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-12264354-B2
Application number	US-201715488320-A
Country	US
Kind code	B2
Filing date	Apr 14, 2017
Priority date	Aug 27, 2012
Publication date	Apr 1, 2025
Grant date	Apr 1, 2025

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 method of producing an intermediate of a metabolic pathway that can be used in the formation of biomass, comprising culturing a non-naturally occurring microbial organism under conditions and for a sufficient period of time to produce the intermediate,
wherein the non-naturally occurring microbial organism has a methanol metabolic pathway and comprises at least three exogenous nucleic acids each 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 formaldehyde, wherein said methanol metabolic pathway comprises:
(i) a methanol methyltransferase, which catalyzes the conversion of methanol to methyltetrahydrofolate (methyl-THF),
(ii) a methylenetetrahydrofolate reductase, which catalyzes the conversion of methyl-THF to methylene-THF, and
(iii) a methylenetetrahydrofolate dehydrogenase, which catalyzes the conversion of methylene-THF to methenyl-THF; and
wherein the non-naturally occurring microbial organism has a formaldehyde assimilation pathway and comprises at least two exogenous nucleic acids each encoding a formaldehyde assimilation pathway enzyme expressed in a sufficient amount to produce an intermediate of 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 synthase and a dihydroxyacetone kinase.

2. The method of claim 1 , wherein the intermediate of a metabolic pathway is:
(i) a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof; or
(ii) a dihydroxyacetone, a dihydroxyacetone phosphate, or a combination thereof.

3. The method of claim 1 , wherein the methanol metabolic pathway comprises:
(i) a methenyltetrahydrofolate cyclohydrolase, and a formyltetrahydrofolate deformylase;
(ii) a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase;
(iii) a methanol dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase;
(iv) a methanol 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 methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase;
(ix) a methanol dehydrogenase, a formaldehyde activating enzyme, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase;
(x) a methanol dehydrogenase;
(xi) a methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate deformylase; or
(xii) a methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase.

4. The method of claim 3 , 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.

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

6. The method of claim 1 , wherein the non-naturally occurring microbial organism further has a 1,4-butanediol (BDO) pathway and comprises at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO.

7. The method of claim 6 , where the BDO pathway 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 4-hydroxybutyryl-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) 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-hydroxybutyryl-phosphate 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; or
(xviii) a succinate reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase, and a 1,4-butanediol dehydrogenase.

8. The method of claim 7 , wherein the BDO pathway further comprises a succinyl-CoA transferase or a succinyl-CoA synthetase.

9. The method of claim 6 , wherein the non-naturally occurring microbial organism comprises four, five, six or seven exogenous nucleic acids, each encoding a BDO Pathway enzyme.

10. The method of claim 1 , wherein the non-naturally occurring microbial organism further comprises one or more gene disruptions, wherein said

Assignees

• Genomatica Inc

Inventors

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

Classifications

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12Y402/01

  Hydro-lyases (4.2.1) · CPC title

• C12Y201/0109

  Methanol—corrinoid protein Co-methyltransferase (2.1.1.90) · CPC title

• C12Y101/01224

  Mannose-6-phosphate 6-reductase (1.1.1.224) · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title