dehydrogenase (2-methylpropanoyl-transferring) (1.2.4.4), i.e. branched-chain-alpha-ketoacid dehydrogenase
• CPC Class Codes: C12Y 202/01006 Acetolactate synthase (2.2.1.6)
• CPC Class Codes: C12Y 401/01072 : Branched-chain-2-oxoacid decarboxylase (4.1.1.72)
• CPC Class Codes: C12Y 402/01009 : Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase
• 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
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

Methods for the fermentative production of four carbon alcohols is provided. Specifically, butanol, preferably isobutanol is produced by the fermentative growth of a recombinant bacterium expressing an isobutanol 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:

i) pyruvate to acetolactate (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate (pathway step b)

iii) 2,3-dihydroxyisovalerate to α

-ketoisovalerate (pathway step c)

iv) α

-ketoisovalerate to isobutyraldehyde, (pathway step d), and

v) isobutyraldehyde to isobutanol;

(pathway step e)

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

4. A host cell according to any of claims 1, 2 or 3 wherein the polypeptide that catalyzes a substrate to product conversion of pyruvate to acetolactate is acetolactate synthase.

5. A host cell according to any of claims 1, 2 or 3 wherein the polypeptide that catalyzes a substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate is acetohydroxy acid isomeroreductase.

6. A host cell according to any of claims 1, 2 or 3 wherein the polypeptide that catalyzes a substrate to product conversion of 2,3-dihydroxyisovalerate to α

-ketoisovalerate is acetohydroxy acid dehydratase.

7. A host cell according to any of claims 1, 2 or 3 wherein the polypeptide that catalyzes a substrate to product conversion of isobutyraldehyde to isobutanol is branched-chain alcohol dehydrogenase.

8. A host cell according to claim 1 wherein the polypeptide that catalyzes a substrate to product conversion of α

-ketoisovalerate to isobutyraldehyde is branched-chain α

-keto acid decarboxylase.

15. A host cell according to any of claims 1, 2 or 3 wherein the cell is selected from the group consisting of:

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

16. A host cell according to claim 15 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.

17. A host cell according to claim 16 wherein the cell is Escherichia coli.

18. A host cell according to claim 16 wherein the cell is Alcaligenes eutrophus.

19. A host cell according to claim 16 wherein the cell is Bacillus licheniformis.

20. A host cell according to claim 16 wherein the cell is Paenibacillus macerans.

21. A host cell according to claim 16 wherein the cell is Rhodococcus erythropolis.

22. A host cell according to claim 16 wherein the cell is Pseudomonas putida.

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

24. A host cell according to claim 16 wherein the cell is Lactobacillus plantarum.

25. A host cell according to claim 16 wherein the cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium, and Enterococcus faecalis.

26. A host cell according to claim 16 wherein the cell is Saccharomyces cerevisiae.

27. A host cell according to claim 4 wherein the acetolactate synthase has an amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

178, and SEQ ID NO;

180.

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

43, SEQ ID NO;

181, SEQ ID NO;

183, and SEQ ID NO;

185.

29. A host cell according to claim 6 wherein the acetohydroxy acid dehydratase has an amino acid sequence selected from the group consisting of SEQ ID NO:

6, SEQ ID NO;

186, SEQ ID NO;

188, and SEQ ID NO;

190.

30. A host cell according to claim 7 wherein the branched-chain alcohol dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

10, SEQ ID NO;

199, SEQ ID NO;

201, SEQ ID NO;

203, and SEQ ID NO;

204.

31. A host cell according to claim 8 wherein the branched-chain α

-keto acid decarboxylase has an amino acid sequence selected from the group consisting of SEQ ID NO;

8, SEQ ID NO;

193, SEQ ID NO;

195, and SEQ ID NO;

197.

38. A host cell according to any of claims 1, 2 or 3 wherein the host cell is a facultative anaerobe.

2. 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:

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α

-ketoisovalerate, (pathway step c)

iv) α

-ketoisovalerate to isobutyryl-CoA, (pathway step f)

v) isobutyryl-CoA to isobutyraldehyde, (pathway step g), and

vi) isobutyraldehyde to isobutanol;

(pathway step e)

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

9. A host cell according to claim 2 wherein the polypeptide that catalyzes a substrate to product conversion of α

-ketoisovalerate to isobutyryl-CoA is branched-chain keto acid dehydrogenase.

10. A host cell according to claim 2 wherein the polypeptide that catalyzes a substrate to product conversion of isobutyryl-CoA to isobutyraldehyde is acylating aldehyde dehydrogenase.

32. A host cell according to claim 9 wherein the branched-chain keto acid dehydrogenase comprises four subunits and the amino acid sequences of said subunits are selected from the group consisting of SEQ ID NO:

214, SEQ ID NO;

216, SEQ ID NO;

218, SEQ ID NO;

220, SEQ ID NO;

210, SEQ ID NO;

208, SEQ ID NO;

206, and SEQ ID NO;

212.

33. A host cell according to claim 10 wherein the acylating aldehyde dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

222, SEQ ID NO;

224, SEQ ID NO;

226, SEQ ID NO;

228, and SEQ ID NO;

230.

3. 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:

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α

-ketoisovalerate, (pathway step c)

iv) α

-ketoisovalerate to valine, (pathway step h)

v) valine to isobutylamine, (pathway step i)

vi) isobutylamine to isobutyraldehyde, (pathway step j), and

vii) isobutyraldehyde to isobutanol;

(pathway step e)

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

11. A host cell according to claim 3 wherein the polypeptide that catalyzes a substrate to product conversion of α

-ketoisovalerate to valine is transaminase.

12. A host cell according to claim 3 wherein the polypeptide that catalyzes a substrate to product conversion of α

-ketoisovalerate to valine is valine dehydrogenase.

13. A host cell according to claim 3 wherein the polypeptide that catalyzes a substrate to product conversion of valine to isobutylamine is valine decarboxylase.

14. A host cell according to claim 3 wherein the polypeptide that catalyzes a substrate to product conversion of isobutylamine to isobutyraldehyde is omega transaminase.

34. A host cell according to claim 11 wherein the transaminase has an amino acid sequence selected from the group consisting of SEQ ID NO:

232, SEQ ID NO;

234, SEQ ID NO;

236, SEQ ID NO;

238, and SEQ ID NO;

240.

35. A host cell according to claim 12 wherein the valine dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

242 and SEQ ID NO;

244.

36. A host cell according to claim 13 wherein the valine decarboxylase has an amino acid sequence as set forth in SEQ ID NO:

246.

37. A host cell according to claim 14 wherein the omega transaminase has an amino acid sequence selected from the group consisting of SEQ ID NO:

248, SEQ ID NO;

250, SEQ ID NO;

252, and SEQ ID NO;

254.

39. A method for the production of isobutanol comprising:

1) 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;

i) pyruvate to acetolactate (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate (pathway step b)

iii) 2,3-dihydroxyisovalerate to α

-ketoisovalerate (pathway step c)

iv) α

-ketoisovalerate to isobutyraldehyde, (pathway step d), and

v) isobutyraldehyde to isobutanol;

(pathway step e)

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

2) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isobutanol is produced.

42. A method according to any of claims 39, 40 or 41 wherein the fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides.

43. A method according to any of claims 39, 40 or 41 wherein the carbon substrate is selected from the group consisting of glucose, sucrose, and fructose.

44. A method according to any of claims 39, 40 or 41 wherein the conditions whereby isobutanol is produced are anaerobic.

45. A method according to any of claims 39, 40 or 41 wherein the conditions whereby isobutanol is produced are microaerobic.

46. A method according to any of claims 39, 40 or 41 wherein the host cell is contacted with the carbon substrate in a minimal medium.

47. A method according to any of claims 39, 40 or 41 wherein the polypeptide that catalyzes a substrate to product conversion of pyruvate to acetolactate is acetolactate synthase.

48. A method according to any of claims 39, 40 or 41 wherein the polypeptide that catalyzes a substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate is acetohydroxy acid isomeroreductase.

49. A method according to any of claims 39, 40 or 41 wherein the polypeptide that catalyzes a substrate to product conversion of 2,3-dihydroxyisovalerate to α

-ketoisovalerate is acetohydroxy acid dehydratase.

50. A method according to any of claims 39, 40 or 41 wherein the polypeptide that catalyzes a substrate to product conversion of isobutyraldehyde to isobutanol is branched-chain alcohol dehydrogenase.

51. A method according to claim 39 wherein the polypeptide that catalyzes a substrate to product conversion of α

-ketoisovalerate to isobutyraldehyde is branched-chain α

-keto acid decarboxylase.

58. A method according to any of claims 39, 40 or 41 wherein the host cell is selected from the group consisting of:

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

70. A method according to claim 47 wherein the acetolactate synthase has the amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

178, and SEQ ID NO;

180.

71. A method according to claim 48 the acetohydroxy acid isomeroreductase has the amino acid sequence selected from the group consisting of SEQ ID NO:

43, SEQ ID NO;

181, SEQ ID NO;

183, and SEQ ID NO;

185.

72. A method according to claim 49 wherein the acetohydroxy acid dehydratase has the amino acid sequence selected from the group consisting of SEQ ID NO:

6, SEQ ID NO;

186, SEQ ID NO;

188, and SEQ ID NO;

190.

73. A method according to claim 50 wherein branched-chain alcohol dehydrogenase has the amino acid sequence selected from the group consisting of SEQ ID NO:

10, SEQ ID NO;

199, SEQ ID NO;

201, SEQ ID NO;

203, and SEQ ID NO;

204.

74. A method according to claim 51 wherein the branched-chain α

-keto acid decarboxylase has the amino acid sequence selected from the group consisting of SEQ ID NO;

8, SEQ ID NO;

193, SEQ ID NO;

195, and SEQ ID NO;

197.

81. A method according to any of claims 39, 40 or 41 wherein the host cell is a facultative anaerobe.

82. An isobutanol containing fermentation medium produced by the method of any of claims 39, 40 or 41.

40. A method for the production of isobutanol comprising:

1) 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;

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α

-ketoisovalerate, (pathway step c)

iv) α

-ketoisovalerate to isobutyryl-CoA, (pathway step f)

v) isobutyryl-CoA to isobutyraldehyde, (pathway step g), and

vi) isobutyraldehyde to isobutanol;

(pathway step e)

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

2) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isobutanol is produced.

52. A method according to claim 40 wherein the polypeptide that catalyzes a substrate to product conversion of α

-ketoisovalerate to isobutyryl-CoA is branched-chain keto acid dehydrogenase.

53. A method according to claim 40 wherein the polypeptide that catalyzes a substrate to product conversion of isobutyryl-CoA to isobutyraldehyde is acylating aldehyde dehydrogenase.

75. A method according to claim 52 wherein the branched-chain keto acid dehydrogenase comprises four subunits and the amino acid sequences of said subunits are selected from the group consisting of SEQ ID NO:

214, SEQ ID NO;

216, SEQ ID NO;

218, SEQ ID NO;

220, SEQ ID NO;

210, SEQ ID NO;

208, SEQ ID NO;

206, and SEQ ID NO;

212.

76. A method according to claim 53 wherein the acylating aldehyde dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:

222, SEQ ID NO;

224, SEQ ID NO;

226, SEQ ID NO;

228, and SEQ ID NO;

230.

41. A method for the production of isobutanol comprising:

1) 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;

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α

-ketoisovalerate, (pathway step c)

iv) α

-ketoisovalerate to valine, (pathway step h)

v) valine to isobutylamine, (pathway step i)

vi) isobutylamine to isobutyraldehyde, (pathway step j), and

vii) isobutyraldehyde to isobutanol;

(pathway step e)

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

2) contacting the host cell of (i) with a fermentable carbon in a fermentation medium substrate under conditions whereby isobutanol is produced.

54. A method according to claim 41 wherein the polypeptide that catalyzes a substrate to product conversion of α

-ketoisovalerate to valine is transaminase.

55. A method according to claim 41 wherein the polypeptide that catalyzes a substrate to product conversion of α

-ketoisovalerate to valine is valine dehydrogenase.

56. A method according to claim 41 wherein the polypeptide that catalyzes a substrate to product conversion of valine to isobutylamine is valine decarboxylase.

57. A method according to claim 41 wherein the polypeptide that catalyzes a substrate to product conversion of isobutylamine to isobutyraldehyde is omega transaminase.

59. A method according to claim 55 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.

60. A method according to claim 59 wherein the host cell is Escherichia coli.

61. A method according to claim 59 wherein the host cell is Alcaligenes eutrophus.

62. A method according to claim 59 wherein the host cell is Bacillus licheniformis.

63. A method according to claim 59 wherein the host cell is Paenibacillus macerans.

64. A method according to claim 59 wherein the host cell is Rhodococcus erythropolis.

65. A method according to claim 59 wherein the host cell is Pseudomonas putida.

66. A method according to claim 59 wherein the host cell is Bacillus subtilis.

67. A method according to claim 59 wherein the host cell is Lactobacillus plantarum.

68. A method according to claim 59 wherein the host cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium, and Enterococcus faecalis.

69. A method according to claim 59 wherein the host cell is Saccharomyces cerevisiae.

77. A method according to claim 54 wherein the transaminase has an amino acid sequence selected from the group consisting of SEQ ID NO:

232, SEQ ID NO;

234, SEQ ID NO;

236, SEQ ID NO;

238, and SEQ ID NO;

240.

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

242 and SEQ ID NO;

244.

79. A method according to claim 56 wherein the valine decarboxylase has an amino acid sequence as set forth in SEQ ID NO:

246.

80. A method according to claim 57 wherein the omega transaminase has an amino acid sequence selected from the group consisting of SEQ ID NO:

248, SEQ ID NO;

250, SEQ ID NO;

252, and SEQ ID NO;

254.