Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing adipate, 6-aminocaproate, hexamethylenediamine or caprolactam related thereto

Patent metadata

Field	Value
Publication number	US-10150976-B2
Application number	US-201314107832-A
Country	US
Kind code	B2
Filing date	Dec 16, 2013
Priority date	Dec 17, 2012
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

Abstract

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as adipate, 6-aminocaproate, hexamethylenediamine or caprolactam. Also provided herein are methods for using such an organism to produce adipate, 6-aminocaproate, hexamethylenediamine or caprolactam.

First claim

What is claimed is:

1. A non-naturally occurring microbial organism comprising:
(A) a methanol metabolic pathway, wherein said non-naturally occurring microbial organism comprises at least two exogenous nucleic acids encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said methanol metabolic pathway comprises:
(i) a methanol methyltransferase and a methylenetetrahydrofolate reductase;
(ii) a methanol dehydrogenase; or
(iii) a methanol dehydrogenase and a formaldehyde activating enzyme; and

(B) one of:
(i) an adipate pathway, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding one or more adipate pathway enzymes 2A, 2B, 2C, 2D and 2L expressed in a sufficient amount to produce adipate;
(ii) a 6-aminocaproate pathway, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding one or more 6-aminocaproate pathway enzymes 2A, 2B, 2C, 2D, 2E and 2F expressed in a sufficient amount to produce 6-aminocaproate,
(iii) a hexamethylenediamine pathway, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding one or more hexamethylenediamine pathway enzymes 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2J, and 2K expressed in a sufficient amount to produce hexamethylenediamine; or
(iv) a caprolactam pathway, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding one or more caprolactam pathway enzymes 2A, 2B, 2C, 2D, 2E, 2F, and 2G and 21, or 2H expressed in a sufficient amount to produce caprolactam,
wherein:
2A is a 3-oxoadipyl-CoA thiolase that enzymatically converts succinyl-CoA or Acetyl-CoA to 3-oxoadipyl-CoA;
2B is a 3-oxoadipyl-CoA reductase that enzymatically converts 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA;
2C is a 3-hydroxyadipyl-CoA dehydratase that enzymatically converts 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA;
2D is a 5-carboxy-2-pentenoyl-CoA reductase that enzymatically converts 5-carboxy-2-pentenoyl-CoA to adipyl-CoA;
2E is an adipyl-CoA reductase (aldehyde forming) that enzymatically converts adipyl-CoA to adipate semialdehyde;
2F is a 6-aminocaproate transaminase or a 6-aminocaproate dehydrogenase that enzymatically converts adipate semialdehyde to 6-aminocaproate;
2G is a 6-aminocaproyl-CoA/acyl-CoA transferase or a 6-aminocaproyl-CoA synthase that enzymatically converts 6-aminocaproate to 6-aminocaproyl-CoA;
2H is an amidohydrolase that enzymatically converts 6-aminocaproate to caprolactam;
2I reflects spontaneous cyclization;
2J is a 6-aminocaproyl-CoA reductase (aldehyde forming) that enzymatically converts 6-aminocaproyl-CoA to 6-aminocaproate semialdehyde;
2K is a HMDA transaminase or a HMDA dehydrogenase that enzymatically converts 6-aminocaproate semialdehyde to hexamethylenediamine; and
2L is an adipyl-CoA hydrolase, an adipyl-CoA ligase, an adipyl-CoA transferase or a phosphotransadipylase/adipate kinase that enzymatically converts adipyl-CoA to adipate.

2. The non-naturally occurring microbial organism of claim 1 , wherein the methanol metabolic pathway comprises:
(i) a methanol methyltransferase, a methylenetetrahydrofolate reductase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase, and a formyltetrahydrofolate deformylase;
(ii) a methanol methyltransferase, a methylenetetrahydrofolate reductase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase;
(iii) a methanol dehydrogenase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase;
(iv) a methanol dehydrogenase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase;
(v) a methanol dehydrogenase and a formaldehyde dehydrogenase;
(vi) a methanol dehydrogenase, a S-(hydroxymethyl)glutathione synthase, a glutathione-dependent formaldehyde dehydrogenase and a S-formylglutathione hydrolase;
(vii) a methanol dehydrogenase, a glutathione-dependent formaldehyde dehydrogenase and a S-formylglutathione hydrolase;
(viii) a methanol dehydrogenase, a formaldehyde activating enzyme, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase; or
(ix) a methanol dehydrogenase, a formaldehyde activating enzyme, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase.

3. The non-naturally occurring microbial organism of claim 1 , wherein:
said non-naturally occurring microbial organism comprises three, four, five, six or seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme.

4. The non-naturally occurring microbial organism of claim 1 , further comprising a formaldehyde assimilation pathway, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of biomass, wherein the formaldehyde assimilation pathway comprises:
(i) a hexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase; or
(ii) a dihydroxyacetone (DHA) synthase and a DHA kinase.

5. The non-naturally occurring microbial organism of claim 1 , wherein
said non-naturally occurring microbial organism is a species of bacteria, yeast, or fungus.

6. The non-naturally occurring microbial organism of claim 1 , wherein the non-naturally occurring microbial organism comprising an adipate pathway comprises two, three, four or five exogenous nucleic acids, each encoding an adipate pathway enzyme.

7. The non-naturally occurring microbial organism of claim 6 , wherein the at least one exogenous nucleic acid encoding an adipate pathway enzyme is a heterologous nucleic acid.

8. The non-naturally occurring microbial organism of claim 1 , wherein the non-naturally occurring microbial organism comprising a 6-aminocaproate pathway comprises two, three, four, five, or six exogenous nucleic acids, each encoding a 6-aminocaproate pathway enzyme.

9. The non-naturally occurring microbial organism of claim 8 , wherein the at least one exogenous nucleic acid encoding a 6-aminocaproate pathway enzyme is a heterologous nucleic acid.

10. The non-naturally occurring microbial organism of claim 1 , wherein the non-naturally occurring microbial organism comprising a hexamethylenediamine pathway comprises two, three, four, five, six, seven, eight or nine exogenous nucleic acids, each encoding a hexamethylenediamine-pathway enzyme.

11. The non-naturally occurring microbial organism of claim 10 , wherein the at least one exogenous nucleic acid encoding a hexamethylenediamine pathway enzyme is a heterologous nucleic acid.

12. The non-naturally occurring microbial organism of claim 1 , wherein the non-naturally occurring microbial organism comprising a caprolactam pathway comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a caprolactam pathway enzyme.

13. The non-naturally occurring microbial organism of claim 12 , wherein the at least one exogenous nucleic acid encoding a caprolactam pathway enzyme is a heterologous nucleic acid.

14. The non-naturally occurring microbial organism of claim 1 , wherein the at least one exogeno

Assignees

• Genomatica Inc

Inventors

• Burgard Anthony P
• Osterhout Robin E
• Van Dien Stephen J
• Tracewell Cara Ann
• Pharkya Priti
• Andrae Stefan

Classifications

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C08G69/16

  Preparatory processes · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12P17/10 Primary

  Nitrogen as only ring hetero atom · CPC title

• C12P13/005

  Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids · CPC title