Recycling system for manipulation of intracellular NADH availability

• US 20030175903A1
• Filed: 11/01/2002
• Published: 09/18/2003
• Est. Priority Date: 11/02/2001
• Status: Active Grant

• Current Assignee: Rice University
• Original Assignee: Rice University
• Inventors: Bennett, George N., San, Ka-Yiu, Berrios-Rivera, Susana J.
• Granted Patent: US 7,256,016 B2
• Time in Patent Office: Days
• US Class Current: 435/90
• CPC Class Codes: C12N 9/0036 acting on NADH or NADPH (1.6)
• CPC Class Codes: C12P 1/00 Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• CPC Class Codes: C12P 7/625 Polyesters of hydroxy carboxylic acids

Abstract

The present invention describes a novel recombinant NADH recycling system that is used as a process for producing reduced compounds. In a specific embodiment, the reduced compounds include ethanol, succinate, lactate, a vitamin, a pharmaceutical and a biodegraded organic molecule. The NADH recycling system effects metabolic flux of reductive pathways in aerobic and anaerobic environments.

Claims

1. A method of increasing the availability of NADH in a cell, comprising the transformation of a cell with a nucleotide sequence encoding an NAD+-dependent dehydrogenase and growth of said cell under conditions in which said NAD+-dependent dehydrogenase increases the intracellular availability of NADH.

2. The method of claim 1, wherein said nucleotide sequence encoding an NAD+-dependent dehydrogenase is a nucleotide sequence encoding an NAD+-dependent formate dehydrogenase.

3. The method of claim 1, wherein said nucleotide sequence encoding an NAD+-dependent dehydrogenase is a yeast nucleotide sequence encoding an NAD+-dependent dehydrogenase.

4. The method of claim 1, wherein said nucleotide sequence encoding an NAD+-dependent dehydrogenase is a yeast nucleotide sequence encoding an NAD+-dependent formate dehydrogenase.

5. The method of claim 1, wherein said nucleotide sequence encoding an NAD+-dependent dehydrogenase is a nucleotide sequence from Candida boidinii.

6. The method of claim 1, wherein said nucleotide sequence encoding an NAD+-dependent dehydrogenase is a yeast nucleotide sequence encoding a NAD+-dependent formate dehydrogenase from Candida boidinii.

7. The method of claim 1, wherein said nucleotide sequence encoding an NAD+-dependent dehydrogenase is a yeast nucleotide sequence encoding a NAD+-dependent formate dehydrogenase from Candida boidinii comprising SEQ ID NO:

1.

8. The method of claim 1, wherein said cell is a bacterium.

9. The method of claim 1, wherein said cell is E. coli.

10. The method of claim 1, wherein said cell is a member of the genus Rhodococcus.

11. A cell comprising a recombinant NADH-recycling system, wherein said system comprises a heterologous nucleotide sequence encoding an NAD+-dependent dehydrogenase.

12. The cell of claim 11, wherein said heterologous nucleotide sequence encoding an NAD+-dependent dehydrogenase is a nucleotide sequence encoding an NAD+-dependent formate dehydrogenase.

13. The cell of claim 11, wherein said heterologous nucleotide sequence encoding an NAD+-dependent dehydrogenase is a yeast nucleotide sequence encoding an NAD+-dependent dehydrogenase.

14. The cell of claim 11, wherein said heterologous nucleotide sequence encoding an NAD+-dependent dehydrogenase is a yeast nucleotide sequence encoding an NAD+-dependent formate dehydrogenase.

15. The cell of claim 11, wherein said heterologous nucleotide sequence encoding an NAD+-dependent dehydrogenase is a nucleotide sequence from Candida boidinii.

16. The cell of claim 11, wherein said heterologous nucleotide sequence encoding an NAD+-dependent dehydrogenase is a nucleotide sequence from Candida boidinii comprising SEQ ID NO:

1.

17. The cell of claim 11, wherein said cell is a bacterium.

18. The cell of claim 11, wherein said cell is E. coli.

19. The cell of claim 11, wherein said cell is a member of the genus Rhodococcus.

20. A method for producing a compound comprising growing a culture of cells in a culture medium, wherein the culture comprises at least one cell comprising a recombinant NADH recycling system, and obtaining said compound from said culture.

21. The method of claim 20, wherein the recombinant NADH-recycling system comprises a heterologous nucleotide sequence encoding an NAD+-dependent dehydrogenase.

22. The method of claim 20, wherein said compound is an amino acid, organic acid, hydroxy acid, ester, alcohol, vitamin, antimicrobial, pharmaceutical, polypeptide, biopolymer, mineral, or biodegraded organic compound.

23. The method of claim 20, wherein said culture medium is supplemented with a substrate for the dehydrogenase component of said recombinant NADH recycling system.

24. The method of claim 20, wherein said culture medium is supplemented with formate.

25. The method of claim 20, wherein said culture medium is supplemented with a concentration of formate of at least about 100 mM.

26. The method of claim 20, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for aerobic growth.

27. The method of claim 20, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for anaerobic growth.

28. The method of claim 20, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for microaerophilic growth.

29. A method for biodegrading a compound comprising growing a culture of cells in a culture medium, wherein the culture comprises at least one cell comprising a recombinant NADH recycling system, and obtaining said biodegraded compound from said culture.

30. The method of claim 29, wherein the recombinant NADH-recycling system comprises a heterologous nucleotide sequence encoding an NAD+-dependent dehydrogenase.

31. The method of claim 29, wherein the compound is an aromatic hydrocarbon, aromatic heterocycle, halogenated organic compound, petroleum hydrocarbon, polycyclic aromatic hydrocarbon, or polyaromatic sulfur heterocycle.

32. The method of claim 29, wherein said culture medium is supplemented with a substrate for the dehydrogenase component of said recombinant NADH recycling system.

33. The method of claim 29, wherein said culture medium is supplemented with formate.

34. The method of claim 29, wherein said culture medium is supplemented with a concentration of formate of at least about 100 mM.

35. The method of claim 29, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for aerobic growth.

36. The method of claim 29, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for anaerobic growth.

37. The method of claim 29, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for microaerophilic growth.

38. A method for altering metabolic flux in a cell comprising growing a culture of cells in a culture medium, wherein the culture comprises at least one cell comprising a recombinant NADH recycling system, wherein the increased intracellular availability of NADH results in a redistribution of metabolism towards a more anaerobic state.

39. The method of claim 38, wherein said method results in decreased intracellular acetate levels.

40. The method of claim 38, wherein said method is used to produce amino acids, organic acids, hydroxy acids, esters, alcohols, vitamins, antimicrobials, pharmaceuticals, polypeptides, biopolymers, minerals, or biodegraded organic compounds.

41. The method of claim 38, wherein said method is used to biodegrade a compound, wherein said compound is an aromatic hydrocarbon, aromatic heterocycle, halogenated organic compound, petroleum hydrocarbon, polycyclic aromatic hydrocarbon, or polyaromatic sulfur heterocycle.

42. The method of claim 38, wherein said culture medium is supplemented with a substrate for the dehydrogenase component of said recombinant NADH recycling system.

43. The method of claim 38, wherein said culture medium is supplemented with formate.

44. The method of claim 38, wherein said culture medium is supplemented with a concentration of formate of at least about 100 mM.

45. The method of claim 38, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for aerobic growth.

46. The method of claim 38, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for anaerobic growth.

47. The method of claim 38, wherein said growing of a culture of cells is carried out under atmospheric conditions suitable for microaerophilic growth.