Fermentive production of four carbon alcohols
View Patent ↗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.
1. A recombinant microbial host cell comprising heterologous DNA molecules encoding polypeptides that catalyze substrate to product conversions for each step below:
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; and
e) butyryl-CoA to butyraldehyde,
wherein said microbial host cell produces 1-butanol by the action of an endogenous alcohol dehydrogenase, wherein said microbial host cell produces 1-butanol through the substrate to product conversions of (a) to (e) under aerobic conditions.
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 cell is selected from the group consisting of: a bacterium, a cyanobacterium , a filamentous fungus and a yeast.
8. A host cell according to claim 7 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.
9. A host cell according to claim 8 wherein the cell is Escherichia coli.
10. A host cell according to claim 8 wherein the cell is Alcaligenes eutrophus.
11. A host cell according to claim 8 wherein the cell is Bacillus licheniformis.
12. A host cell according to claim 8 wherein the cell is Paenibacillus macerans.
13. A host cell according to claim 8 wherein the cell is Rhodococcus erythropolis.
14. A host cell according to claim 8 wherein the cell is Pseudomonas putida.
15. A host cell according to claim 8 wherein the cell is Bacillus subtilis.
16. A host cell according to claim 8 wherein the cell is Lactobacillus plantarum.
17. A host cell according to claim 8 wherein the cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium , and Enterococcus faecalis.
18. A host cell according to claim 8 wherein the cell is Saccharomyces cerevisiae.
19. A host cell according to claim 1 wherein the host cell is a facultative anaerobe.
20. A method for the production of 1-butanol comprising:
i) providing the recombinant microbial host cell of claim 1 ; and
ii) contacting the host cell of (i) with a fermentable carbon substrate under conditions whereby 1-butanol is produced.
21. A method according to claim 20 wherein the fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides.
22. A method according to claim 20 wherein the carbon substrate is selected from the group consisting of glucose, sucrose, and fructose.
23. A method according to claim 20 wherein the conditions whereby 1-butanol is produce are anaerobic.
24. A method according to claim 20 wherein the conditions whereby 1-buanol is produced are microaerobic.
25. A method according to claim 20 wherein the host cell is contacted with the carbon substrate in minimal media.
26. A method according to claim 20 wherein the polypeptide that catalyzes a substrate to product conversion of acetyl-CoA to acetoacetyl-CoA is acetyl-CoA acetyltransferase.
27. A method according to claim 20 wherein the polypeptide that catalyzes a substrate to product conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA is 3-hydroxybutyryl-CoA dehydrogenase.
28. A method according to claim 20 wherein the polypeptide that catalyzes a substrate to product conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA is crotonase.
29. A method according to claim 20 wherein the polypeptide that catalyzes a substrate to product conversion of crotonyl-CoA to butyryl-CoA is butyryl-CoA dehydrogenase.
30. A method according to claim 20 wherein the polypeptide that catalyzes a substrate to product conversion of butyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase.
31. A method according to claim 20 wherein the host cell is selected from the group consisting of: a bacterium, a cyanobacterium , a filamentous fungus and a yeast.
32. A method according to claim 31 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.
33. A method according to claim 32 wherein the host cell is Escherichia coli.
34. A method according to claim 32 wherein the host cell is Alcaligenes eutrophus.
35. A method according to claim 32 wherein the host cell is Bacillus licheniformis.
36. A method according to claim 32 wherein the host cell is Paenibacillus macerans.
37. A method according to claim 32 wherein the host cell is Rhodococcus erythropolis.
38. A method according to claim 32 wherein the host cell is Pseudomonas putida.
39. A method according to claim 32 wherein the host cell is Bacillus subtilis.
40. A method according to claim 32 wherein the host cell is Lactobacillus plantarum.
41. A method according to claim 32 wherein the host cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium , and Enterococcus faecalis.
42. A method according to claim 32 wherein the host cell is Saccharomyces cerevisiae.
43. A method according to claim 26 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.
44. A method according to claim 27 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.
45. A method according to claim 28 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.
46. A method according to claim 29 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 NO:149, SEQ ID NO:151, and SEQ ID NO:187.
47. A method according to claim 30 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.
48. A method according to claim 20 wherein the host cell is a facultative anaerobe.
49. A host cell according to claim 1 further comprising a heterologous DNA molecule that encodes a polypeptide that catalyzes the substrate to product conversion of butyraldehyde to 1-butanol.
50. The method according to claim 20 wherein the host cell further comprises a heterologous DNA molecule that encodes a polypeptide that catalyzes the substrate to product conversion of butyraldehyde to 1-butanol.
51. A host cell according to claim 1 wherein the host cell produces more butanol than a control cell lacking the heterologous DNA molecules encoding polypeptides that catalyze the substrate to product conversions of steps (a) to (e).
52. A host cell according to claim 50 wherein the polypeptide that catalyzes the substrate to product conversion of butyraldehyde to 1-butanol is butanol dehydrogenase.
53. A method according to claim 50 wherein the polypeptide that catalyzes the substrate to product conversion of butyraldehyde to 1-butanol is butanol dehydrogenase.
54. A method according to claim 53 wherein the butanol dehydrogenase has an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ NO:153, SEQ ID NO:155, and SEQ ID NO:157.