Compositions and methods for succinate production

Patent metadata

Field	Value
Publication number	US-9605285-B2
Application number	US-201213981517-A
Country	US
Kind code	B2
Filing date	Jan 25, 2012
Priority date	Jan 25, 2011
Publication date	Mar 28, 2017
Grant date	Mar 28, 2017

Abstract

The present application provides genetically modified yeast cell comprising an active succinate fermentation pathway, as well as methods of using these cells to produce succinate.

First claim

What is claimed is:

1. A genetically modified yeast cell having an active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate, wherein the active succinate fermentation pathway comprises an exogenous malate dehydrogenase gene encoding a polypeptide comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:172.

2. The genetically modified yeast cell of claim 1 , wherein the active succinate fermentation pathway includes the reactions:
(a) pyruvate to oxaloacetate;
(b) oxaloacetate to malate;
(c) malate to fumarate; and
(d) fumarate to succinate.

3. The genetically modified yeast cell of claim 2 , wherein the active succinate fermentation pathway further includes:
(e) export of succinate from inside the cell to the extracellular environment.

4. The genetically modified yeast cell of claim 3 , wherein the cell comprises one or more exogenous genes that encode an enzyme that catalyzes any of reactions (a) through (e).

5. The genetically modified yeast cell of claim 4 , wherein the cell comprises one or more copies of one or more exogenous genes selected from the group consisting of pyruvate carboxylase, fumarate reductase, and succinate exporter genes.

6. The genetically modified yeast cell of claim 5 , wherein said exogenous pyruvate carboxylase gene is derived from a fungal pyruvate carboxylase source gene and encodes a polypeptide with at least 95% sequence identity to a polypeptide encoded by the fungal pyruvate carboxylase source gene, wherein the fungal pyruvate carboxylase source gene is not derived from R. oryzae.

7. The genetically modified yeast cell of claim 5 , wherein said exogenous fumarase gene is derived from a yeast fumarase source gene and encodes a polypeptide with at least 95% sequence identity to a polypeptide encoded by the yeast fumarase source gene.

8. The genetically modified yeast cell of claim 7 , wherein said exogenous fumarase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

9. The genetically modified yeast cell of claim 5 , wherein said exogenous fumarate reductase gene is derived from a protozoan fumarate reductase source gene and encodes a polypeptide with at least 95% sequence identity to a polypeptide encoded by the protozoan fumarate reductase source gene.

10. The genetically modified yeast cell of claim 9 , wherein said exogenous fumarate reductase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, or SEQ ID NO:180.

11. The genetically modified yeast cell of claim 2 , wherein the active succinate fermentation pathway comprises the reaction phosphoenolpyruvate to oxaloacetate in addition to reaction (a).

12. The genetically modified yeast cell of claim 11 , wherein the cell comprises one or more copies of one or more exogenous phosphoenolpyruvate carboxylase genes.

13. The genetically modified yeast cell of claim 2 , wherein the cell comprises a deletion or disruption of an endogenous malic enzyme gene.

14. The genetically modified yeast cell of claim 2 , wherein the cell comprises a deletion or disruption of an endogenous pyruvate decarboxylase gene.

15. The genetically modified yeast cell of claim 1 , wherein the yeast cell is from the Pichia fermentans/Issatchenkia orientalis clade.

16. A method of producing succinate comprising culturing the genetically modified yeast cell of claim 1 in the presence of at least one carbon source and isolating succinate from the culture.

17. The A-genetically modified yeast cell of claim 1 , wherein the yeast cell comprises the species Issatchenkia orientalis.

18. The genetically modified yeast cell of 15 , wherein the yeast cell is succinate resistant.

19. The genetically modified yeast cell of claim 15 , wherein the genetically modified yeast cell comprises one or more copies of an exogenous pyruvate carboxylase gene derived from a yeast pyruvate carboxylase source gene and encodes a polypeptide with at least 95% sequence identity to a polypeptide encoded by the yeast pyruvate carboxylase source gene.

20. The genetically modified yeast cell of claim 19 , wherein said exogenous pyruvate carboxylase gene encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

Assignees

• Finley Kenneth R
• Huryta Jeanette M
• Mastel Beth M
• Mcmullin Thomas W
• Poynter Gregory M
• Rush Brian J
• Watts Kevin T
• Fosmer Arlene M
• Mcintosh Jr Vernon L
• Brady Keith M
• Cargill Inc

Inventors

• Finley Kenneth R
• Huryta Jeanette M
• Mastel Beth M
• Mcmullin Thomas W
• Poynter Gregory M
• Rush Brian J
• Watts Kevin T
• Fosmer Arlene M
• Mcintosh Jr Vernon L
• Brady Keith M

Classifications

• C12N15/815 Primary

  for yeasts other than Saccharomyces · CPC title

• C12N9/88

  Lyases (4.) · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C12Y103/01006

  Fumarate reductase (NADH) (1.3.1.6) · CPC title

• C12Y402/01002

  Fumarate hydratase (4.2.1.2) · CPC title