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

• US 9,932,611 B2
• Filed: 10/21/2013
• Issued: 04/03/2018
• Est. Priority Date: 10/22/2012
• Status: Active Grant

• Current Assignee: Genomatica Incorporated
• Original Assignee: Genomatica Incorporated
• Inventors: Burgard, Anthony P., Osterhout, Robin E., Van Dien, Stephen J., Tracewell, Cara Ann, Pharkya, Priti, Andrae, Stefan
• Primary Examiner(s): Desai, Anand U
• Application Number:
  US14/059,285
• Publication Number:
  US 20140235815A1
• Time in Patent Office: 1,625 Days
• CPC Class Codes: C07C 55/10 Succinic acid
• CPC Class Codes: C07C 59/235 Saturated compounds containing more than one carboxyl group
• CPC Class Codes: C08G 63/00 Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule polyester-amides C08G69/44; polyester-imides C08G73/16
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/77 for Corynebacterium; for Brevibacterium
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0028 with NAD or NADP as acceptor (1.5.1)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12P 7/24 containing a carbonyl group
• CPC Class Codes: C12P 7/46 Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
• CPC Class Codes: C12Y 101/01244 Methanol dehydrogenase (1.1.1.244)
• CPC Class Codes: C12Y 105/0102 Methylenetetrahydrofolate reductase [NAD(P)H] (1.5.1.20)

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 succinate. Also provided herein are methods for using such an organism to produce succinate.

Claims

1. A non-naturally occurring microbial organism comprising:

(a) a methanol metabolic pathway, wherein said non-naturally occurring microbial organism comprises at least one exogenous nucleic acid 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 succinate, 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 succinate pathway comprising exogenous nucleic acids wherein at least one of said exogenous nucleic acids is a heterologous nucleic acid encoding a succinate pathway enzyme expressed in a sufficient amount to produce succinate, said succinate pathway comprising;

(i) a phosphoenolpyruvate (PEP) carboxylase or a PEP carboxykinase, a malate dehydrogenase, a fumarase, and a fumarate reductase;

(ii) a pyruvate carboxylase, a malate dehydrogenase, a fumarase, and a fumarate reductase;

or

(iii) a malic enzyme, a fumarase, and a fumarate reductase.

2. The non-naturally occurring microbial organism of claim 1, wherein the non-naturally occurring microbial organism comprises two, three, or four heterologous nucleic acids, each encoding a succinate pathway enzyme.

3. 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.

4. The non-naturally occurring microbial organism of claim 1, wherein the methanol metabolic pathway further comprises a formate dehydrogenase, a formate hydrogen lyase, or hydrogenase.

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

6. The non-naturally occurring microbial organism of claim 1, wherein said at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme is a heterologous nucleic acid.

7. 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, CO₂, and/or amino acids by said microbial organism, and wherein said one or more gene disruptions confer increased production of succinate in said microbial organism.

8. The non-naturally occurring microbial organism of claim 7, wherein said protein(s) or enzyme(s) is a pyruvate decarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, a glycerol-3-phosphatase, a glycerol-3-phosphate dehydrogenase, a lactate dehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a pyruvate:

quinone oxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, a lactate dehydrogenase, a pyruvate dehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, a monocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase, a pyruvate kinase, or any combination thereof.

9. The non-naturally occurring microbial organism of claim 1, wherein one or more endogenous enzymes involved in native production of ethanol, glycerol, acetate, lactate, formate, CO2 and/or amino acids by said microbial organism has attenuated enzyme activity or expression levels.

10. The non-naturally occurring microbial organism of claim 9, wherein said enzyme is a pyruvate decarboxylase, an ethanol dehydrogenase, a glycerol dehydrogenase, a glycerol-3-phosphatase, a glycerol-3-phosphate dehydrogenase, a lactate dehydrogenase, an acetate kinase, a phosphotransacetylase, a pyruvate oxidase, a pyruvate:

quinone oxidoreductase, a pyruvate formate lyase, an alcohol dehydrogenase, a lactate dehydrogenase, a pyruvate dehydrogenase, a pyruvate formate-lyase-2-ketobutyrate formate-lyase, a pyruvate transporter, a monocarboxylate transporter, a NADH dehydrogenase, a cytochrome oxidase, a pyruvate kinase, or any combination thereof.

11. 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, and wherein said formaldehyde assimilation pathway comprises

(i) a hexulose-6-phosphate synthase and a 6-phospho-3-hexuloisomerase, wherein optionally the intermediate is a hexulose-6-phosphate, a fructose-6-phosphate, or a combination thereof;

or

(ii) a dihydroxyacetone synthase and a dihydroxyacetone-phosphate kinase, wherein optionally the intermediate is a dihydroxyacetone, a dihydroxyacetone phosphate, or a combination thereof.

12. The non-naturally occurring microbial organism of claim 11, wherein the non-naturally occurring microbial organism comprises two exogenous nucleic acids, each encoding a formaldehyde assimilation pathway enzyme.

13. The non-naturally occurring microbial organism of claim 1, wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.

14. A method for producing succinate, comprising culturing the organism of claim 1 under conditions and for a sufficient period of time to produce succinate

wherein optionally the organism is a Crabtree positive, eukaryotic organism, and wherein the organism is cultured in a culture medium comprising glucose.

15. A method of producing formaldehyde, comprising culturing the organism of claim 1 under conditions and for a sufficient period of time to produce formaldehyde, and optionally wherein the formaldehyde is consumed to provide a reducing equivalent or to incorporate into succinate or target product.

16. 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 organism of claim 11 under conditions and for a sufficient period of time to produce the intermediate, and optionally wherein the intermediate is consumed to provide a reducing equivalent or to incorporate into succinate or target product.

17. The non-naturally occurring microbial organism of claim 11, wherein at least one of said exogenous nucleic acids is a heterologous nucleic acid.

18. The non-naturally occurring microbial organism of claim 12, wherein the two exogenous nucleic acids are heterologous nucleic acids, each encoding a formaldehyde assimilation pathway enzyme.

19. The non-naturally occurring microorganism of claim 1, wherein said non-naturally occurring microbial organism is selected from the group consisting of:

(i) 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, and Pseudomonas putida;

or

(ii) Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, and Rhizopus oryzae.