Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

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.

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 - 43 . (canceled)  
     
     
         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 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 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;    (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 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.