Microorganisms for the production of methacrylic acid

Patent metadata

Field	Value
Publication number	US-9951355-B2
Application number	US-201414557188-A
Country	US
Kind code	B2
Filing date	Dec 1, 2014
Priority date	May 1, 2008
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

The invention provides a non-naturally occurring microbial organism having a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid pathway. The invention additionally provides a method for producing 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid. The method can include culturing a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid producing microbial organism expressing at least one exogenous nucleic acid encoding a 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid pathway enzyme in a sufficient amount and culturing under conditions and for a sufficient period of time to produce 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid or methacrylic acid.

First claim

What is claimed is:

1. A non-naturally occurring microbial organism having a methacrylic acid pathway comprising at least one exogenous nucleic acid encoding a methacrylic acid pathway enzyme expressed in a sufficient amount to produce methacrylic acid, said methacrylic acid pathway comprising methylmalonyl-CoA mutase, methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase, and aspartate ammonia lyase.

2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a methacrylic acid pathway enzyme.

3. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a methacrylic acid pathway enzyme.

4. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a methacrylic acid pathway enzyme.

5. The non-naturally occurring microbial organism of claim 4 , wherein said four exogenous nucleic acids encode methylmalonyl-CoA mutase, methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase, and aspartate ammonia lyase.

6. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises five exogenous nucleic acids each encoding a methacrylic acid pathway enzyme.

7. The non-naturally occurring microbial organism of claim 6 , wherein said methacrylic acid pathway further comprises methylmalonyl-CoA epimerase.

8. The non-naturally occurring microbial organism of claim 7 , wherein said five exogenous nucleic acids encode methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase, and aspartate ammonia lyase.

9. The non-naturally occurring microbial organism of claim 1 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

10. The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

11. The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism further comprises a genetic modification that increases the activity of at least one enzyme selected from citrate synthase, aconitase, isocitrate lyase, malate synthase, pyruvate dehydrogenase, pyruvate ferredoxin oxidoreductase and phosphoenolpyruvate carboxykinase, wherein the increase in activity is relative to the absence of said genetic modification.

12. A method for producing methacrylic acid, comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce methacrylic acid.

13. The method of claim 12 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

14. The method of claim 12 , wherein said microbial organism comprises two exogenous nucleic acids each encoding a methacrylic acid pathway enzyme.

15. The method of claim 12 , wherein said microbial organism comprises three exogenous nucleic acids each encoding a methacrylic acid pathway enzyme.

16. The method of claim 12 , wherein said microbial organism comprises four exogenous nucleic acids each encoding a methacrylic acid pathway enzyme.

17. The method of claim 16 , wherein said four exogenous nucleic acids encode methylmalonyl-CoA mutase, methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase, and 3-amino-2-methylpropionate ammonia lyase.

18. The method of claim 12 , wherein said microbial organism comprises five exogenous nucleic acids each encoding a methacrylic acid pathway enzyme.

19. The non-naturally occurring microbial organism of claim 18 , wherein said methacrylic acid pathway further comprises methylmalonyl-CoA epimerase.

20. The method of claim 19 , wherein said five exogenous nucleic acids encode methylmalonyl-CoA mutase, methylmalonyl-CoA epimerase, methylmalonyl-CoA reductase, 3-amino-2-methylpropionate transaminase, and 3-amino-2-methylpropionate ammonia lyase.

21. The method of claim 12 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

Assignees

• Genomatica Inc

Inventors

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

Classifications

• C12N9/0008

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12Y206/01022

  (S)-3-Amino-2-methylpropionate transaminase (2.6.1.22) · CPC title

• C12Y403/01

  Ammonia-lyases (4.3.1) · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C12N9/88

  Lyases (4.) · CPC title