Claims
- 1. A soluble cytochrome P450 reductase (CPR) from Candida sp. wherein the CPR has an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region.
- 2. The CPR of claim 1 which is capable of complementing a membrane-associated cytochrome P450 anchored in a microsomal membrane.
- 3. The CPR of claim 1 lacking all or a part of the N-terminal hydrophobic domain.
- 4. The CPR of claim 1 wherein hydrophobic amino acids in the N-terminal region are substituted with hydrophilic amino acids.
- 5. The CPR of claim 1 comprising an amino acid sequence as set forth in at least one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:6.
- 6. The CPR of claim 5 further comprising a methionine residue before the first amino acid in any of the sequences set forth in SEQ ID Nos 2, 3, 5, or 6.
- 7. The CPR of claim 4 further comprising at the N-terminal end, a heterologous peptide sequence such as a purification moiety or secretion sequence.
- 8. The CPR of claim 7 wherein the purification moiety is a his-tag.
- 9. A host cell comprising the CPR of any of claims 1-7.
- 10. The host cell of claim 8 which is a prokaryotic cell such as a bacterial cell.
- 11. The host cell of claim 10 wherein the bacterial cell is E. coli.
- 12. The host cell of claim 9 which is a eukaryotic cell such as a yeast, insect, animal, or plant cell.
- 13. An Isolated nucleic acid molecule encoding a soluble cytochrome P450 reductase (CPR) wherein the soluble CPR comprises an amino acid sequence as set forth in any one of SEQ ID NOs:2, 3, 5, or 6.
- 14. The isolated nucleic acid molecule of claim 13 comprising a nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:4.
- 15. The isolated nucleic acid molecule of claim 13 further comprising hydrophilic amino acid residues at the N-terminal end.
- 16. A vector comprising the nucleic acid molecule of any of claims 13-15.
- 17. The vector of claim 16 wherein the vector is a plasmid, phagemid, phage or cosmid.
- 18. The vector of claim 16 wherein the vector is a linear vector.
- 19. A host cell transfected or transformed with the nucleic acid of any of claims 13-15.
- 20. A host cell transfected or transformed with a vector according to claim 16.
- 21. A host cell transfected or transformed with the vector of claim 17.
- 22. A host cell transfected or transformed with the vector of claim 18.
- 23. The host cell of claim 19 which is a prokaryotic cell such as a bacterial cell.
- 24. The host cell of claim 20 which is a prokaryotic cell such as a bacterial cell.
- 25. The host cell of claim 21 which is a prokaryotic cell such as a bacterial cell.
- 26. The host cell of claim 22 wherein the bacterial cells are E. coli cells.
- 27. The host cell of claim 23 wherein the bacterial cells are E. coli cells.
- 28. The host cell of claim 24 wherein the bacterial cells are E. coli cells.
- 29. The host cell of claim 25 wherein the bacterial cells are E. coli cells.
- 30. The host cell of claim 19 which is a eukaryotic cell such as a yeast, insect, animal, or plant cell.
- 31. The host cell of claim 20 which is a eukaryotic cell such as a yeast, insect, animal, or plant cell.
- 32. The host cell of claim 21 which is a eukaryotic cell such as a yeast, insect, animal, or plant cell.
- 33. The host cell of claim 22 which is a eukaryotic cell such as a yeast, insect, animal, or plant cell.
- 34. The host cell according to claim 30 wherein the yeast cell is from Candida sp.
- 35. The host cell according to claim 31 wherein the yeast cell is from Candida sp.
- 36. The host cell according to claim 32 wherein the yeast cell is from Candida sp.
- 37. The host cell according to claim 33 wherein the host cell is from Candida tropicalis.
- 38. A method for producing a soluble cytochrome P450 reductase (CPR), said method comprising:
(a) transforming a suitable host cell with a nucleotide sequence encoding a Candida sp. CPR having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region, wherein the nucleotide sequence is operably linked to a promoter which functions in the host cell and a codon for a translational start signal; and (b) culturing the host cell under conditions favorable for expression of the soluble CPR.
- 39. The method of claim 38 wherein the nucleotide sequence comprises SEQ ID NO:1 or SEQ ID NO:4.
- 40. The method of claim 39 wherein the nucleotide sequence further encodes a heterologous peptide sequence located at the N-terminal end of the truncated CPR protein.
- 41. The method of claim 40 wherein the heterologous peptide sequence comprises a purification moiety such as a his-tag.
- 42. The method of claim 40 wherein the heterologous peptide sequence is a sequence which enhances secretion of the CPR from the host cell.
- 43. The method of claim 42 wherein the secretion sequence is an OmpA secretion sequence or a signal peptide from an extracellular enzyme such as a protease, amylase, cellulase, xylenase or lipase.
- 44. A method for increasing production of a dicarboxylic acid, said method comprising:
(a) providing a host cell having one or more genes for a cytochrome P450; (b) introducing into the host cell, one or more coding sequences for a Candida sp. soluble CPR having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region, wherein said coding sequence is operatively linked to a promoter which functions in the host cell and a translational initiation codon; and (c) culturing the host cell under conditions favorable for expression of the soluble CPR.
- 45. The method of claim 44 wherein the host cell further comprises one or more genes for a microsomal CPR.
- 46. The method of claim 38 or 44 wherein the soluble CPR comprises the amino acid sequence set forth in any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:6.
- 47. The method of claim 38 or 44 wherein the coding sequence for a soluble CPR comprises the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:4.
- 48. A method for detecting a cytochrome P450, said method comprising:
(a) isolating a microsomal preparation from an organism; (b) adding a soluble P450 cytochrome reductase (CPR) to the microsomal preparation wherein said soluble CPR comprises the amino acid sequence set forth in any of SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:5, or SEQ ID NO:6; (c) measuring oxidation of of NADPH; and (d) correlating an increase in oxidation of NADPH with the detection of a cytochrome P450.
- 49. A method for producing a carboxylic acid comprising: culturing Candida sp. in a fermentation medium containing a substrate of the formula R(CH2)nCH3, wherein n=≧1 and R is selected from the group consisting of epoxide, alkoxy, ether, saturated primary alcohol, cyloalky, aryl, diol and diol ester, wherein at least one terminal methyl group of the substrate is oxidized to a carboxylic acid, and wherein the Candida sp. expresses one or more copies of a gene for a soluble CPR.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of U.S. Provisional Application No. 60/328,752 filed Oct. 12, 2001, which application is incorporated by reference herein.
Provisional Applications (1)
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Number |
Date |
Country |
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60328752 |
Oct 2001 |
US |