Production of malonyl-CoA derived products via anaerobic pathways

Patent metadata

Field	Value
Publication number	US-11162125-B2
Application number	US-201816166274-A
Country	US
Kind code	B2
Filing date	Oct 22, 2018
Priority date	Aug 6, 2010
Publication date	Nov 2, 2021
Grant date	Nov 2, 2021

Abstract

The present invention provides for novel metabolic pathways to convert biomass and other carbohydrate sources to malonyl-CoA derived products, such as hydrocarbons and other bioproducts, under anaerobic conditions and with the net production of ATP. More specifically, the invention provides for a recombinant microorganism comprising one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to achieve conversion of a carbohydrate source to, e.g., long-chain hydrocarbons and hydrocarbon derivatives, wherein the one or more native and/or heterologous enzymes is activated, upregulated, downregulated, or deleted. The invention also provides for processes to convert biomass to malonyl-CoA derived products which comprise contacting a carbohydrate source with a recombinant microorganism of the invention.

First claim

What is claimed is:

1. A recombinant yeast microorganism comprising one or more engineered metabolic pathways to convert a carbohydrate source to a hydrocarbon, wherein the one or more engineered metabolic pathways comprises
(a) the conversion of phosphoenolpyruvate to oxaloacetate by a phosphoenolpyruvate carboxykinase and
(b) the conversion of oxaloacetate and acetyl-CoA to malonyl-CoA and pyruvate by a heterologous transcarboxylase Enzyme Commission Number 2.1.3.1;
wherein the one or more engineered metabolic pathways further comprises downregulation or deletion of native pyruvate decarboxylase, and
wherein the one or more engineered metabolic pathway further comprises a heterologous pyruvate formate lyase, pyruvate dehydrogenase, pyruvate:ferredoxin oxidoreductase or pyruvate:NADP+oxidoreductase.

2. The recombinant microorganism of claim 1 , wherein the conversion of a carbohydrate source to a hydrocarbon is under anaerobic or microaerophilic conditions.

3. The recombinant microorganism of claim 1 , wherein said hydrocarbon is selected from the group consisting of:
(a) an alkane;
(b) an alkene;
(c) an alkyne;
(d) a hydrocarbon derivative; and
(e) combinations of (a)-(d).

4. The recombinant microorganism of claim 3 , wherein said hydrocarbon derivative is selected from the group consisting of:
(a) an aldehyde;
(b) an alcohol;
(c) an ester;
(d) a fatty acid;
(e) an unsaturated fatty acid;
(f) a branched-chain fatty acid;
(g) a branched methoxy fatty acid;
(h) a multi-methyl branched acid;
(i) a divinyl-ether fatty acid;
(j) a w-phenylalkanoic acid;
(k) a dicarboxylic acid; and
(l) combinations of (a)-(k).

5. The recombinant microorganism of claim 4 , wherein said hydrocarbon or hydrocarbon derivative comprises a carbon backbone of C4-C40.

6. The recombinant microorganism of claim 5 , wherein said hydrocarbon or hydrocarbon derivative comprises a carbon backbone selected from the group consisting of:
(a) C6-C36;
(b) C8-C32;
(C) C10-C28;
(d) C12-C24;
(e) C14-C22;
(f) C16-C20; and
(g) combinations of (a)-(f).

7. The recombinant microorganism of claim 5 , wherein said hydrocarbon or hydrocarbon derivative comprises a carbon backbone selected from the group consisting of: (a) C12; (b) C14; (c) C16; (d) C18; (e) C20; (f) C22; (g) C24; and (h) combinations of (a)-(g).

8. The recombinant microorganism of claim 7 , wherein one of said engineered metabolic pathways further comprises the conversion of pyruvate and CoA-SH into acetyl-CoA and CO2 and NAD(P)H.

9. The recombinant microorganism of claim 1 , wherein one of said engineered metabolic pathways further comprises at least one of the following steps:
(a) conversion of malonyl-CoA to malonyl-ACP;
(b) conversion of malonyl-ACP to an acyl-ACP;
(c) conversion of an acyln-ACP to a β-keto ester.n+2-ACP;
(d) conversion of a β-keto estern+2-ACP to a β-D-hydroxyacyln+2-ACP;
(e) conversion of a β-D-hydroxyacyln+2-ACP to a trans-2-unsaturated acyl.n+2-ACP; or
(f) conversion of a trans-2-unsaturated acyln+2-ACP to an acyln+2-ACP.

10. The recombinant microorganism of claim 1 , wherein said phosphoenolpyruvate carboxykinase is encoded by a polynucleotide from a Thermoanaerobacter species, E. coli, S. cerevisiae or C. thermocellum.

11. The recombinant microorganism of claim 1 , wherein said transcarboxylase is encoded by a polynucleotide from a Thermoanaerobacter species, P. freudenreichii, P. acnes, C. thermocellum, C. bescii, C. cellulolyticum, C. kroppenstedtii, B. fragilis, V. parvula V. gazogenes, P. thermopropionicum, Candidatus, Cloacamonas acidaminovorans, G. bemidjiensis or D. propionicus.

12. The recombinant microorganism of claim 1 , wherein said downregulated or deleted native enzyme is selected from the group consisting of:
(a) a pyruvate kinase;
(b) a hydrogenase;
(c) a lactate dehydrogenase;
(d) a phosphotransacetylase;
(e) an acetate kinase;
(f) an acetaldehyde dehydrogenase;
(g) an alcohol dehydrogenase;
(h) a pyruvate formate lyase;
(i) a pyruvate decarboxylase;
(j) an enzyme involved in degradation of fatty acids and their derivatives;
and (k) combinations of (a)-(j).

13. The recombinant microorganism of claim 1 , wherein said downregulated or deleted native enzyme is selected from the group consisting of:
(a) a lactate dehydrogenase;
(b) a phosphate acetyltransferase;
(c) an acetaldehyde dehydrogenase/alcohol dehydrogenase;
(d) a pyruvate kinase;
(e) a malate dehydrogenase;
(f) a PEP-protein phosphotransferase of PTS system; and
(g) combinations of (a)-(f).

14. A process for converting a carbohydrate source to a hydrocarbon comprising contacting the carbohydrate source with a recombinant microorganism B according to claim 1 .

15. An engineered metabolic pathway for producing a 1 hydrocarbon in a consolidated bioprocessing (CBP) organism according to claim 1 .

16. The recombinant microorganism of claim 1 , wherein the conversion of a carbohydrate source to a hydrocarbon is redox neutral.

17. The recombinant microorganism of claim 1 , wherein one of said engineered metabolic pathways further comprises the conversion of acyln+2-ACP to a fatty acid.

18. The recombinant microorganism of claim 17 , wherein said acyln+2-ACP is converted to a fatty acid by a chain termination enzyme.

19. The recombinant microorganism of claim 18 , wherein said chain termination enzyme is selected from an E. coli codon optimized C12 acyl-ACP thioesterase, an E. coli codon optimized C16 acyl-ACP thioesterase, an acyl-ACP reductase and an acyl-ACP reductase homolog.

20. The recombinant yeast microorganism of claim 1 , wherein said yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe, Candida albicans , and Schwanniomyces occidentalis.

Assignees

• Lallemand Hungary Liquidity Man Llc

Inventors

• Sillers William Ryan
• Tripathi Shital A
• Shaw Iv Arthur J
• Argyros Aaron
• Hogsett David A

Classifications

• C12N1/22 Primary

  Processes using, or culture media containing, cellulose or hydrolysates thereof · CPC title

• C12P17/182 Primary

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title

• C12N9/1018

  Carboxy- and carbamoyl transferases (2.1.3) · CPC title

• C12P7/6409

  Fatty acids · CPC title

• C12N9/88

  Lyases (4.) · CPC title