Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds

Inventors: Priti Pharkya (San Diego, CA); Anthony P. Burgard (Bellefonte, PA); Stephen J. Van Dien (Encinitas, CA); Robin E. Osterhout (San Diego, CA); Mark J. Burk (San Diego, CA); John D. Trawick (La Mesa, CA); Michael P. Kuchinskas (Escondido, CA); Brian Steer (San Diego, CA)

Assignee: Genomatica, Inc.

C12N1/20 C07C31/207 C07C59/01 C08G18/284 C08G18/3206 C08G63/06 C08G63/16 C08G63/181 C08G69/02 C12N1/14 C12N1/16 C12N9/18 C12N15/52 C12N15/63 C12P7/18 C12P7/42 C12Y301/01 Y02P20/52

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Quick Facts

Patent No.: US 11,085,015
App. No.: 15/585,078
Granted: Aug 10, 2021
Kind: B2

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Abstract

The invention provides non-naturally occurring microbial organisms having a 4-hydroxybutyrate pathway and being capable of producing 4-hydroxybutyrate, wherein the microbial organism comprises one or more genetic modifications. The invention additionally provides methods of producing 4-hydroxybutyrate or related products using the microbial organisms.

Claims (49)

1. A non-naturally occurring microbial organism, said microbial organism having a 4-hydroxybutyrate pathway and being capable of producing 4-hydroxybutyrate, wherein said microbial organism comprises a genetic modification that decreases expression of an acyl coenzyme A thioesterase.

2. The microbial organism of claim 1 , wherein the microbial organism has decreased production of gamma-butyrolactone relative to a parent microbial organism in the absence of said genetic modification.

3. The microbial organism of claim 1 , wherein said microbial organism further comprises a 4-hydroxybutyryl-CoA pathway.

4. A method for producing 4-hydroxybutyrate, comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce 4-hydroxybutyrate.

5. The method of claim 4 , wherein the microbial organism has decreased production of gamma-butyrolactone relative to a parent microbial organism in the absence of said genetic modification.

6. The method of claim 4 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

7. The microbial organism of claim 1 , wherein the microbial organism has a genetic modification comprising at least two genetic modifications that decrease expression of at least two acyl coenzyme A thioesterases.

8. The microbial organism of claim 1 , wherein the microbial organism further comprises a genetic modification selected from:

(A) a genetic modification that increases expression of phosphoenolpyruvate carboxylase;

(B) a genetic modification that increases expression of alpha-ketoglutarate dehydrogenase;

(D) a genetic modification that increases expression of a gamma-butyrolactone esterase;

(E) a genetic modification that decreases expression of succinyl-CoA synthetase;

(F) a genetic modification that decreases expression of an alcohol dehydrogenase;

(G) a genetic modification that decreases expression of a non-energy-producing NADH dehydrogenase;

(H) a genetic modification that decreases expression of a cytochrome oxidase; and

(I) a combination of two or more of the genetic modifications of parts (A)-(H).

9. The microbial organism of claim 8 , wherein:

(J) the microbial organism of part (A), (B) or (C) has decreased production of ethanol, acetate, pyruvate or alanine, or a combination thereof, relative to a parent microbial organism in the absence of said genetic modification;

(K) the microbial organism of part (B) has decreased production of glutamate relative to a parent microbial organism in the absence of said genetic modification;

(L) the microbial organism of part (C) has a genetic modification comprising increased expression of a permease, glucokinase, or a glucose facilitator, or a combination thereof;

(M) the microbial organism of part (D) has decreased production of gamma-butyrolactone relative to a parent microbial organism in the absence of said genetic modification;

(N) the microbial organism of part (E) has increased production of 4-hydroxybutyrate relative to a parent microbial organism in the absence of said genetic modification;

(O) the microbial organism of part (F) has decreased backflux from a downstream product of the 4-hydroxybutyrate pathway relative to a parent microbial organism in the absence of said genetic modification;

(P) the microbial organism of part (G) has suppressed depletion of the NADH pool or increased energy efficiency in the microbial organism, or a combination thereof, relative to a parent microbial organism in the absence of said genetic modification;

(Q) the microbial organism of part (H) has increased energy efficiency relative to a parent microbial organism in the absence of said genetic modification; or

(R) the microbial organism of part (H) has increased tolerance to a range of oxygen concentrations relative to a parent microbial organism in the absence of said genetic modification.

10. The microbial organism of claim 8 , wherein the microbial organism of part (D) further comprises a 4-hydroxybutyryl-CoA pathway.

11. The microbial organism of claim 8 , wherein the microbial organism further comprises (A) a genetic modification that increases expression of phosphoenolpyruvate carboxylase.

12. The microbial organism of claim 8 , wherein the microbial organism further comprises (B) a genetic modification that increases expression of alpha-ketoglutarate dehydrogenase.

13. The microbial organism of claim 8 , wherein the microbial organism further comprises (C) a genetic modification that increases expression of a non-phosphotransferase (PTS) glucose uptake system.

14. The microbial organism of claim 8 , wherein the microbial organism further comprises (D) a genetic modification that increases expression of a gamma-butyrolactone esterase.

15. The microbial organism of claim 8 , wherein the microbial organism further comprises (E) a genetic modification that decreases expression of succinyl-CoA synthetase.

16. The microbial organism of claim 8 , wherein the microbial organism further comprises (F) a genetic modification that decreases expression of an alcohol dehydrogenase.

17. The microbial organism of claim 8 , wherein the microbial organism further comprises (G) a genetic modification that decreases expression of a non-energy-producing NADH dehydrogenase.

18. The microbial organism of claim 8 , wherein the microbial organism further comprises (H) a genetic modification that decreases expression of a cytochrome oxidase.

19. The microbial organism of claim 8 , wherein the microbial organism further comprises (I) a combination of two or more of the genetic modifications of parts (A)-(H).

20. The method of claim 4 , wherein the microbial organism has a genetic modification comprising at least two genetic modifications that decrease expression of at least two acyl coenzyme A thioesterases.

21. The method of claim 4 , wherein the microbial organism further comprises a genetic modification selected from: