FERMENTIVE PRODUCTION OF FOUR CARBON ALCOHOLS

• US 20070292927A1
• Filed: 04/30/2007
• Published: 12/20/2007
• Est. Priority Date: 05/02/2006
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Nagarajan, Vasantha, Tomb, Jean-Francois, Nakamura, Charles, Eliot, Andrew, Donaldson, Gail
• Granted Patent: US 8,962,298 B2
• Time in Patent Office: Days
• US Class Current: 435/160
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/78 acting on carbon to nitrogen bonds other than peptide bonds (3.5)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/26 Ketones
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Methods for the fermentive production of four carbon alcohols are provided. Specifically, butanol, preferably 2-butanol is produced by the fermentive growth of a recombinant bacteria expressing a 2-butanol biosynthetic pathway. The recombinant microorganisms and methods of the invention can also be adapted to produce 2-butanone, an intermediate in the 2-butanol biosynthetic pathways disclosed herein.

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 alpha-acetolactate;

ii) alpha-acetolactate to acetoi;

iii) acetoin to 2,3-butanediol;

iv) 2,3-butanediol to 2-butanone; and

v) 2-butanone to 2-butanol;

wherein the at least one DNA molecule is heterologous to said microbial host cell and wherein said microbial host cell produces 2-butanol.

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

4. A host cell according to claims 1 or 2 wherein the polypeptide that catalyzes a substrate to product conversion of alpha-acetolactate to acetoin is acetolactate decarboxylase.

5. A host cell according to claims 1 or 2 wherein the polypeptide that catalyzes a substrate to product conversion of acetoin to 2,3-butanediol is butanediol dehydrogenase.

6. A host cell according to claims 1 or 2 wherein the polypeptide that catalyzes a substrate to product conversion of 2,3-butanediol to 2-butanone is diol dehydratase or glycerol dehydratase.

7. A host cell according to claim 1 wherein the polypeptide that catalyzes a substrate to product conversion of 2-butanone to 2-butanol is butanol dehydrogenase.

8. A host cell according to claims 1 or 2 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, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

10. A host cell according to claim 3 wherein the acetolactate synthase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

4, SEQ ID NO;

77, and SEQ ID NO;

79 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

11. A host cell according to claim 4 wherein the acetolactate decarboxylase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

81, and SEQ ID NO;

83 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=1 0, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

12. A host cell according to claim 5 wherein the butanediol dehydrogenase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

6, SEQ ID NO;

85, SEQ ID NO;

87, and SEQ ID NO;

89 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

13. A host cell according to claim 6 wherein the diol dehydratase or glycerol dehydratase comprise full length large, medium and small subunits that each give an E-value parameter of 0.01 or less when queried using a Profile Hidden Markov Model prepared using the large subunits of SEQ ID NOs:

8, 99, 105, 135, 138, 141, 146, and 164;

the medium subunits of SEQ ID NOs;

10, 101, 107, 136, 139, 142, 148, and 165; and

the small subunits of SEQ ID NOs;

12, 103, 109, 137, 140, 143, 150, and 166;

each query being carried out using the hmmsearch algorithm wherein the Z parameter is set to 1 billion.

14. A host cell according to claim 6 wherein the diol dehydratase or the glycerol dehydratase identified by a process comprising the steps of:

a) generating a Profile Hidden Markov Model from the alignment of the amino acid sequences corresponding to the large, medium and small subunits of diol and glycerol dehydratase enzymes wherein;

i) the large subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

8, 99, 105, 135, 138, 141, 146, and 164;

ii) the medium subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

10, 101, 107, 136, 139, 142, 148, and 165; and

iii) the small subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

12, 103, 109, 137, 140, 143, 150, and 166;

b) querying at least one public database of protein sequences containing sequences of diol and glycerol dehydratases with the Profile Hidden Markov Model of (a) using the hmmsearch algorithm wherein the Z parameter is set to 1 billion and the E-value parameter is set to 0.01, to identify a first data set of diol and glycerol dehydratase amino acid sequences; and

c) removing any partial sequences from the first data set of (b) to generate a second data set of diol and glycerol dehydratase amino acid sequences, wherein diol dehydratase and the glycerol dehydratase enzymes are identified.

15. A host cell according to claim 6 wherein the diol dehydratase or glycerol dehydratase comprises a large subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:

8, 93, 99, 105, 135, 138, 141, 146, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 130, 243, 254, 255, 256, 257, 258 and 259, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

16. A host cell according to claim 6 wherein the diol dehydratase or glycerol dehydratase comprises a medium subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:

10, 95, 101, 107, 136, 139, 142, 148, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 244, 250, 252, 260, 261, 262, 263, 364, 265, 266, and 167 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=1 0, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

17. A host cell according to claim 6 wherein the diol dehydratase or glycerol dehydratase comprises a small subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:

12, 97, 103, 109, 137, 140, 143, 150, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 234, 236, 238, 240, 242, 245, 248, 249, 251, 253, 268, 270, 271, 272, 273, and 274, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

18. A host cell according to claim 6 wherein the diol dehydratase or glycerol dehydratase comprises fused large, medium and small subunits comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:

233, 235, 237, 239, 241, 246, and 247, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

19. A host cell according to claim 6 wherein the diol dehydratase or glycerol dehydratase comprises fused large, medium and small subunits and has at least 95% identity to an amino acid sequence comprising all three of the amino acid sequences encoding large, medium and small subunits, selected from the group consisting of:

a) SEQ ID NO;

8, SEQ ID NO;

10, and SEQ ID NO;

12;

b) SEQ ID NO;

93, SEQ ID NO;

95, and SEQ ID NO;

97;

c) SEQ ID NO;

99, SEQ ID NO;

101, and SEQ ID NO;

103;

d) SEQ ID NO;

105, SEQ ID NO;

107, and SEQ ID NO;

109;

e) SEQ ID NO;

135, SEQ ID NO;

136, and SEQ ID NO;

137;

f) SEQ ID NO;

138, SEQ ID NO;

139, and SEQ ID NO;

140;

g) SEQ ID NO;

146, SEQ ID NO;

148, and SEQ ID NO;

150;

h) SEQ ID NO;

141, SEQ ID NO;

142, and SEQ ID NO;

143; and

i) SEQ ID NO;

164, SEQ ID NO;

165, and SEQ ID NO;

166;

based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

20. A host cell according to claim 8 wherein the butanol dehydrogenase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

14, SEQ ID NO;

72, SEQ ID NO;

75, and SEQ ID NO;

91 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=1 0, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

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 alpha-acetolactate;

ii) alpha-acetolactate to acetoin;

iii) acetoin to 2,3-butanediol; and

iv) 2,3-butanediol to 2-butanone;

wherein the at least one DNA molecule is heterologous to said microbial host cell and wherein said microbial host cell produces 2-butanone.

21. A method for the production of 2-butanol 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 alpha-acetolactate;

ii) alpha-acetolactate to acetoin;

iii) acetoin to 2,3-butanediol;

iv) 2,3-butanediol to 2-butanone; and

v) 2-butanone to 2-butanol;

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

2) contacting the host cell of (1) with a fermentable carbon substrate in a fermentation medium under conditions whereby 2-butanol is produced.

23. A method according to claims 21 or 22 wherein the fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides.

24. A method according to claims 21 or 22 wherein the polypeptide that catalyzes a substrate to product conversion of pyruvate to alpha-acetolactate is acetolactate synthase.

25. A method according to claims 21 or 22 wherein the polypeptide that catalyzes a substrate to product conversion of alpha-acetolactate to acetoin is acetolactate decarboxylase.

26. A method according to claims 21 or 22 wherein the polypeptide that catalyzes a substrate to product conversion of acetoin to 2,3-butanediol is butanediol dehydrogenase.

27. A method according to claims 21 or 22 wherein the polypeptide that catalyzes a substrate to product conversion of 2,3-butanediol to 2-butanone is diol dehydratase or glycerol dehydratase.

28. A method according to claim 21 wherein the polypeptide that catalyzes a substrate to product conversion of 2-butanone to 2-butanol is butanol dehydrogenase.

29. A method according to claims 21 or 22 wherein the cell is selected from the group consisting of:

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

30. A method according to claim 29 wherein the cell is a member of a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

31. A method according to claim 24 wherein the acetolactate synthase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

4, SEQ ID NO;

77, and SEQ ID NO;

79 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

32. A method according to claim 25 wherein the acetolactate decarboxylase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

81, and SEQ ID NO;

83 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

33. A method according to claim 26 wherein the butanediol dehydrogenase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

6, SEQ ID NO;

85, SEQ ID NO;

87, and SEQ ID NO;

89 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

34. A method according to claim 27 wherein the diol dehydratase or glycerol dehydratase comprise full length large, medium and small subunits that each give an E-value parameter of 0.01 or less when queried using a Profile Hidden Markov Model prepared using the large subunits of SEQ ID NOs:

8, 99, 105, 135, 138, 141, 146, and 164;

the medium subunits of SEQ ID NOs;

10, 101, 107, 136, 139, 142, 148, and 165; and

the small subunits of SEQ ID NOs;

12, 103, 109, 137, 140, 143, 150, and 166;

each query being carried out using the hmmsearch algorithm wherein the Z parameter is set to 1 billion.

35. A method according to claim 27 wherein the diol dehydratase or the glycerol dehydratase identified by a process comprising the steps of:

a) generating a Profile Hidden Markov Model from the alignment of the amino acid sequences corresponding to the large, medium and small subunits of diol and glycerol dehydratase enzymes wherein;

i) the large subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

8, 99, 105, 135, 138, 141, 146, and 164;

ii) the medium subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

10, 101, 107, 136, 139, 142, 148, and 165; and

iii) the small subunit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs;

12, 103, 109, 137, 140, 143, 150, and 166;

b) querying at least one public database of protein sequences containing sequences of diol and glycerol dehydratases with the Profile Hidden Markov Model of (a) using the hmmsearch algorithm wherein the Z parameter is set to 1 billion and the E-value parameter is set to 0.01, to identify a first data set of diol and glycerol dehydratase amino acid sequences; and

c) removing any partial sequences from the first data set of (b) to generate a second data set of diol and glycerol dehydratase amino acid sequences, wherein diol dehydratase and the glycerol dehydratase enzymes are identified.

36. A method according to claim 27 wherein the diol dehydratase or glycerol dehydratase comprises a large subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:

8, 93, 99, 105, 135, 138, 141, 146, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 130, 243, 254, 255, 256, 257, 258 and 259, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

37. A method according to claim 27 wherein the diol dehydratase or glycerol dehydratase comprises a medium subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:

10, 95, 101, 107, 136, 139, 142, 148, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 244, 250, 252, 260, 261, 262, 263, 364, 265, 266, and 167 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

38. A method according to claim 27 wherein the diol dehydratase or glycerol dehydratase comprises a small subunit comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:

12, 97, 103, 109, 137, 140, 143, 150, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 234, 236, 238, 240, 242, 245, 248, 249, 251, 253, 268, 270, 271, 272, 273, and 274, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

39. A method according to claim 27 wherein the diol dehydratase or glycerol dehydratase comprises fused large, medium and small subunits comprising an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:

233, 235, 237, 239, 241, 246, and 247, based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

40. A method I according to claim 27 wherein the diol dehydratase or glycerol dehydratase comprises fused large, medium and small subunits and has at least 95% identity to an amino acid sequence comprising all three of the amino acid sequences encoding large, medium and small subunits, selected from the group consisting of:

a) SEQ ID NO;

8, SEQ ID NO;

10, and SEQ ID NO;

12;

b) SEQ ID NO;

93, SEQ ID NO;

95, and SEQ ID NO;

97;

c) SEQ ID NO;

99, SEQ ID NO;

101, and SEQ ID NO;

103;

d) SEQ ID NO;

105, SEQ ID NO;

107, and SEQ ID NO;

109;

e) SEQ ID NO;

135, SEQ ID NO;

136, and SEQ ID NO;

137;

f) SEQ ID NO;

138, SEQ ID NO;

139, and SEQ ID NO;

140;

g) SEQ ID NO;

146, SEQ ID NO;

148, and SEQ ID NO;

150;

h) SEQ ID NO;

141, SEQ ID NO;

142, and SEQ ID NO;

143; and

i) SEQ ID NO;

164, SEQ ID NO;

165, and SEQ ID NO;

166;

based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

41. A method according to claim 28 wherein the butanol dehydrogenase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:

14, SEQ ID NO;

72, SEQ ID NO;

75, and SEQ ID NO;

91 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

42. A 2-butanol containing fermentation product medium produced by the method of claim 21.

22. A method for the production of 2-butanone 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 alpha-acetolactate;

ii) alpha-acetolactate to acetoin;

iii) acetoin to 2,3-butanediol; and

iv) 2,3-butanediol to 2-butanone;

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

2) contacting the host cell of (1) with a fermentable carbon substrate in a fermentation medium under conditions whereby 2-butanone is produced.

43. A 2-butanone containing fermentation product medium produced by the method of claim 22.