MICROORGANISMS AND METHODS FOR ENHANCING THE AVAILABILITY OF REDUCING EQUIVALENTS IN THE PRESENCE OF METHANOL, AND FOR PRODUCING SUCCINATE RELATED THERETO

• US 20160083752A1
• Filed: 10/21/2013
• Published: 03/24/2016
• 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
• Granted Patent: US 11,535,874 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 (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.

Claims

1. A non-naturally occurring microbial organism (NNOMO) comprising:

(a) a methanol metabolic pathway (MMP), wherein said organism comprises at least one exogenous nucleic acid encoding a MMP enzyme (MMPE) 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); and

(b) a succinate pathway (SucP).

2. The organism of claim 1, wherein said 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) (i) a phosphoenolpyruvate (PEP) carboxylase (ES1A) or a PEP carboxykinase (ES1B);

(ii) a malate dehydrogenase (ES3);

(iii) a fumarase (ES5); and

(iv) a fumarate reductase (ES6);

(b) (i) a pyruvate carboxylase (ES2);

(ii) a ES3;

(iii) a ES5; and

(iv) a ES6;

or

(c) (i) a malic enzyme (ES4);

(ii) a ES5; and

(iii) a ES6;

wherein said organism optionally comprises one, two, three, or four nucleic acids, each encoding a SucPE;

wherein said at least one nucleic acid encoding a succinate enzyme optionally is an exogenous nucleic acid; and

/or

wherein said at least one exogenous nucleic acid encoding a succinate enzyme optionally is a heterologous nucleic acid.

3. The organism of claim 1, wherein:

(a) the MMP comprises;

(i) an EM9, a methylenetetrahydrofolate dehydrogenase (EM3), a methenyltetrahydrofolate cyclohydrolase (EM4) and a formyltetrahydrofolate deformylase (EM5);

(ii) an EM9, an EM3, an EM4 and a formyltetrahydrofolate synthetase (EM6);

(iii) an EM9 and a formaldehyde dehydrogenase (EM11);

(iv) an EM9, a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EM13) and a S-formylglutathione hydrolase (EM14);

or

(v) an EM9, an EM13 and an EM14;

(vi) an EM9, an EM10, an EM3, an EM4 and an EM5;

or

(vii) an EM9, an EM10, an EM3, an EM4 and an EM6;

or

(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 the MMP optionally further comprises a formate dehydrogenase (EM8), a formate hydrogen lyase (EM15) or a hydrogenase (EM16); and

/or

wherein said organism optionally comprises two, three, four, five, six or seven exogenous nucleic acids, each encoding a MMPE.

4. The organism of claim 1, wherein

(a) said organism further comprises 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;

wherein said protein(s) or enzyme(s) optionally 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; and

/or

(b) 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.

wherein said enzyme optionally 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.

5. The organism of claim 1, further comprising a formaldehyde assimilation pathway (FAP), wherein said organism comprises at least one exogenous nucleic acid encoding a 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 biomass, and wherein

(a) said FAP optionally comprises a hexulose-6-phosphate synthase (EF1) and a 6-phospho-3-hexuloisomerase (EF2);

(b) said FAP optionally comprises a dihydroxyacetone synthase (EF3) or a dihydroxyacetone kinase (EF4);

(c) the intermediate optionally 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; and

/or

(d) the organism optionally comprises two exogenous nucleic acids, each encoding a FAPE.

6. The organism of claim 1, wherein

(a) said at least one exogenous nucleic acid is a heterologous nucleic acid;

(b) said organism is in a substantially anaerobic culture medium; and

/or

(c) said microbial organism is a species of bacteria, yeast, or fungus.

7. The NNOMO of claim 1;

wherein the host microbial organism is a recombinant microbial organism having increased succinate production as compared to the wild-type host microbial organism;

wherein optionally;

(a) the succinate production is generated by introduction of one or more gene disruptions of a host microbial organism gene and/or an exogenous nucleic acid;

(b) the host microbial organism is a rumen bacteria,

(c) the host microbial organism includes a gene disruption in one or more genes selected from a lactate dehydrogenase gene (ldhA), a pyruvate formate-lyase gene (pfl), a phosphotransacetylase gene (pta), and an acetate kinase gene (ackA);

(d) the host microbial organism includes a gene disruption in a gene encoding ldhA, pta, and ackA, without disrupting a gene encoding pfl.

(e) the host microbial organism is an Actinobacillus species, a Bacteroides species, a Succinimonas species, a Succinivibrio species, or an Anaerobiospirillum species;

wherein optionally the Actinobacillus species is Actinobacillus succinogenes; and

/or

wherein optionally the Anaerobiospirillum species is Anaerobiospirillum succiniciproducens;

(f) the host microbial organism (a) has gene disruptions in genes encoding IdhA, pfl and a phosphopyruvate carboxylase (ppc);

(b) has gene disruptions in genes encoding a glucose phosphotransferase (ptsG) and a pyruvate kinase (pykA and pykF);

(c) has gene disruptions in genes encoding a succinic semialdehyde dehydrogenase (GabD, sad), and/or (d) has introduction or amplification of a nucleic acid encoding a C4-dicarboxylate transport protein (DctA), which is associated with transport of succinate;

(g) the host microbial organism is a Lumen bacteria, a Corynebacterium species, a Brevibacterium species or an Escherichia species;

wherein optionally the Escherichia species is strain W3110GFA;

(h) the microbial organism having increased succinate production is generated by introducing an exogenous nucleic acid encoding an enzyme or protein that increases production of succinate;

wherein optionally the nucleic acid encoding a malic enzyme B, a fumarate hydratase C, a formate dehydrogenase D, PEP carboxykinase, pyruvate carboxylase, or a formate dehydrogenase E;

(i) the host microbial organism is an organism that can produce succinate using glycerol as a carbon source;

(j) the host microbial organism is an organism that simultaneously use sucrose and glycerol as carbon sources to produce succinate, optionally by weakening a catabolic inhibition mechanism of the glycerol by sucrose;

(k) the host microbial organism is a bacterial strain of the genus Pasteurella, which is optionally catalase- and/or oxidase-positive;

wherein optionally the Pasteurella strain is Pasteurella strain DD1, or a variant thereof; and

/or

wherein optionally the DD1 variant is LU15348 (DD1 with deletion of pfl gene);

LU15050 (DD1 deletion of ldh gene);

or LU15224 (DD1 with deletion of both pfl and ldh genes);

(l) the host microbial organism is a Mannheimia species,

wherein optionally the Mannheimia species is Mannheimia sp. LPK, Mannheimia sp. LPK4, Mannheimia sp. LPK7, Mannheimia sp. LPK (KCTC 10558BP), Mannheimia succiniciproducens MBEL55E (KCTC 0769BP), Mannheimia succiniciproducens PALK (KCTC10973BP), Mannheimia succiniciproducens ALK, or Mannheimia succiniciproducens ALKt);

Mannheimia succiniciproducens, Mannheimia succiniciproducens MBEL55E, or a variant thereof; and

/or

(m) the host microbial organism is a genetically modified yeast strain, comprising modifications to enhance succinate production and/or export and/or select for succinate tolerance of increased intracellular and/or extracellular succinate concentrations;

wherein optionally the genetically modified yeast strain belongs to a genus selected from the group consisting of Issatchenkia, Candida, Pichia, Zygosaccharomyces, Kluyveromyces, Saccharomyces, Debaryomyces, and Saccharomycopsis; and

/or

wherein optionally, the genetically modified yeast strain is a species selected from the group consisting of Issatchenkia orientalis, Candida lambica, Candida sorboxylosa, Candida zemplinina, Candida geochares, Pichia membranifaciens, Zygosaccharomyces kombuchaensis, Candida sorbosivorans, Kluyveromyces marxianus, Candida vanderwaltii, Candida sorbophila, Zygosaccharomyces bisporus, Zygosaccharomyces lentus, Saccharomyces bayanus, Saccharomyces bulderi, Debaryomyces castellii, Candida boidinii, Candida etchellsii, Kluyveromyces lactis, Pichia jadinii, Pichia anomala, Saccharomycopsis crataegensis, and Pichia jadinii;

or the Pichia fermentans/lssatchenkia orientalis clade.

8. 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 said method optionally further comprises separating the BDO from other components in the culture,

wherein the separation optionally 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; and

/or

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

9. A bioderived succinate, or an intermediate thereof, produced according to the method of claim 8;

wherein said bioderived succinate optionally has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source; and

/or

said bioderived succinate optionally has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.

10. A culture medium comprising the bioderived succinate of claim 9;

wherein said bioderived succinate optionally has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source;

said bioderived succinate has an Fm value of at least 80%, at least 85%, at least 90%, at least 95% or at least 98%; and

/or

wherein said culture medium is optionally separated from the NNOMO having the SucP.

11. A composition comprising said bioderived succinate of a claim 9, and a compound other than said bioderived succinate;

wherein said compound other than said bioderived succinate optionally is a trace amount of a cellular portion of a NNOMO having a SucP.

12. A biobased product comprising said bioderived succinate, or an intermediate thereof, of claim 9.

13. The biobased product of claim 12, wherein said product is selected from the group consisting of a butanediol, tetrahydrofuran, pyrrolidone, solvent, paint, deicer, plastic, fuel additive, fabric, carpet, pigment, detergent, metal plating, polymer, polybutylene succinate polymer, biodegradable plastic, flexible packaging, agricultural film, compostable bag;

a combination of polybutylene succinate with polymers such as polypropylene (PP), polystyrene (PS) and polycarbonate (PC), and with plastics such as polylactic acid, polyhydroxyalkanoate, and poly-3-hydroxy butyrateco-valerate; and

a combination of polybutylene succinate with fibers or fillers, automotive interiors, nonwovens, construction materials, and consumer goods.

14. The biobased product of claim 12, wherein said biobased product comprises

(a) at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% bioderived succinate;

and/or

(b) a portion of said bioderived succinate as a repeating unit.

15. A molded product obtained by molding the biobased product of claim 12.

16. A process for producing the biobased product of claim 12, comprising chemically reacting said bioderived succinate with itself or another compound in a reaction that produces said biobased product.

17. A polymer comprising or obtained by converting the bioderived succinate of claim 9.

18. A method for producing a polymer, comprising chemically of enzymatically converting the bioderived succinate of claim 9 to the polymer.

19. A composition comprising the bioderived succinate of claim 9, or a cell lysate or culture supernatant thereof.

20. 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.

21. 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 5 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.

22. The method of claim 20, wherein the 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.