At 24 and 72 h of cultivation, the expression of this gene was between 2 and 5 times higher in the 385-cyp61 hph /cyp61 zeo , CBS-cyp61 hph and Av2-cyp61 zeo strains than in the respective parental strains (Figure  8). Discussion Cytochrome P450 monooxygenases are involved in the oxidative metabolism of an enormous diversity of substrates, taking part in primary, secondary and xenobiotic metabolism. CYP51 and CYP61 are structurally and functionally conserved fungal P450s involved VX-770 mouse in membrane ergosterol biosynthesis [36], and the role

of CYP61 as a C22-desaturase in fungal membrane sterol synthesis has been elucidated in S. cerevisiae[24] and Candida glabrata[37]. In this study, we isolated and characterized a gene, CYP61, from X. dendrorhous that has nine exons, encodes a putative 526-residue polypeptide and shares significant similitude and identity with the C22-sterol desaturase from S. cerevisiae[25]. We could predict several P450 characteristic secondary structural elements, www.selleckchem.com/products/3-methyladenine.html and we identified three residues in CYP61 that are completely conserved in P450s. Together, these observations support the hypothesis that the X. dendrohous CYP61 gene encodes the cytochrome P450 CYP61. As in other organisms [25], the CYP61 gene is not essential

for the X. dendrorhous viability, even though we demonstrated that it is involved in ergosterol biosynthesis. Disruption of the CYP61 gene prevents ergosterol biosynthesis and leads to the accumulation

of other intermediary sterols including ergosta-5,8-dien-3-ol and ergosta-5,8,22-trien-3-ol. Contrary to our findings, the specific mutation of ERG5 in S. cerevisiae results in the predominant accumulation of ergosta-5,7-dien-3-ol, although the C22-desaturase substrate is ergosta-5,7,24-trien-3-ol [25, 38]. Like in X. dendrohous, ergosta-5,8,22-trien-3-ol accumulation has been observed in other fungi, such as C. neoformans, after the inhibition of the ERG6-encoding enzyme [39] and in nystatin-resistant Neurospora crassa strains that are unable to produce ergosterol [40]. Although our second found intermediary, ergosta-5,8-dien-3-ol, is an atypical sterol, it has Succinyl-CoA been detected in fungi strains that are unable to synthetize ergosterol that in turn are resistant to fungicidal polyenes, such as nystatin and primaricin; polyenes bind ergosterol in the fungal cell membrane, creating channels that disrupt the transmembrane potential and its functions [41]. This phenomenon was observed in a nystatin-resistant S. cerevisiae strain [42] and primaricin-resistant Aspergillus nidulans strains [43]. Clearly, these observations and our results indicate the existence of alternative sterol biosynthesis pathways, which require further studies.