Molecular characterization and isolation of cytochrome P450 genes from the filamentous fungus Aspergillus oryzae.

We explored the molecular diversity of cytochrome P450 genes in the filamentous fungus Aspergillus oryzae using bioinformatic and experimental approaches. Based on bioinformatic annotation, we found 155 putative genes of cytochromes P450 in the whole genome sequence; however, 13 of 155 appeared to be pseudogenes due to sequence deletions and/or inframe stop codon(s). There are 87 families of A. oryzae cytochromes P450 (AoCYPs), indicating considerable phylogenetic diversity. To characterize A. oryzae AoCYPs, we attempted to isolate cDNAs using RT-PCR and determined their transcriptional capabilities. To date, we have confirmed gene expression of 133 AoCYPs and cloned 121 AoCYPs as full-length cDNAs encoding a mature open reading frame. Using experimentally deduced sequences and intron-exon organization, we analyzed AoCYPs phylogenetically. We also identified intronic consensus sequences in AoCYPs genes. The experimentally validated exonic and intronic sequences will be a powerful advantage in identification and characterization of novel P450s from various ascomycetous fungi.