Cloning and partial sequencing of the NADPH-cytochrome P450 reductase gene of Candida bombicola.
Inge N.A. Van Bogaert, Sofie De Maeseneire, Erick J. Vandamme, Wim Soetaert
Lab. Ind. Microb. & Biocat., Ghent University, Coupure Links 653, Ghent, 9000, Belgium
Candida bombicola is a non pathogenic yeast capable of growing on alkanes when provided as the sole carbon source. This species is also known to synthesise sophorolipids, a bio-surfactant group consisting of the disaccharide sophorose to which a hydroxy fatty acid is linked. In both biochemical pathways the activity of cytochrome P450 monooxygenase is required. The enzymes must hydroxylate the alkane or fatty acid in order to respectively initiate the beta-oxidation or the synthesis of the sophorolipid. Cytochrome P450 monooxygenases form a small electron transfer chain together with the NADPH-cytochrome P450 reductase (CPR). Both enzymes are N-terminal anchored to the endoplasmatic reticulum and its related structures. NADPH-cytochrome P450 reductase receives electrons from NADPH and transfers them via FAD and FMN to the cytochrome P450 monooxygenase as required to activate molecular oxygen. Part of the CPR-gene was amplified by the use of degenerated oligonucleotides designed on the conserved amino acid sequences of the Saccharomycotina. The derived amino acid sequence was closely related to the CPR-enzyme of Yarrowia lipolytica. Also for other known yeast CPR-enzymes, high homology was observed. Overall alignments with all other known eukaryotic CPR-proteins revealed, as expected, the presence of highly conserved regions for the binding of FAD and FMN. This work was supported by a grant from the Bijzonder Onderzoeksfonds, Ghent University.