8 rRNA gene and ITS2 DNA sequences . Phenotypic analyses of cyp61 mutant strains To compare the phenotypic differences between wild-type and CYP61 mutant strains, phenotypic analyses were performed. The strains were grown in YM medium, and growth curves were constructed including the analyses of total carotenoid yield and composition, ergosterol production and relative mRNA expression of the HMGR gene at three timepoints. For these analyses, the seven X. dendrorhous strains (UCD 67–385, 385-cyp61(+/−), 385-cyp61(−/−), CBS 6938, CBS-cyp61(−), AVHN2 and selleck chemicals Av2-cyp61(−)) were cultivated in triplicate 600 ml YM cultures in Erlenmeyer flasks at 22°C with constant agitation. The yeast growth was determined by the OD at 600
nm, which was measured in V-630 UV–vis Spectrophotometer from JASCO. Culture samples of 75 ml
were taken after 24, 72 and 120 h of growth and segregated for analysis as follows: 5 ml to determine the dry weight of the yeast, BTK animal study 30 ml for RNA, 30 ml for pigment and 10 ml for sterol extractions. In each case, the cell pellet was washed with distilled water, frozen with liquid nitrogen and stored at −80°C until further processing. Carotenoid extraction and RP-HPLC Carotenoids were extracted from cellular pellets according to the acetone extraction method . Total carotenoids were quantified by absorbance at 465 nm using an absorption coefficient of A1% = 2,100 and normalized to the dry weight of the yeast. Carotenoids were separated by RP-HPLC using a reverse phase RP-18 Lichrocart125-4 (Merck) column with acetonitrile: methanol: isopropyl (85:10:5 v/v) as the selleck products mobile phase with a 1 ml/min flux under isocratic conditions. The elution spectra were recovered using a diode array detector, and carotenoids were identified by their PJ34 HCl spectra and retention time according to standards. Sterol extraction and identification Sterol extraction was adapted from  and . Briefly, 4 g of KOH and 16 ml of 60% (v/v) ethanol/water were added to the cell pellets, which were mixed and saponified at 80 ± 2°C for 2 h. Non-saponificable
sterols were extracted with 10 ml of petroleum and dried. Sterols were separated by RP-HPLC with a C-18 column, using methanol/water (97:3, v/v) as the mobile phase at 1 ml/min. The elution spectra were recovered using a diode array detector, and sterols were visualized in the 280 nm channel. Standard ergosterol was purchased at Sigma-Aldrich (catalogue number 57-87-4). Sterols were quantified spectrophotometrically at 280 nm . The identification of the sterols was performed by an external service (Corthorn Quality; http://www.corthorn.cl/) by GC/MS (Agilent 5970N gas chromatographer/Agilent 5890N mass spectrometer). An RTX5 sil MS (Restk) 30 m × 250 μm × 0.25 μm column was used with the following oven conditions: 270°C for 10 s, raised to 280°C at 30°C/min and maintained for 2 min. The injector temperature was 270°C, and the ion source was kept at 70 eV.