Complementation of a Saccharomyces cerevisiae ERG11/CYP51 (sterol 14α-demethylase) doxycycline-regulated mutant and screening of the azole sensitivity of Aspergillus fumigatus isoenzymes CYP51A and CYP51B

Abstract

Aspergillus fumigatus sterol 14α-demethylase isoenzymes CYP51A and CYP51B were heterologously expressed in a Saccharomyces cerevisiae mutant (YUG37-erg11), wherein native ERG11/CYP51 expression is controlled using a doxycycline-regulatable promoter. When cultured in the presence of doxycycline, recombinant YUG37-pcyp51A and YUG37-pcyp51B yeasts were able to synthesize ergosterol and grow; a control strain harboring reverse-oriented cyp51A could not. YUG37-pcyp51A and YUG37-pcyp51B constructs showed identical sensitivity to itraconazole, posaconazole, clotrimazole, and voriconazole. Conversely, YUG37-pcyp51A withstood 16-fold-higher concentrations of fluconazole than YUG37-pcyp51B (8 and 0.5 μg ml⁻¹, respectively).

FIG. 1.

Example GC-MS chromatograms for the YUG37-pCTRL construct (A) and the complementing YUG37-pcyp51A construct (B) cultured using _gal/rafYM^+dox induction medium. 1, ergosterol; 2, 14α-methyl fecosterol; 3, 4,14α-dimethyl cholesta 8,24-dienol; 4, 14α-methyl ergosta 8,24(28) dien-3β-6α-diol; 5, lanosterol and/or obtusifoliol.