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 in membrane ergosterol biosynthesis
, and the role of CYP61 as a C22-desaturase in fungal membrane sterol synthesis has been elucidated in S. cerevisiae and Candida glabrata. 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. We could predict several P450 characteristic secondary structural elements, 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
, 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
 and in nystatin-resistant Neurospora crassa strains that are unable to produce ergosterol
. Although our second found intermediary, ergosta-5,8-dien-3-ol, is an atypical sterol, it has 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
. This phenomenon was observed in a nystatin-resistant S. cerevisiae strain
 and primaricin-resistant Aspergillus nidulans strains
. Clearly, these observations and our results indicate the existence of alternative sterol biosynthesis pathways, which require further studies.
Because the cyp61
mutant strains were viable without significant changes in their growth (at least in our experimental conditions), ergosta-5,8,22-trien-3-ol and/or ergosta-5,8-dien-3-ol may play roles similar to ergosterol in the X. dendrorhous cell membrane. Finally, even though the cyp61
mutant strains were not able to produce ergosterol, their sterol content was higher compared to the corresponding parental strains, suggesting an ergosterol-mediated feedback regulatory mechanism in the sterol biosynthesis pathway of X. dendrorhous.
In addition to the alterations in sterol content and composition, the cyp61
mutant X. dendrorhous strains exhibited color phenotypes dissimilar to their parental strains (Figure
7). Carotenoid analyses revealed that the mutant strains produced more carotenoids (Table
4), demonstrating that the CYP61 gene mutation affected carotenoid biosynthesis. Major differences were observed after 72 and 120 h of culture, which coincide with the early and late stationary phases of growth (Figure
8). Wozniak and co-workers reported that maximum expression levels of carotenogenic genes are reached by the end of the exponential and beginning of the stationary phase of X. dendrorhous growth
, coinciding with the induction of carotenogenesis
. It is expected that greater differences in the carotenoid content would be observed once carotenogenesis is induced.
Similar to our results, other studies have demonstrated an increase in astaxanthin production in Phaffia rhodozyma (anamorphic state of X. dendrorhous) when the ergosterol levels were reduced by fluconazole treatment
. A possible explanation for the increased carotenoids in the cyp61
mutants could be the greater availability of carotenoid precursors in absence of the ergosterol negative feedback regulation. This reasoning is also supported by the fact that in the cyp61
mutants, the total sterol content was also increased. For example, supplementation of P. rhodozyma cultures with MVA resulted in an increase in carotenoid production
. Likewise, deletion of the squalene synthase-encoding gene (ERG9) in combination with the overexpression of the catalytic domain of HMGR in a recombinant C. utilis strain that produces carotenoids caused an increase of in lycopene biosynthesis
IPP is the isoprenoid building block; in most eukaryotes, it is derived from the MVA pathway
. Many of the regulatory aspects of isoprenoid biosynthesis involve elements of this pathway; the expression of HMGR (Figure
1) is a critical regulatory step
. The alteration of HMGR expression in the X. dendrorhous cyp61
mutants could explain the increased carotenoid and sterol content. We quantified the HMGR transcript levels, and at all of the growth phases analyzed, it was greater than in the corresponding parental strain. Similarly, an increase in the HMGR transcript level corresponded to an increase in the carotenoid content when the fungus Blaskelea tripora was treated with ketoconazole, which is a specific ergosterol biosynthesis inhibitor
. In S. cerevisiae, HMG-CoA reductase is encoded by two isogenes, HMG1 and HMG2, and the expression of HMG1 is controlled at the transcriptional level by ergosterol
. The overexpression of HMG1 combined with ketoconazole treatment in a S. cerevisiae recombinant strain resulted in an increase in beta-carotene production
. Finally, our results are similar to those reported in the astaxanthin over-producing X. dendrorhous mutant strain with lower ergosterol and a higher HMGR transcript level than the parental strain after 72 h of cultivation
. However, the astaxanthin over-producing strain was obtained by random chemical mutagenesis, while we specifically blocked ergosterol biosynthesis by disrupting the CYP61 gene.