Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis
AssigneeCognis Corporation
InventorMark Donnelly
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Abstract
The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.
Claims49
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.
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.
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.
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.
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.
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.
Dependencies are inferred from the claim text.