Producing Itaconic Acid in Yeast Using Glycerol as the Substrate

• US 20110053232A1
• Filed: 08/25/2009
• Published: 03/03/2011
• Est. Priority Date: 08/25/2009
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Industrial Technology Research Institute
• Inventors: Teng, Kelly, Wang, Jia-Hung, Tsai, Shu-Hsien
• Granted Patent: US 8,192,965 B2
• Time in Patent Office: Days
• US Class Current: 435/142
• CPC Class Codes: C12N 1/32 Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/44 Polycarboxylic acids

Abstract

Method for producing itaconic acid in yeast cells using glycerol as the substrate. The yeast cells express cis-aconitic acid decarboxylase and optionally, citrate synthase and/or aconitase at high levels.

Claims

1. A method of producing itaconic acid in yeast, comprising

providing a genetically modified yeast host cell, the host cell containing a first expression cassette including a first yeast promoter operably linked to a nucleotide sequence encoding a cis-aconitic acid decarboxylase,

culturing the yeast host cell in a medium containing glycerol at a concentration of 5-700 g/L, wherein the yeast host cell converts glycerol to itaconic acid, and collecting the medium for isolation of itaconic acid.

2. The method of claim 1, wherein the genetically modified yeast host cell further contains a second expression cassette including a second yeast promoter operably linked to a nucleotide sequence encoding a citrate synthase or an aconitase.

3. The method of claim 2, wherein the nucleotide sequence in the second expression cassette encodes a citrate synthase and the genetically modified yeast host cell further contains a third expression cassette composed of a third yeast promoter operably linked to a nucleotide sequence encoding an aconitase.

4. The method of claim 1, wherein the glycerol is the sole substrate for producing itaconic acid.

5. The method of claim 4, wherein the medium contains glycerol at a concentration of 5-250 g/L.

6. The method of claim 1, wherein the first yeast promoter is selected from the group consisting of hp4d, pTEF, pRPS7, and pG3P.

7. The method of claim 2, wherein the first yeast promoter is pTEF and the second promoter is pRPS7.

8. The method of claim 3, wherein the first yeast promoter is pTEF, the second yeast promoter is pRPS7, and the third yeast promoter is pG3P.

9. The method of claim 1, wherein the first expression cassette further includes a leader sequence upstream to the nucleotide sequence encoding the cis-aconitate dearbosylase.

10. The method of claim 9, wherein the leader sequence encodes the amino acid sequence of MSAILSTTSKSFLSRGSTRQCQNMQKALFALLNARHYS or MKLATAFTILTAVLA.

11. The method of claim 1, wherein the yeast host cell is a Yarrowia lipolytica cell.

12. The method of claim 11, wherein the yeast promoter is selected from the group consisting of hp4d, pTEF, pRPS7, and pG3P.

13. The method of claim 11, wherein the Yarrowia lipolytica cell further contains a second expression cassette including a second yeast promoter operably linked to a nucleotide sequence encoding a citrate synthase or an aconitase.

14. The method of claim 13, wherein the first yeast promoter is pTEF and the second promoter is pRPS7.

15. The method of claim 11, wherein the nucleotide sequence in the second expression cassette encodes a citrate synthase and the Yarrowia lipolytica cell further contains a third expression cassette composed of a third yeast promoter and a nucleotide sequence encoding an aconitase.

16. The method of claim 15, wherein the first yeast promoter is pTEF, the second yeast promoter is pRPS7, and the third yeast promoter is pG3P.

17. The method of claim 11, wherein the glycerol is the sole substrate for producing itaconic acid.

18. The method of claim 17, wherein the medium contains glycerol at a concentration of 5-250 g/L.

19. The method of claim 11, wherein the first expression cassette further includes a leader sequence upstream to the nucleotide sequence encoding the cis-aconitate dearbosylase.

20. The method of claim 19, wherein the leader sequence encodes the amino acid sequence of MSAILSTTSKSFLSRGSTRQCQNMQKALFALLNARHYS or MKLATAFTILTAVLA.