Aerobic succinate production in bacteria

• US 20050170482A1
• Filed: 11/12/2004
• Published: 08/04/2005
• Est. Priority Date: 11/14/2003
• Status: Active Grant

• Current Assignee: Rice University
• Original Assignee: Rice University
• Inventors: Bennett, George N., San, Ka-Yiu, Lin, Henry
• Granted Patent: US 7,244,610 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 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12P 7/46 Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid

Abstract

Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.

Claims

1. A method of producing succinic acid in an aerobic culture comprising:

a) supplying a mutant strain of E. coli bacteria;

b) contacting the bacteria with a glucose substrate;

c) allowing the bacteria to metabolize the glucose under aerobic conditions; and

d) isolating and recovering the succinic acid from the culture, wherein the bacteria produces succinic acid from the glucose substrate in a ratio of at least 0.6;

1 succinic acid to substrate.

2. The method of claim 1, wherein the bacteria accumulates to between approximately 10⁸ to 10¹² cells per milliliter.

3. The method of claim 1, wherein the temperature of the culture is selected from between approximately 25°

C. and 45°

C.

4. The method of claim 1, wherein the pH of the culture is selected from between approximately 5 and 9.

5. The method of claim 1, wherein the bacteria produces succinic acid from the glucose substrate in a ratio of at least 0.8:

1 succinic acid to substrate.

6. The method of claim 1, wherein the bacteria produces succinic acid from the glucose substrate in a ratio of about 1:

1 succinic acid to substrate.

7. A method of producing succinic acid in an aerobic culture comprising:

a) supplying a mutant strain of E. coli bacteria, said organism containing a combination of mutations for the genes sdh, poxB, ackA-pta, icd, iclR, and ptsG wherein the mutations cause the organism to contain a mutant succinate dehydrogenase, a mutant pyruvate oxidase, a mutant acetate kinase-phosphotransacetylase, a mutant isocitrate dehydrogenase and a mutant aceBAK operon repressor system;

b) contacting said bacteria with a glucose substrate;

c) allowing the bacteria to metabolize the glucose under aerobic conditions; and

d) isolating and recovering the succinic acid from the culture, wherein the bacteria produces succinic acid from the glucose substrate in a ratio of at least 0.6;

1 succinic acid to substrate.

8. The method of claim 7, wherein the mutant strains are HL51276k, HL512769k, HL2765k, HL27659k.

9. The method of claim 7, wherein the mutant succinate dehydrogenase, the mutant pyruvate oxidase, the mutant acetate kinase-phosphotransacetylase, the mutant isocitrate dehydrogenase, the mutant aceBAK operon repressor system, and the mutant glucose phosphotransferase system result from knockout mutations to the genes sdh, poxB, ackA-pta, icd, iclR, and ptsG.

10. The method of claim 7, wherein the mutant succinate dehydrogenase, the mutant pyruvate oxidase, the mutant acetate kinase-phosphotransacetylase, the mutant isocitrate dehydrogenase, the mutant aceBAK operon repressor system, and the mutant glucose phosphotransferase system result from deletion mutations to the genes sdh, poxB, ackA-pta, icd, iclR, and ptsG.

11. The method of claim 7, wherein the mutant succinate dehydrogenase, the mutant pyruvate oxidase, the mutant acetate kinase-phosphotransacetylase, the mutant isocitrate dehydrogenase, the mutant aceBAK operon repressor system, and the mutant glucose phosphotransferase system result from point mutations to the genes sdh, poxB, ackA-pta, icd, iclR, and ptsG.

12. The method of claim 7, wherein the bacteria are capable of utilizing more than one substrate simultaneously to produce succinic acid simultaneously.

13. The method of claim 7, wherein the yield of succinic acid production is increased by the expression of an isolated polynucleotide comprising a gene encoding phosphoenolpyruvate carboxylase.

14. The method of claim 13, wherein the phosphoenolpyruvate carboxylase is from the genus Sorghum.

15. The method of claim 7, wherein the yield of succinic acid production is increased by the increased expression of enzymes selected from the group consisting of pyruvate carboxylase, malic enzyme, citrate synthase, aconitase, isocitrate lyase, malate synthase, and acetyl-CoA synthetase.

16. The method of claim 7, wherein the bacteria produces at least 0.8:

1 succinic acid to substrate.

17. The method of claim 7, wherein the bacteria produces about 1:

1 succinic acid to substrate.

18. Mutant bacterial strains characterized in that they produce a mixture of succinic acid and acetic acid under aerobic conditions, and said strains lack functional genes such as a combination of succinate dehydrogenase, pyruvate oxidase, acetate kinase-phosphotransacetylase, isocitrate dehydrogenase, aceBAK operon repressor, and glucose phosphotransferase system.

19. The mutant bacterial strains of claim 18, wherein succinic acid is the major product.