Methods for enhancing microbial production of specific length fatty alcohols in the presence of methanol

Patent metadata

Field	Value
Publication number	US-12398396-B2
Application number	US-202117516424-A
Country	US
Kind code	B2
Filing date	Nov 1, 2021
Priority date	Nov 25, 2013
Publication date	Aug 26, 2025
Grant date	Aug 26, 2025

Abstract

The invention provides non-naturally occurring microbial organisms having a formaldehyde fixation pathway, a formate assimilation pathway, and/or a methanol metabolic pathway in combination with a fatty alcohol, fatty aldehyde, fatty acid or isopropanol pathway, wherein the microbial organisms selectively produce a fatty alcohol, fatty aldehyde or fatty acid of a specified length or isopropanol. The microbial organisms provided advantageously enhance the production of substrates and/or pathway intermediates for the production of chain length specific fatty alcohols, fatty aldehydes, fatty acids or isopropanol. In some aspects, the microbial organisms of the invention have select gene disruptions or enzyme attenuations that increase production of fatty alcohols, fatty aldehydes or fatty acids. The invention additionally provides methods of using the above microbial organisms to produce a fatty alcohol, a fatty aldehyde, a fatty acid or isopropanol.

First claim

What is claimed is:

1. A non-naturally occurring microbial organism having:
(i) a formaldehyde fixation pathway; and
(ii) a methanol metabolic pathway, and
a fatty acyl-ACP elongation (FAACPE) cycle in combination with a termination pathway,
wherein said formaldehyde fixation pathway comprises a 3-hexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase
wherein said methanol metabolic pathway comprises a methanol dehydrogenase,
wherein said FAACPE cycle comprises one or more β-ketoacyl-ACP synthase, one or more β-ketoacyl-ACP reductase, one or more β-hydroxyacyl-ACP reductase, and one or more enoyl ACP-reductase,
wherein said termination pathway comprises a thioesterase
wherein an enzyme of the formaldehyde fixation pathway, the methanol metabolic pathway, the FAACPE cycle or the termination pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce a compound of Formula (I):

wherein R 1 is C 1-24 linear alkyl; R 2 is H; and represents a single,
wherein the substrate of each of said enzymes of the FAACPE cycle and the termination pathway are independently selected from a compound of Formula (II) or malonyl-ACP:

wherein R 1 is C 1-24 linear alkyl; R 3 is H, OH, or oxo (═O)); R 4 is S-CoA, ACP, OH or H; and represents a single bond with the proviso that the valency of the carbon atom to which R 3 is attached is four,
wherein said one or more enzymes of the FAACPE cycle are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no greater than the number of carbon atoms at R 1 of said compound of Formula (I), and
wherein said one or more enzymes of the termination pathway are each selective for a compound of Formula (II) having a number of carbon atoms at R 1 that is no less than the number of carbon atoms at R 1 of said compound of Formula (I).

2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises a formate assimilation pathway.

3. The non-naturally occurring microbial organism of claim 1 , wherein the microbial organism further comprises an acetoacetyl-ACP pathway of:
(1) 12A, 12B, and 12C; or (2) 12A, 12B, and 12D,
wherein 12A is an acetyl-CoA carboxylase, wherein 12B is malonyl-CoA ACP transacylase, wherein 12C is an acetoacetyl-ACP synthase, and wherein 12D is a β-ketoacyl-ACP synthase, or
wherein the microbial organism further comprises a 3-oxovalery-ACP pathway comprising an acetyl-CoA carboxylase, a malonyl-CoA ACP transacylase, and a β-ketoacyl-ACP synthase.

4. The non-naturally occurring microbial organism of 3 , wherein an enzyme of the acetoacetyl-ACP pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce acetoacetyl-ACP, wherein an enzyme of the 3-oxovalery-ACP pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to produce 3-oxovalery-ACP, and wherein the acetoacetyl-ACP or the 3-oxovalery-ACP is a β-ketoacyl-ACP of the FAACPE cycle.

5. The non-naturally occurring microbial organism of claim 1 , wherein R 1 is C 9 linear alkyl, C 10 linear alkyl, Cn, linear alkyl, C 12 linear alkyl or Cis linear alkyl.

6. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises two, three, or four exogenous nucleic acids each encoding an enzyme of said FAACPE cycle or said termination pathway.

7. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises one, two, or three exogenous nucleic acids each encoding a formaldehyde fixation pathway enzyme, or a methanol metabolic pathway enzyme.

8. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes 3-hexulose-6-phosphate synthase, 6-phospho-3-hexuloisomerase, and methanol dehydrogenase.

9. The non-naturally occurring microbial organism of claim 1 , wherein said organism comprises at least one exogenous nucleic acid encoding the methanol dehydrogenase.

10. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises 3H or 3P, wherein 3H is a hydrogenase, wherein 3P a carbon monoxide dehydrogenase.

11. The non-naturally occurring microbial organism of claim 1 , wherein said at least one exogenous nucleic acid encoding said formaldehyde fixation pathway enzyme, said methanol metabolic pathway enzyme, said FAACPE cycle enzyme, or said termination pathway enzyme is a heterologous nucleic acid.

12. The non naturally occurring microbial organism of claim 1 , wherein said enzyme of the formaldehyde fixation pathway, methanol metabolic pathway, FAACPE cycle or termination pathway is expressed in a sufficient amount to produce a compound of Formula (XIV):

wherein
R 1 is C 9 linear alkyl, C 10 linear alkyl, C 11 , linear alkyl, C 12 linear alkyl or Cis linear alkyl.

13. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises an acetyl-CoA pathway and at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce or enhance carbon flux through acetyl-CoA, wherein said acetyl-CoA pathway comprises a pathway selected from:
(1) 3A and 3B; (2) 3A, 3C, and 3D; (3) 3H; (4) 3G and 3D; (5) 3E, 3F and 3B; (6) 3E and 31; (7) 3J, 3F and 3B; (8) 3J and 31; (9) 4A, 4B, and 4C; (10) 4A, 4B, 4J, 4K, and 4D; (11) 4A, 4B, 4G, and 4D; (12) 4A, 4F, and 4D; (13) 4N, 4H, 4B and 4C; (14) 4N, 4H, 4B, 4J, 4K, and 4D; (15) 4N, 4H, 4B, 4G, and 4D; (16) 4N, 4H, 4F, and 4D; (17) 4L, 4M, 4B and 4C; (18) 4L, 4M, 4B, 4J, 4K, and 4D; (19) 4L, 4M, 4B, 4G, and 4D; (20) 4L, 4M, 4F, and 4D; (21) 5A, 5B, 5D, 5H, 5I, and 5J; (22) 5A, 5B, 5E, 5F, 5H, 5I, and 5J; (23) 5A, 5B, 5E, 5K, 5L, 5H, 5I, and 5J; (24) 5A, 5C, 5D, 5H, and 5J; (25) 5A, 5C, 5E, 5F, 5H, and 5J; (26) 5A, 5C, 5E, 5K, 5L, 5H, and 5J; (27) 6A, 6B, 6D, and 6G; (28) 6A, 6B, 6E, 6F, and 6G; (29) 6A, 6B, 6E, 6K, 6L, and 6G; (30) 6A, 6C, and 6D; (31) 6A, 6C, 6E, and 6F; (32) 6A, 6C, 6E, 6K, and 6L; (33) 1T and 1V; (34) 1T, 1W, and 1X; (35) 1U and 1V; and (36) 1U, 1W, and 1X,
wherein 3A is a pyruvate oxidase (acetate-forming), wherein 3B is an acetyl-CoA synthetase, an acetyl-CoA ligase or an acetyl-CoA transferase, wherein 3C is an acetate kinase, wherein 3D is a phosphotransacetylase, wherein 3E is a pyruvate decarboxylase, wherein 3F is an acetaldehyde dehydrogenase, wherein 3G is a pyruvate oxidase (acetyl-phosphate forming), wherein 3H is a pyruvate dehydrogenase, a pyruvate: ferredoxin oxidoreductase, a pyruvate: NAD(P)H oxidoreductase or a pyruvate formate lyase, wherein 31 is an acetaldehyde dehydrogenase (acylating), wherein 3J is a threonine aldolase, wherein 4A is a phosphoenolpyruvate (PEP) carboxylase or a PEP carboxykinase, wherein 4B is an oxaloacetate decarboxylase, wherein 4C is a malonate semialdehyde dehydrogenase (acetylating), wherein 4D is an acetyl-CoA carboxylase or a malonyl-CoA decarboxylase, wherein 4F is an oxaloacetate dehydrogenase or an oxaloacetate oxidoreductase, wherein 4G is a malonate semialdehyde dehydrogenase (acylating), wherein 4H is a pyruvate carboxylase, wherein 4J is a malonate semialdehyde

Assignees

• Genomatica Inc

Inventors

• Osterhout Robin E
• Burgard Anthony P
• Pharkya Priti
• Andrae Stefan

Classifications

• C12Y503/01027

  6-Phospho-3-hexuloisomerase (5.3.1.27) · CPC title

• C12Y401/02043

  3-Hexulose-6-phosphate synthase (4.1.2.43) · CPC title

• C12Y202/01003

  Formaldehyde transketolase (2.2.1.3) · CPC title

• C12Y101/01244

  Methanol dehydrogenase (1.1.1.244) · CPC title

• C12P7/04

  acyclic · CPC title