Microorganisms and methods for the biosynthesis of butadiene
View Patent ↗The invention provides non-naturally occurring microbial organisms having a butadiene pathway. The invention additionally provides methods of using such organisms to produce butadiene.
1. A non-naturally occurring microbial organism comprising a biosynthetic pathway for the production of crotyl alcohol wherein said biosynthetic pathway comprises at least one exogenous nucleic acid encoding at least one metabolic pathway enzyme expressed in a sufficient amount to produce crotyl alcohol, wherein said biosynthetic pathway comprises converting crotonyl-CoA to crotyl alcohol using one or more metabolic pathway enzymes, wherein said metabolic pathway enzymes are selected from the group consisting of:
(i) a crotonyl-CoA reductase converting crotonyl-CoA to crotonaldehyde, and a crotonaldehyde reductase converting crotonaldehyde to crotyl alcohol;
(ii) a crotonyl-CoA reductase converting crotonyl-CoA to crotyl alcohol; and
(iii) a crotonyl-CoA hydrolase, a crotonyl-CoA synthetase, or a crotonyl-CoA transferase converting crotonyl-CoA to crotonate, a crotonate reductase converting crotonate to crotonaldehyde, and a crotonaldehyde reductase converting crotonaldehyde to crotyl alcohol, and
wherein said biosynthetic pathway further comprises:
(i) converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase;
(ii) converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase;
(iii) converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase;
(iv) converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase; or
(v) converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase.
2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises one exogenous nucleic acid encoding a metabolic pathway enzyme.
3. The non-naturally occurring microbial organism of claim 1 , wherein said biosynthetic pathway comprises a crotonyl-CoA reductase converting crotonyl-CoA to crotonaldehyde and a crotonaldehyde reductase converting crotonaldehyde to crotyl alcohol.
4. The non-naturally occurring microbial organism of claim 3 , wherein said biosynthetic pathway further comprises converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase.
5. The non-naturally occurring microbial organism of claim 3 , wherein said biosynthetic pathway further comprises converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase.
6. The non-naturally occurring microbial organism of claim 3 , wherein said biosynthetic pathway further comprises converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase.
7. The non-naturally occurring microbial organism of claim 3 , wherein said biosynthetic pathway further comprises converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase.
8. The non-naturally occurring microbial organism of claim 3 , wherein said biosynthetic pathway further comprises converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase.
9. The non-naturally occurring microbial organism of claim 1 , wherein said biosynthetic pathway comprises a crotonyl-CoA reductase converting crotonaldehyde to crotyl alcohol.
10. The non-naturally occurring microbial organism of claim 9 , wherein said biosynthetic pathway further comprises converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase.
11. The non-naturally occurring microbial organism of claim 9 , wherein said biosynthetic pathway further comprises converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase.
12. The non-naturally occurring microbial organism of claim 9 , wherein said biosynthetic pathway further comprises converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase.
13. The non-naturally occurring microbial organism of claim 9 , wherein said biosynthetic pathway further comprises converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase.
14. The non-naturally occurring microbial organism of claim 9 , wherein said biosynthetic pathway further comprises converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase.
15. The non-naturally occurring microbial organism of claim 1 , said biosynthetic pathway comprises a crotonyl-CoA hydrolase, crotonyl-CoA synthetase or crotonyl-CoA transferase converting crotonyl-CoA to crotonate, a crotonate reductase converting crotonate to crotonaldehyde, and crotonaldehyde reductase converting crotonaldehyde to crotyl alcohol.
16. The non-naturally occurring microbial organism of claim 15 , wherein said biosynthetic pathway further comprises converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase.
17. The non-naturally occurring microbial organism of claim 15 , wherein said biosynthetic pathway further comprises converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase.
18. The non-naturally occurring microbial organism of claim 15 , wherein said biosynthetic pathway further comprises converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase.
19. The non-naturally occurring microbial organism of claim 15 , wherein said biosynthetic pathway further comprises converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase.
20. The non-naturally occurring microbial organism of claim 15 , wherein said biosynthetic pathway further comprises converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase.
21. The non-naturally occurring microbial organism of claim 1 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.
22. The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
23. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism is a bacteria, a yeast, or a fungus.
24. The non-naturally occurring microbial organism of claim 23 , wherein said bacteria is Escherichia coli.
25. A method for producing crotyl alcohol comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce crotyl alcohol.
26. The method of claim 25 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
27. The method of claim 25 , wherein said method further comprises separating the butadiene from other components in the culture.
28. The method of claim 27 , wherein the separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, distillation, centrifugation, extractive filtration, ion exchange chromatography, absorption chromatography, or ultrafiltration.
29. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a metabolic pathway enzyme.
30. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a metabolic pathway enzyme.
31. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a metabolic pathway enzyme.
32. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises five exogenous nucleic acids each encoding a metabolic pathway enzyme.
33. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises six exogenous nucleic acids each encoding a metabolic pathway enzyme.
34. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism is a yeast.
35. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism is a fungus.
36. The non-naturally occurring microbial organism of claim 34 , wherein said yeast is Saccharomyces cerevisiae.