Genetically Modified Microorganisms for Producing Itaconic Acid with High Yields

• US 20100285546A1
• Filed: 05/11/2009
• Published: 11/11/2010
• Est. Priority Date: 05/11/2009
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Industrial Technology Research Institute
• Inventors: Liao, James C., Chang, Pei-Ching
• Granted Patent: US 8,143,036 B2
• Time in Patent Office: Days
• US Class Current: 435/145
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/46 Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid

Abstract

Genetically modified microorganisms that produce itaconic acid at high yields and uses thereof.

Claims

1. A genetically modified microorganism, comprising

a first exogenous nucleotide sequence encoding a cis-aconitic acid decarboxylase (CAD), the first exogenous nucleotide sequence being operably linked to a promoter;

and

a mutated endogenous icd gene encoding isocitrate dehydrogenase, the mutated endogenous icd gene expressing a lower level of isocitrate dehydrogenase relative to its wild-type counterpart.

2. The genetically modified microorganism of claim 1, wherein the microorganism is selected from the group consisting of Aspergillus, Citrobacter, Corynebacterium, Dekkera, Enterobacter, Enterococcus, Escherichia, Erwinia, Klebsiella, Kluyveromyces, Lactobacillus, Lactococcus, Morganella, Pantoea, Pectobacterium, Penicillium, Pichia, Proteus, Pseudomonas, Pseudozyma, Rhodotorula, Salmonella, Serratia, Shigella, Saccharomyces, Ustilago, and Yarrowia.

3. The genetically modified microorganism of claim 2, wherein the microorganism is Aspergillus niger, Aspergillus terreus, Escherichia coli, Pseudozyma Antarctica, Yarrowia lipotica, and Saccharomyces cerevisiae.

4. The genetically modified microorganism of claim 1, further comprising a second exogenous nucleotide sequence encoding a phosphoenolpyruvate carboxylase, a citrate synthase, a 2-methylcitrate synthase, a citrate lyase, an aconitase, or a 2-methylcitrate dehydratase, the second exogenous nucleotide sequence being operably linked to a promoter.

5. The genetically modified microorganism of claim 4, wherein the microorganism is Escherichia coli;

the second exogenous nucleotide sequence encodes the phosphoenolpyruvate carboxylase, the citrate synthase, or the aconitase, and the aconitase is an aconitase A or an aconitase B.

6. The genetically modified microorganism of claim 4, wherein the second exogenous nucleotide sequence encodes the phosphoenolpyruvate carboxylase and the microorganism further contains a third exogenous nucleotide sequence encoding a citrate synthase, a 2-methylcitrate synthase, a citrate lyase, an aconitase, or a 2-methylcitrate dehydratase, the third exogenous nucleotide sequence being operably linked to a promoter.

7. The genetically modified microorganism of claim 6, wherein the microorganism is Escherichia coli;

the third exogenous nucleotide sequence encodes the citrate synthase or the aconitase; and

the aconitase is an aconitase A or an aconitase B.

8. The genetically modified microorganism of claim 4, wherein the second exogenous nucleotide sequence encodes the citrate synthase, the 2-methylcitrate synthase, or the citrate lyase and the microorganism further contains a third exogenous nucleotide sequence encoding an aconitase or a 2-methylcitrate dehydratase, the third exogenous nucleotide sequence being operably linked to a promoter.

9. The genetically modified microorganism of claim 8, wherein the microorganism is Escherichia coli;

the second exogenous nucleotide sequence encodes the citrate synthase;

the third exogenous nucleotide sequence encodes the aconitase; and

the aconitase is an aconitase A or an aconitase B.

10. The genetically modified microorganism of claim 6, wherein the third exogenous nucleotide sequence encodes the citrate synthase, the 2-methylcitrate synthase, or the citrate lyase and the microorganism further contains a fourth exogenous nucleotide sequence encoding an aconitase or a 2-methylcitrate dehydratase, the fourth exogenous nucleotide sequence being operably linked to a promoter.

11. The genetically modified microorganism of claim 1O, wherein the microorganism is Escherichia coli;

the third exogenous nucleotide sequence encodes the citrate synthase;

the fourth exogenous nucleotide sequence encodes the aconitase; and

the aconitase is an aconitase A or an aconitase B.

22. A method for producing itaconic acid in a microorganism, comprising

providing the genetically modified microorganism of claim 1,

cultivating the genetically modified microorganism in a medium to produce itaconic acid, and

collecting the medium for isolation of the itaconic acid thus produced.

23. The method of claim 22, wherein the medium contains glucose at a concentration of 5-80 g/L.

24. The method of claim 23, wherein the glucose concentration is 10-40 g/L.

25. The method of claim 22, wherein the genetically modified microorganism is permeabilized and the medium contains citrate at a concentration of 5-80 g/L.

26. The method of claim 25, wherein the citrate concentration is 10-40 g/L.

12. A genetically modified microorganism, comprising

a first exogenous nucleotide sequence encoding a cis-aconitic acid decarboxylase and

a second exogenous nucleotide sequence encoding a phosphoenolpyruvate carboxylase, a citrate synthase, a 2-methylcitrate synthase, or a citrate lyase,

each of the first and second exogenous nucleotide sequences being operably linked to a promoter.

13. The genetically modified microorganism of claim 12, wherein the microorganism is selected from the group consisting of Aspergillus, Citrobacter, Corynebacterium, Dekkera, Enterobacter, Enterococcus, Escherichia, Erwinia, Klebsiella, Kluyveromyces, Lactobacillus, Lactococcus, Morganella, Pantoea, Pectobacterium, Penicillium, Pichia, Proteus, Pseudomonas, Pseudozyma, Rhodotorula, Salmonella, Serratia, Shigella, Saccharomyces, Ustilago, and Yarrowia.

14. The genetically modified microorganism of claim 13, wherein the microorganism is Aspergillus niger, Aspergillus terreus, Escherichia coli, Pseudozyma Antarctica, Yarrowia lipotica, and Saccharomyces cerevisiae.

15. The genetically modified microorganism of claim 12, wherein the second exogenous nucleotide sequence encodes the phosphoenolpyruvate carboxylase and the microorganism further contains a third exogenous nucleotide sequence encoding a citrate synthase, a 2-methylcitrate synthase, a citrate lyase, an aconitase, or a 2-methylcitrate dehydratase, the third exogenous nucleotide sequence being operably linked to a promoter.

16. The genetically modified microorganism of claim 12, wherein the second exogenous nucleotide sequence encodes the citrate synthase, the 2-methylcitrate synthase, or the citrate lyase and the microorganism further contains a third exogenous nucleotide sequence encoding an aconitase or a 2-methylcitrate dehydratase, the third exogenous nucleotide sequence being operably linked to a promoter.

17. The genetically modified microorganism of claim 15, wherein the third exogenous nucleotide sequence encoding the citrate synthase, the 2-methylcitrate synthase, or the citrate lyase and the microorganism further contains a fourth exogenous nucleotide sequence encoding an aconitase or a 2-methylcitrate dehydratase, the fourth exogenous nucleotide sequence being operably linked to a promoter.

18. The genetically modified microorganism of claim 14, wherein the microorganism is Escherichia coli;

the second exogenous nucleotide sequence is operably linked to an E. coli promoter and encodes a phosphoenolpyruvate carboxylase, or a citrate synthase.

19. The genetically modified microorganism of claim 18, wherein the second exogenous nucleotide sequence encodes the phosphoenolpyruvate carboxylase;

the microorganism further contains a third exogenous nucleotide sequence encoding a citrate synthase or an aconitase and the aconitase is an aconitase A or an aconitase B, the third exogenous nucleotide sequence being operably linked to an E. coli promoter.

20. The genetically modified microorganism of claim 18, wherein the second exogenous nucleotide sequence encodes the citrate synthase;

the microorganism further contains a third exogenous nucleotide sequence encoding an aconitase and the aconitase is an aconitase A or an aconitase B, the third exogenous nucleotide sequence being operably linked to an E. coli promoter.

21. The genetically modified microorganism of claim 19, wherein the second exogenous nucleotide sequence encodes the phosphoenolpyruvate carboxylase;

the third exogenous nucleotide sequence encodes an citrate synthase;

the microorganism further contains a fourth exogenous nucleotide sequence encoding an aconitase and the aconitase is an aconitase A or an aconitase B, the fourth exogenous nucleotide sequence being operably linked to an E. coli promoter.

27. A method for producing itaconic acid in a microorganism, comprising

providing the genetically modified microorganism of claim 12,

cultivating the genetically modified microorganism in a medium to produce itaconic acid, and

collecting the medium for isolation of the itaconic acid thus produced.

28. The method of claim 27, wherein the medium contains glucose at a concentration of 5-80 g/L.

29. The method of claim 28, wherein the glucose concentration is 10-40 g/L.

30. The method of claim 27, wherein genetically modified microorganism is permeabilized and the medium contains citrate at a concentration of 5-80 g/L.

31. The method of claim 30, wherein the citrate concentration is 10-40 g/L.