Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 1,4-butanediol related thereto

Patent metadata

Field	Value
Publication number	US-9657316-B2
Application number	US-201313975678-A
Country	US
Kind code	B2
Filing date	Aug 26, 2013
Priority date	Aug 27, 2012
Publication date	May 23, 2017
Grant date	May 23, 2017

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 1,4-butanediol (BDO). Also provided herein are methods for using such an organism to produce BDO.

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 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, or metabolize methanol as a carbon source for biosynthesis of 1,4-butanediol (BDO), 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) a 1,4-butanediol (BDO) pathway comprising at least two exogenous nucleic acids each encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO.

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

4. The non-naturally occurring microbial organism of claim 2 , wherein said non-naturally occurring microbial organism comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a methanol metabolic pathway enzyme.

5. The non-naturally occurring microbial organism of claim 1 , wherein the BDO pathway of (B) comprises:
(i) a succinyl-CoA reductase (aldehyde forming), a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanediol dehydrogenase;
(ii) a succinyl-CoA reductase (aldehyde forming), a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanediol dehydrogenase;
(iii) a succinyl-CoA reductase (aldehyde forming), a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase, and a 4hydroxybutyryl-CoA reductase (alcohol forming);
(iv) a succinyl-CoA reductase (aldehyde forming), a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyryl-CoA reductase (alcohol forming);
(v) a succinyl-CoA reductase (aldehyde forming), a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase, and a 1,4-butanediol dehydrogenase;
(vi) a succinyl-CoA reductase (aldehyde forming), a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a 4-hydroxybutyrylphosphate reductase, and a 1,4-butanediol dehydrogenase;
(vii) a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanediol dehydrogenase;
(viii) a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyryl-CoA reductase (alcohol forming);
(ix) a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanediol dehydrogenase;
(x) a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase, and a 4-hydroxybutyryl-CoA reductase (alcohol forming);
(xi) a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyrate reductase, and a 1,4-butanediol dehydrogenase;
(xii) a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase and a 1,4-butanediol dehydrogenase;
(xiii) a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanediol dehydrogenase;
(xiv) a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), and a 1,4-butanediol dehydrogenase;
(xv) a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase or a 4-hydroxybutyryl-CoA synthetase, and a 4-hydroxybutyryl-CoA reductase (alcohol forming);
(xvi) a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyryl-CoA reductase (alcohol forming);
(xvii) a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase, and a 1,4-butanediol dehydrogenase; and
(xviii) a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase, and a 1,4-butanediol dehydrogenase.

6. The non-naturally occurring microbial organism of claim 5 , wherein the BDO pathway further comprises:
(i) a succinyl-CoA transferase; or
(ii) a succinyl-CoA synthetase.

7. The non-naturally occurring microbial organism of claim 5 , wherein the non-naturally occurring microbial organism comprises four, five, six or seven exogenous nucleic acids, each encoding a BDO pathway enzyme.

8. The non-naturally occurring microbial organism of claim 1 , further comprising one or more gene disruptions, wherein said one or more gene disruptions occur in one or more endogenous genes encoding protein(s) or enzyme(s) involved in native production of ethanol, glycerol, acetate, lactate, formate, CO 2 , and/or amino acids, by said non-naturally occurring microbial organism, and wherein said one or more gene disruptions confers increased production of BDO in said non-naturally occurring organism microbial organism.

9. The non-naturally occurring microbial organism of claim 8 , wherein one or more endogenous enzymes involved in: native production of ethanol, glycerol, acetate, lactate, formate, CO 2 and/or amino acids by said non-naturally occurring microbial organism, has attenuated enzyme activity or expression levels.

10. The non-naturally occurring microbial organism of claim 1 , further comprising a formaldehyde assim

Assignees

• Genomatica Inc

Inventors

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

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12N9/0006 Primary

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

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title

• C12Y101/01244

  Methanol dehydrogenase (1.1.1.244) · CPC title