Microorganisms and methods for improving product yields on methanol using acetyl-CoA synthesis

Patent metadata

Field	Value
Publication number	US-10808262-B2
Application number	US-201415039221-A
Country	US
Kind code	B2
Filing date	Nov 25, 2014
Priority date	Dec 3, 2013
Publication date	Oct 20, 2020
Grant date	Oct 20, 2020

Abstract

The invention provides non-naturally occurring microbial organisms containing enzymatic pathways and/or metabolic modifications for enhancing carbon flux through acetyl-CoA. In some embodiments, the microbial organisms having such pathways also include pathways for generating reducing equivalents, formaldehyde fixation and/or formate assimilation. The enhanced carbon flux through acetyl-CoA, in combination with pathways for generating reducing equivalents, formaldehyde fixation and/or formate assimilation can, in some embodiments, be used for production of a bioderived compound. Accordingly, in some embodiments, the microbial organisms of the invention can include a pathway capable of producing a bioderived compound of the invention. The invention still further provides a bioderived compound produced by a microbial organism of the invention, culture medium having the bioderived compound of the invention, compositions having the bioderived compound of the invention, a biobased product comprising the bioderived compound of the invention, and a process for producing a bioderived compound of the invention.

First claim

What is claimed is:

1. A non-naturally occurring microbial organism having a methanol metabolic pathway and an acetyl-CoA pathway, wherein said methanol metabolic pathway comprises 2A or 2J, wherein 2A is a methanol methyltransferase, wherein 2J is a methanol dehydrogenase,
wherein said acetyl-CoA pathway comprises a pathway selected from:
(1) 1T and 1V; (2) 1T, 1W, and 1X; (3) 1U and 1V; (4) 1U, 1W, and 1X;
wherein 1T is a fructose-6-phosphate phosphoketolase, wherein 1U is a xylulose-5-phosphate phosphoketolase, wherein 1V is a phosphotransacetylase, wherein 1W is an acetate kinase, wherein 1X is an acetyl-CoA transferase, an acetyl-CoA synthetase, or an acetyl-CoA ligase,
wherein an enzyme of the methanol metabolic pathway or the acetyl-CoA pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said microbial organism is a species of bacteria or yeast.

2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises a formaldehyde fixation pathway, wherein said formaldehyde fixation pathway comprises:
(1) 1D and 1Z; (2) 1D; or (3) 1B and 1C,
wherein 1B is a 3-hexulose-6-phosphate synthase, wherein 1C is a 6-phospho-3-hexuloisomerase, wherein 1D is a dihydroxyacetone synthase, wherein 1Z is a fructose-6-phosphate aldolase.

3. The non-naturally occurring microbial organism of claim 2 , wherein an enzyme of the formaldehyde fixation pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to enhance carbon flux through acetyl-CoA.

4. The non-naturally occurring microbial organism of claim 1 , wherein said methanol metabolic pathway comprises a pathway selected from:
(1) 2A and 2B; (2) 2A, 2B and 2C; (3) 2J, 2K and 2C; (4) 2J, 2M, and 2N; (5) 2J and 2L; (6) 2J, 2L, and 2G; (7) 2J, 2L, and 2I; (8) 2A, 2B, 2C, 2D, and 2E; (9) 2A, 2B, 2C, 2D, and 2F; (10) 2J, 2K, 2C, 2D, and 2E; (11) 2J, 2K, 2C, 2D, and 2F; (12) 2J, 2M, 2N, and 2O; (13) 2A, 2B, 2C, 2D, 2E, and 2G; (14) 2A, 2B, 2C, 2D, 2F, and 2G; (15) 2J, 2K, 2C, 2D, 2E, and 2G; (16) 2J, 2K, 2C, 2D, 2F, and 2G; (17) 2J, 2M, 2N, 2O, and 2G; (18) 2A, 2B, 2C, 2D, 2E, and 2I; (19) 2A, 2B, 2C, 2D, 2F, and 2I; (20) 2J, 2K, 2C, 2D, 2E, and 2I; (21) 2J, 2K, 2C, 2D, 2F, and 2I; and (22) 2J, 2M, 2N, 2O, and 2I,
wherein 2A is a methanol methyltransferase, wherein 2B is a methylenetetrahydrofolate reductase, wherein 2C is a methylenetetrahydrofolate dehydrogenase, wherein 2D is a methenyltetrahydrofolate cyclohydrolase, wherein 2E is a formyltetrahydrofolate deformylase, wherein 2F is a formyltetrahydrofolate synthetase, wherein 2G is a formate hydrogen lyase, wherein 2I is a formate dehydrogenase, wherein 2J is a methanol dehydrogenase, wherein 2K is a formaldehyde activating enzyme or spontaneous, wherein 2L is a formaldehyde dehydrogenase, wherein 2M is a S-(hydroxymethyl)glutathione synthase or spontaneous, wherein 2N is a glutathione-dependent formaldehyde dehydrogenase, wherein 2O is a S-formylglutathione hydrolase.

5. The non-naturally occurring microbial organism of claim 4 , wherein said microbial organism comprises:
(a) one, two, three, four, five, or six exogenous nucleic acids each encoding a methanol metabolic pathway enzyme; or
(b) exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from (1)-(22).

6. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises:
(a) one, two, or three exogenous nucleic acids each encoding an acetyl-CoA pathway enzyme; or
(b) exogenous nucleic acids encoding each of the enzymes of at least one of the pathways selected from the group consisting of the pathway (1) to pathway (4).

7. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism further comprises a formate assimilation pathway, wherein said formate assimilation pathway comprises a pathway selected from:
(1) 1E; (2) 1F, and 1G; (3) 1H, 1I, 1J, and 1K; (4) 1H, 1I, 1J, 1L, 1M, and 1N; (5) 1E, 1H, 1I, 1J, 1L, 1M, and 1N; (6) 1F, 1G, 1H, 1I, 1J, 1L, 1M, and 1N; (7) 1K, 1H, 1I, 1J, 1L, 1M, and 1N; and (8) 1H, 1I, 1J, 1O, and 1P,
wherein 1E is a formate reductase, IF is a formate ligase, a formate transferase, or a formate synthetase, wherein 1G is a formyl-CoA reductase, wherein 1H is a formyltetrahydrofolate synthetase, wherein 1I is a methenyltetrahydrofolate cyclohydrolase, wherein 1J is a methylenetetrahydrofolate dehydrogenase, wherein 1K is a formaldehyde-forming enzyme or spontaneous, wherein 1L is a glycine cleavage system, wherein 1M is a serine hydroxymethyltransferase, wherein 1N is a serine deaminase, wherein 1O is a methylenetetrahydrofolate reductase, wherein 1P is an acetyl-CoA synthase.

8. The non-naturally occurring microbial organism of claim 7 , wherein an enzyme of the formate assimilation pathway is encoded by at least one exogenous nucleic acid and is expressed in a sufficient amount to enhance carbon flux through acetyl-CoA.

9. The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism further comprises a pathway capable of producing a bioderived compound.

10. The non-naturally occurring microbial organism of claim 9 , wherein said bioderived compound is an alcohol, a glycol, an organic acid, an alkene, a diene, an organic amine, an organic aldehyde, a vitamin, a nutraceutical or a pharmaceutical.

11. The non-naturally occurring microbial organism of claim 10 , wherein said bioderived compounds is selected from the group consisting of:
(i) 1,4-butanediol or an intermediate thereto, wherein said intermediate is optionally 4-hydroxybutanoic acid (4-HB);
(ii) butadiene (1,3-butadiene) or an intermediate thereto, wherein said intermediate is optionally 1,4-butanediol, 1,3-butanediol, 2,3-butanediol, crotyl alcohol, 3-buten-2-ol (methyl vinyl carbinol) or 3-buten-1-ol;
(iii) 1,3-butanediol or an intermediate thereto, wherein said intermediate is optionally 3-hydroxybutyrate (3-HB), 2,4-pentadienoate, crotyl alcohol or 3-buten-1-ol;
(iv) adipate, 6-aminocaproic acid, caprolactam, hexamethylenediamine, levulinic acid or an intermediate thereto, wherein said intermediate is optionally adipyl-CoA or 4-aminobutyryl-CoA;
(v) methacrylic acid or an ester thereof, 3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or an intermediate thereto, wherein said ester is optionally methyl methacrylate or poly(methyl methacrylate);
(vi) 1,2-propanediol (propylene glycol), 1,3-propanediol, glycerol, ethylene glycol, diethylene glycol, triethylene glycol, dipropylene glycol, tripropylene glycol, neopentyl glycol, bisphenol A or an intermediate thereto;
(vii) succinic acid or an intermediate thereto; and
(viii) a fatty alcohol, a fatty aldehyde or a fatty acid comprising C4 to C27 carbon atoms, C8 to C18 carbon atoms, C12 to C18 carbon atoms, or C12 to C14 carbon atoms, wherein said fatty alcohol is optionally dodecanol (C12; lauryl alcohol), tridecyl alcohol (C13; 1-tridecanol, tridecanol, isotridecanol), myristyl alcohol (C14; 1-tetradecanol), pentadecyl alcohol (C15; 1-pentadecanol, pentadecanol), cetyl alcohol (C16; 1-hexadecanol), heptadecyl alcohol (C17; 1-n-heptadecanol, heptadecanol) and stearyl alcohol (C18; 1-octadecanol) or palmitoleyl alcohol (C16 unsaturated; cis-9-hexadecen-1-ol).

12. The non-naturally occurring microbial organism of claim 1 , wherein said acetyl-CoA pathway comprises 1T and 1V.

13. The non-naturally occurring microbial organism of claim 12 , wherein said formaldehyde fixation pathway comprises 1D and 1Z.

14. The

Assignees

• Genomatica Inc

Inventors

• Osterhout Robin E
• Burgard Anthony P
• Pharkya Priti
• Andrae Stefan

Classifications

• C12N1/14 Primary

  Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor · CPC title

• C12P7/44

  Polycarboxylic acids · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12P5/026 Primary

  Unsaturated compounds, i.e. alkenes, alkynes or allenes · CPC title

• C12P7/18

  polyhydric · CPC title