Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing succinate related thereto

Patent metadata

Field	Value
Publication number	US-11535874-B2
Application number	US-201314436722-A
Country	US
Kind code	B2
Filing date	Oct 21, 2013
Priority date	Oct 22, 2012
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

Provided herein is a non-naturally occurring microbial organism (NNOMO) 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 succinate. Also provided herein are methods for using such an organism to produce succinate.

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 at least one exogenous nucleic acid encoding a MMP enzyme expressed in a sufficient amount for biosynthesis of succinate, and wherein said MMP comprises:
(i) a methanol dehydrogenase (EM9) from bacteria,
(ii) an EM9 from bacteria and a formaldehyde activating enzyme (EM10), or
(iii) a methanol methyltransferase (EM1) and a methylenetetrahydrofolate reductase (EM2), and
wherein EM9 converts methanol to formaldehyde,
wherein EM10 converts formaldehyde to methylene-tetrahydrofolate (THF),
wherein EM1 converts methanol to methyl-THF, and
wherein EM2 converts methyl-THF to methylene-THF;

(b) a succinate pathway (SucP); and
(c) a formaldehyde assimilation pathway (FAP), wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid encoding a FAP enzyme and wherein said FAP comprises:
(i) a hexulose-6-phosphate synthase (EF1) and a 6-phospho-3-hexuloisomerase (EF2), or
(ii) a dihydroxyacetone synthase (EF3) or a dihydroxyacetone kinase (EF4);
wherein EF1 converts formaldehyde and D-ribulose-5-phosphate to hexulose-6-phosphate,
wherein EF2 converts hexulose-6-phosphate to fructose-6-phosphate,
wherein EF3 converts formaldehyde and xylulose-6-phosphate to dihydroxyacetone, and
wherein EF4 converts dihydroxyacetone to dihydroxyacetone-phosphate; and

wherein said non-naturally occurring microbial organism comprises an Escherichia coli or Saccharomyces cerevisiae.

2. The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism comprises at least one nucleic acid encoding a SucP enzyme (SucPE) expressed in a sufficient amount to produce succinate, wherein said SucP comprises:
(a) a phosphoenolpyruvate (PEP) carboxylase (ES1A) or a PEP carboxykinase (ES1 B), a malate dehydrogenase (ES3), a fumarase (ES5), and a fumarate reductase (ES6);
(b) a pyruvate carboxylase (ES2), an ES3, an ES5, and an ES6; or
(c) a malic enzyme (ES4), an ES5, and an ES6;
wherein ES1A and ES1B convert PEP to oxaloacetate,
wherein ES2 converts pyruvate to oxaloacetate,
wherein ES3 converts oxaloacetate to malate,
wherein ES4 converts pyruvate to malate,
wherein ES5 converts malate to fumarate, and
wherein ES6 converts fumarate to succinate.

3. The non-naturally occurring microbial organism of claim 1 , wherein:
(a) said MMP comprises:
(i) an EM9 from bacteria, a methylenetetrahydrofolate dehydrogenase (EM3), a methenyltetrahydrofolate cyclohydrolase (EM4), and a formyltetrahydrofolate deformylase (EM5);
(ii) an EM9 from bacteria, an EM3, an EM4, and a formyltetrahydrofolate synthetase (EM6);
(iii) an EM9 from bacteria and a formaldehyde dehydrogenase (EM11);
(iv) an EM9 from bacteria, a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EM13), and a S-formylglutathione hydrolase (EM14);
(v) an EM9 from bacteria, an EM13, and an EM14;
(vi) an EM9 from bacteria, an EM10, an EM3, an EM4, and an EM5;
(vii) an EM9 from bacteria, an EM10, an EM3, an EM4, and an EM6;
(viii) an EM1, an EM2, an EM3, an EM4, and an EM5; or
(ix) an EM1, an EM2, an EM3, an EM4 and an EM6;
wherein EM3 converts methylene-THF to methenyl-THF,
wherein EM4 converts methenyl-THF to formyl-THF,
wherein EM5 and EM6 convert formyl-THF to formate,
wherein EM11 converts formaldehyde to formate,
wherein EM12 converts formaldehyde to S-hydroxymethylglutathione,
wherein EM13 converts S-hydroxymethylglutathione to S-formylglutathione, and
wherein EM14 converts S-formylglutathione to formate.

4. The non-naturally occurring microbial organism of claim 1 , wherein:
said non-naturally occurring microbial organism comprises two exogenous nucleic acids, each encoding a FAP enzyme.

5. The non-naturally occurring microbial organism of claim 1 , wherein:
(a) said at least one exogenous nucleic acid is a heterologous nucleic acid; and/or
(b) said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

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

7. The method of claim 6 , wherein said method further comprises separating said succinate from other components in said culture.

8. The method of claim 7 , wherein said separating comprises extraction, continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane separation, reverse osmosis, electrodialysis, distillation, crystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, or ultrafiltration.

9. The method of claim 6 , wherein said culturing is in a culture medium comprising glucose.

10. The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism is Saccharomyces cerevisiae and is Crabtree positive.

11. The non-naturally occurring microbial organism of claim 1 , wherein said MMP comprises an EM9 from bacteria.

12. The non-naturally occurring microbial organism of claim 1 , wherein said MMP comprises an EM9 from bacteria and EM10.

13. The non-naturally occurring microbial organism of claim 1 , wherein said MMP comprises an EM1 and an EM2.

14. The non-naturally occurring microbial organism of claim 1 , wherein said FAP comprises an EF1 and an EF2.

15. The non-naturally occurring microbial organism of claim 1 , wherein said FAP comprises an EF3 or an EF4.

16. The non-naturally occurring microbial organism of claim 2 , wherein said non-naturally occurring microbial organism comprises one, two, three, or four nucleic acids, each encoding a SucPE.

17. The non-naturally occurring microbial organism of claim 2 , wherein said at least one nucleic acid encoding a succinate enzyme is an exogenous or heterologous nucleic acid.

18. The non-naturally occurring microbial organism of claim 3 , wherein said MMP further comprises a formate dehydrogenase (EM8), a formate hydrogen lyase (EM15), or a hydrogenase (EM16);
wherein EM8 converts formate to reducing equivalents,
wherein EM15 converts formate to reducing equivalents, and
wherein EM16 converts hydrogen gas to reducing equivalents.

19. The non-naturally occurring microbial organism of claim 3 , wherein said non-naturally occurring microbial organism comprises two, three, four, five, six, or seven exogenous nucleic acids, each encoding a MMP enzyme.

Assignees

• Genomatica Inc

Inventors

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

Classifications

• C12N15/815

  for yeasts other than Saccharomyces · CPC title

• C12N9/0006

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

• C12Y105/0102

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

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title