Production of malonyl-CoA derived products via anaerobic pathways

Patent metadata

Field	Value
Publication number	US-10138504-B2
Application number	US-201113814616-A
Country	US
Kind code	B2
Filing date	Aug 5, 2011
Priority date	Aug 6, 2010
Publication date	Nov 27, 2018
Grant date	Nov 27, 2018

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 malonyl-CoA derived product,
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 heterologous pyruvate formate lyase.

2. The recombinant yeast microorganism of claim 1 , wherein the conversion of a carbohydrate source to a malonyl-CoA derived product is under anaerobic or microaerophilic conditions.

3. The recombinant yeast microorganism of claim 1 , wherein at least one of said engineered metabolic pathways produces net ATP.

4. The recombinant yeast microorganism of claim 1 , wherein said product is a polyketide or an organic acid.

5. The recombinant yeast microorganism of claim 4 , wherein said polyketide is an antibiotic, antitumor, antifungal, or immunosuppressive.

6. The recombinant yeast microorganism of claim 4 , wherein said organic acid is 3-hydroxypropionic acid.

7. The recombinant yeast microorganism of claim 6 , wherein one of said engineered metabolic pathways comprises the following steps: (a) conversion of malonyl-CoA to malonate semialdehyde and coA; and (b) conversion of malonate semialdehyde to 3-hydroxypropanoate.

8. The recombinant yeast microorganism of claim 7 , wherein said malonyl-CoA is converted to malonate semialdehyde and coA by a malonyl-CoA reductase.

9. The recombinant yeast microorganism of claim 8 , wherein said malonyl-CoA reductase is encoded by a polynucleotide from a C. aurantiacus.

10. The recombinant yeast microorganism of claim 7 , wherein said malonate semialdehyde is converted to 3-hydroxypropanoate by a 3-hydroxypropionate dehydrogenase.

11. The recombinant yeast microorganism of claim 6 , wherein said malonyl-CoA is converted to 3-hydroxypropanoate by a bifunctional dehydrogenase.

12. The recombinant yeast microorganism of claim 4 , wherein said organic acid is adipic acid.

13. The recombinant yeast microorganism of claim 1 , wherein said carbohydrate source is a lignocellulosic material.

14. The recombinant yeast microorganism of claim 1 , wherein one of said engineered metabolic pathways further comprises the conversion of pyruvate and CoA-SH into acetyl-CoA and CO.sub.2 and NAD(P)H.

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

16. The recombinant yeast microorganism of claim 1 , wherein the one or more engineered metabolic pathways further comprises downregulation or deletion of native enzymes selected from the group consisting of: (a) a pyruvate carboxykinase; (b) a hydrogenase; (c) a lactate dehydrogenase; (d) a phosphotransacetylase; (e) an acetate kinase; (f) an acetaldehyde dehydrogenase; (g) an alcohol dehydrogenase; (h) an enzyme involved in degradation of fatty acids and their derivatives; and (i) combinations of (a) (i).

17. The recombinant yeast microorganism of claim 1 , wherein the one or more engineered metabolic pathways further comprises downregulation or deletion of native enzymes selected from the group consisting of: (a) a lactate dehydrogenase; (b) a phosphate acetyltransferase; (c) an acetaldehyde dehydrogenase/alcohol dehydrogenase; (d) a pyruvate carboxykinase; (e) a malate dehydrogenase; (f) a PEP-protein phosphotransferase of PTS system; and (g) combinations of (a)-(f).

18. A process for converting a carbohydrate source to a malonyl-CoA derived product comprising contacting the carbohydrate source with a recombinant yeast microorganism according to claim 1 .

19. The process of claim 18 , wherein said carbohydrate source comprises lignocellulosic biomass.

20. An engineered metabolic pathway for producing a malonyl-CoA derived product in a consolidated bioprocessing (CBP) organism according to claim 1 .

21. The recombinant yeast microorganism of claim 1 , wherein the conversion of a carbohydrate source to a malonyl-CoA derived product is redox neutral.

22. The recombinant yeast microorganism of claim 1 , wherein one of said engineered metabolic pathways further comprises one or more formate dehydrogenases capable of converting formate to CO.sub.2 and NAD(P)H.

23. The recombinant yeast microorganism of claim 22 , wherein said formate dehydrogenase is encoded by an S. cerevisiae NAD+FDH1, a B. stabilis NADP+FDH, or both.

24. The recombinant yeast microorganism of claim 1 , wherein one of said engineered metabolic pathways further comprises an enzyme encoding a palmitoyl-acyl carrier protein thioesterase (FatB1).

25. The recombinant yeast microorganism of claim 24 , wherein said palmitoyl-acyl carrier protein thioesterase (FatB1) is from A. thaliana.

26. The recombinant yeast microorganism of claim 1 , wherein the one or more engineered metabolic pathways further comprises downregulation or deletion of native enzymes selected from a glycerol-3-phosphate dehydrogenase 1 (GPD1), a glycerol-3-phosphate dehydrogenase 2 (GPD2), a formate dehydrogenase 1 (FDH1), a formate dehydrogenase 2 (FDH2), or a combination thereof.

27. The recombinant yeast microorganism of claim 26 , wherein said native enzyme is downregulated or deleted by insertion of a heterologous enzyme at the locus of the native enzyme.

28. The recombinant yeast microorganism of claim 1 , wherein said heterologous pyruvate formate lyase enzyme is a pyruvate formate lyase A (PFLA), a pyruvate formate lyase B (PFLB), or a combination thereof.

29. The recombinant yeast microorganism of claim 1 , wherein said pyruvate decarboxylase is a pyruvate decarboxylase 5 (PDC5), a pyruvate decarboxylase 6 (PDC6), a pyruvate decarboxylase 1 (PDC1), or a combination thereof.

30. 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 utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenit, Debaryomyces polymorphus, Schizosaccharomyces pombe, Candida albicans , and Schwanniomyces occidentalis.

Assignees

• Sillers William Ryan
• Tripathi Shital A
• Shaw Iv Arthur J
• Argyros Aaron
• Hogsett David A
• Lallemand Hungary Liquidity Man Llc

Inventors

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

Classifications

• C12N9/88

  Lyases (4.) · CPC title

• C12N9/1018

  Carboxy- and carbamoyl transferases (2.1.3) · CPC title

• C12P7/6409

  Fatty acids · CPC title

• G01N2333/91034

  Carboxyl- and carbamoyl transferases (2.1.3) · 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