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-10626422-B2
Application number	US-201314424404-A
Country	US
Kind code	B2
Filing date	Aug 27, 2013
Priority date	Aug 27, 2012
Publication date	Apr 21, 2020
Grant date	Apr 21, 2020

Abstract

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway (MMP) 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 (MMP), wherein said non-naturally occurring microbial organism comprises exogenous nucleic acids encoding at least two MMP enzymes expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said MMP comprises:
(i) a methanol dehydrogenase (EM9);
(ii) an EM9 and a formaldehyde activating enzyme (EM10); or
(iii) a methanol methyltransferase (EM1) and a methylenetetrahydrofolate reductase (EM2);

(b) a formaldehyde assimilation pathway (FAP), wherein said non-naturally occurring microbial organism comprises exogenous nucleic acids encoding at least one FAP enzyme (FAPE) expressed in a sufficient amount to produce an intermediate of glycolysis and/or a metabolic pathway that can be used in the formation of 1,4-butanediol (BDO); and
(c) a BDO pathway, wherein said non-naturally occurring microbial organism comprises exogenous nucleic acids encoding at least two BDO pathway enzymes expressed in a sufficient amount to produce BDO.

2. The non-naturally occurring microbial organism of claim 1 , wherein:
(A) the MMP of (a)(i) further comprises:
(1) a methylenetetrahydrofolate dehydrogenase (EM3), a methenyltetrahydrofolate cyclohydrolase (EM4) and a formyltetrahydrofolate deformylase (EM5);
(2) an EM3, an EM4 and a formyltetrahydrofolate synthetase (EM6);
(3) a formaldehyde dehydrogenase (EM11);
(4) a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EM13) and a S-formylglutathione hydrolase (EM14); or
(5) an EM13 and an EM14;

(B) the MMP of (a)(ii) further comprises:
(1) an EM3, an EM4 and an EM5; or
(2) an EM3, an EM4 and an EM6; or

(c) the MMP of (a)(iii) further comprises:
(1) an EM3, an EM4, and an EM5; or
(2) an EM3, an EM4 and an EM6.

3. The non-naturally occurring microbial organism of claim 1 , wherein the BDO Pathway (BDOP) comprises:
(i) a succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), and a 1,4-butanediol dehydrogenase (EB8);
(ii) an EB3, an EB4, a 4-hydroxybutyryl-CoA transferase (EB11) or a 4-hydroxybutyryl-CoA synthetase (EB12), an EB7, and an EB8;
(iii) an EB3, an EB4, an EB11 or a 4-hydroxybutyryl-CoA synthetase, and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15);
(iv) an EB3, an EB4, an EB5, an EB6, and an EB15;
(v) an EB3, an EB4, a 4-HB reductase (EB13), and an EB8;
(vi) an EB3, an EB4, an EB5, a 4-hydroxybutyryl-phosphate reductase (EB14), and an EB8;
(vii) a succinyl-CoA reductase (alcohol forming) (EB10), an EB5, an EB6, an EB7, and an EB8;
(viii) an EB10, an EB5, an EB6, and an EB15;
(ix) an EB10, an EB11 or an EB12, an EB7, and an EB8;
(x) an EB10, an EB11 or an EB12, and an EB15;
(xi) an EB10, an EB13, and an EB8;
(xii) an EB10, an EB5, an EB14 and an EB8;
(xiii) a succinate reductase (EB9), an EB4, an EB5, an EB6, an EB7, and an EB8;
(xiv) an EB9, an EB4, an EB11 or an EB12, an EB7, and an EB8;
(xv) an EB9, an EB4, an EB11 or an EB12, and an EB15;
(xvi) an EB9, an EB4, an EB5, an EB6, and an EB15;
(xvii) an EB9, an EB4, an EB13, and an EB8; and
(xviii) an EB9, an EB4, an EB5, an EB14, and an EB8.

4. The non-naturally occurring microbial organism of claim 1 , wherein
(a) said non-naturally occurring microbial organism further comprises two or more gene disruptions, wherein said two 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 two or more gene disruptions confer increased production of BDO in said non-naturally occurring microbial organism; and/or
(b) 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.

5. The non-naturally occurring microbial organism of claim 1 , wherein
(A) the formaldehyde assimilation pathway comprises:
(1) a hexulose-6-phosphate synthase (EF1) and a 6-phospho-3-hexuloisomerase (EF2); or
(2) a dihydroxyacetone synthase (EF3) or a dihydroxyacetone kinase (EF4); and

(B) the intermediate of glycolysis is
(1) a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof; or
(2) a dihydroxyacetone, a dihydroxyacetone phosphate, or a combination thereof.

6. The non-naturally occurring microbial organism of claim 1 , wherein
(A) at least one of said exogenous nucleic acids is a heterologous nucleic acid;
(B) said non-naturally occurring microbial organism is in a substantially anaerobic culture medium; and/or
(C) said non-naturally occurring microbial organism is a species of bacteria, yeast, or fungus.

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

8. A bioderived BDO produced by the non-naturally occurring microbial organism of claim 1 .

9. A culture medium comprising the bioderived BDO of claim 8 ; wherein said culture medium is separated from the non-naturally occurring microbial organism having the BDO Pathway.

10. A composition comprising said bioderived BDO of claim 8 , and a compound other than said bioderived BDO; wherein said compound other than said bioderived BDO is a trace amount of a cellular portion of a non-naturally occurring microbial organism having a BDOP.

11. A polymer comprising or obtained by converting the bioderived BDO of claim 8 .

12. A method for producing a polymer, comprising chemically of enzymatically converting the bioderived BDO of claim 8 to the polymer.

13. A composition comprising the bioderived BDO of claim 8 .

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

15. A method of producing an intermediate of glycolysis and/or an intermediate of a metabolic pathway that can be used in the formation of biomass, comprising culturing the non-naturally occurring microbial organism of claim 5 under conditions and for a sufficient period of time to produce the intermediate.

16. The method of claim 14 , wherein the non-naturally occurring microbial organism is cultured in a medium comprising biomass, glucose, xylose, arabinose, galactose, mannose, fructose, sucrose, starch, glycerol, methanol, carbon dioxide, formate, methane, or any combination thereof as a carbon source.

17. The method of claim 16 , wherein the non-naturally occurring microbial organism is cultured in methanol.

18. The non-naturally occurring microbial organism of claim 1 , wherein the non-naturally occurring microbial organism is a host bacteria selected from the group consisting of: Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens , a

Assignees

• Genomatica Inc

Inventors

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

Classifications

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title

• C12Y402/01

  Hydro-lyases (4.2.1) · CPC title

• C08G18/3206

  aliphatic · CPC title

• C12Y105/0102

  Methylenetetrahydrofolate reductase [NAD(P)H] (1.5.1.20) · CPC title

• C12P7/18 Primary

  polyhydric · CPC title