Organisms for the production of 1,3-butanediol

Patent metadata

Field	Value
Publication number	US-11180780-B2
Application number	US-201715616841-A
Country	US
Kind code	B2
Filing date	Jun 7, 2017
Priority date	Apr 30, 2009
Publication date	Nov 23, 2021
Grant date	Nov 23, 2021

Abstract

A non-naturally occurring microbial organism includes a microbial organism having a 1,3-butanediol (1,3-BDO) pathway having at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme expressed in a sufficient amount to produce 1,3-BDO. The pathway includes an enzyme selected from a 2-amino-4-ketopentanoate (AKP) thiolase, an AKP dehydrogenase, a 2-amino-4-hydroxypentanoate aminotransferase, a 2-amino-4-hydroxypentanoate oxidoreductase (deaminating), a 2-oxo-4-hydroxypentanoate decarboxylase, a 3-hydroxybutyraldehyde reductase, an AKP aminotransferase, an AKP oxidoreductase (deaminating), a 2,4-dioxopentanoate decarboxylase, a 3-oxobutyraldehyde reductase (ketone reducing), a 3-oxobutyraldehyde reductase (aldehyde reducing), a 4-hydroxy-2-butanone reductase, an AKP decarboxylase, a 4-aminobutan-2-one aminotransferase, a 4-aminobutan-2-one oxidoreductase (deaminating), a 4-aminobutan-2-one ammonia-lyase, a butenone hydratase, an AKP ammonia-lyase, an acetylacrylate decarboxylase, an acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), an acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), an acetoacetyl-CoA reductase (ketone reducing), a 3-hydroxybutyryl-CoA reductase (aldehyde forming), a 3-hydroxybutyryl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA dehydratase, and a crotonase. A method for producing 1,3-BDO, includes culturing such microbial organisms under conditions and for a sufficient period of time to produce 1,3-BDO.

First claim

What is claimed is:

1. A non-naturally occurring microbial organism comprising a set of enzymes that convert 4-hydroxbutyryl-CoA to 1,3-butanediol (1,3-BDO),
wherein said microbial organism comprises at least one heterologous nucleic acid encoding a crotonase that converts crotonoyl-CoA to 3-Hydroxybutyryl-CoA, and
wherein the crotonase is expressed in a sufficient amount to produce 1,3-BDO.

2. The non-naturally occurring microbial organism of claim 1 , wherein said crotonase is encoded by one or more genes selected from the group consisting of crt, crt1, ech, paaA, paaB, phaA, phaB, maoC, paaF, paaG, abfD, Msed_1220, fadA, fadB, fadI, fadJ, and fadR, wherein each of said genes encode an enzyme capable of catalyzing hydration of crotonyl-CoA to form 3-hydroxybutyryl-CoA.

3. The non-naturally occurring microbial organism of claim 1 , further comprising at least one exogenous nucleic acid encoding:
(a) a 3-hydroxybutyryl-CoA reductase (aldehyde forming) that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde; or
(b) a 3-hydroxybutyraldehyde reductase that converts 3-hydroxybutyraldehyde to 1,3-butanediol.

4. The non-naturally occurring microbial organism of claim 3 comprising at least one exogenous nucleic acid encoding the 3-hydroxybutyryl-CoA reductase (aldehyde forming) that converts 3-hydroxybutyryl-CoA to 3-hydroxybutyraldehyde and at least one exogenous nucleic acid encoding the 3-hydroxybutyraldehyde reductase that converts 3-hydroxybutyraldehyde to 1,3-butanediol.

5. The non-naturally occurring microbial organism of claim 3 , wherein said 3-hydroxybutyraldehdye reductase is encoded by one or more genes selected from the group consisting of alrA, ADH2, yqhD, bdh I, bdh II, adhA, 4hbd, adhI, P84067, mmsb, dhat, and 3hidh.

6. The non-naturally occurring microbial organism of claim 3 , wherein said 3-hydroxybutyryl-CoA reductase (aldehyde forming) is encoded by one or more genes selected from the group consisting of acr 1, sucD, bphG, bld, adhE, Msed_0709, mcr, asd-2, Sad 2370, Ald, and eutE.

7. The non-naturally occurring microbial organism of claim 1 , further comprising at least one exogenous nucleic acid encoding a 3-hydroxybutyryl-CoA reductase (alcohol forming) that converts 3-hydroxybutyryl-CoA to 1,3-butanediol.

8. The non-naturally occurring microbial organism of claim 7 , wherein said 3-hydroxybutyryl-CoA reductase (alcohol forming) is encoded by one or more genes selected from the group consisting of adhE, adhE2, mcr, Rcas_2929, NAP1_02720, MGP2080_00535, and FAR.

9. The non-naturally occurring microbial organism of claim 1 further comprising at least one exogenous nucleic acid encoding a 4-hydroxybutyryl-CoA dehydratase that converts 4-hydroxybutyryl-CoA to crotonyl-CoA.

10. The non-naturally occurring microbial organism of claim 9 , wherein said 4-hydroxybutyryl-CoA dehydratase is encoded by one or more genes selected from the group consisting of fumA, fumB, fumC, fumH, fum1, MmcB, MmcC, hmd, BACCAP_02294, ANACOL_02527, NtherDRAFT_2368, dmdA, dmdB, crt, crt1, ech paaA, paaB, phaA, phaB, maoC, paaF, paaG, abfD, Msed_1220, fadA, fadB, fadI, fadJ, and fadR.

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

12. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism is a bacterium.

13. The non-naturally occurring microbial organism of claim 12 , wherein said bacteria is E. coli.

14. A culture medium comprising the non-naturally occurring microbial organism of claim 1 .

15. The culture medium of claim 14 further comprising 1,3-BDO.

16. A method for producing 1,3-BDO, comprising culturing the non-naturally occurring microbial of claim 1 under conditions and for a sufficient period of time to produce 1,3-BDO.

17. The method of claim 16 further comprising separating 1,3-BDO from other components in the culture.

18. The method of claim 17 , wherein the separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, absorption chromatography, or ultrafiltration.

19. The method of claim 17 , wherein the separating comprises distillation.

20. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism comprises three exogenous nucleic acids, each encoding an enzyme of the set.

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

22. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism comprises four exogenous nucleic acids, each encoding an enzyme of the set.

Assignees

• Genomatica Inc

Inventors

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

Classifications

• C12N9/1029

  transferring groups other than amino-acyl groups (2.3.1) · CPC title

• C12Y403/01

  Ammonia-lyases (4.3.1) · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title

• C12Y101/01

  with NAD+ or NADP+ as acceptor (1.1.1) · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title