Microorganisms and methods for the biosynthesis of butadiene

Patent metadata

Field	Value
Publication number	US-10487343-B2
Application number	US-201715645880-A
Country	US
Kind code	B2
Filing date	Jul 10, 2017
Priority date	May 5, 2010
Publication date	Nov 26, 2019
Grant date	Nov 26, 2019

Abstract

The invention provides non-naturally occurring microbial organisms having a butadiene pathway. The invention additionally provides methods of using such organisms to produce butadiene.

First claim

What is claimed is:

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 comp

Assignees

• Genomatica Inc

Inventors

• Burk Mark J
• Burgard Anthony P
• Sun Jun
• Osterhout Robin E
• Pharkya Priti

Classifications

• C12P7/04

  acyclic · CPC title

• C12N1/14

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title

• C12P5/026 Primary

  Unsaturated compounds, i.e. alkenes, alkynes or allenes · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N1/18

  Baker's yeast; Brewer's yeast · CPC title