Unveiling the genetic basis of the low pH response in the acidophilic yeast Maudiozyma bulderi (previously Kazachstania bulderi) via transcriptomic analysis
Unveiling the genetic basis of the low pH response in the acidophilic yeast Maudiozyma bulderi (previously Kazachstania bulderi) via transcriptomic analysis
Three Maudiozyma bulderi strains used in this study CBS 8638, CBS 8639, and NRRL Y-27205 were obtained from the Agricultural Research Service (ARS-NRRL) culture collection. The yeasts were cultured and maintained on YPD agar medium (15 g/L agar, 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) or in SD minimal media (Yeast nitrogen base with ammonium sulphate and amino acids 6.7g/L)  Bioreactor fermentations for RNA extraction were conducted using one litre stirred tank bioreactors (Multifors, Infors-HT, Bottmingen, Switzerland). All fermentation experiments were carried out with cells grown in 500 mL of SD minimal media supplemented with 20g/l glucose. Fermentations were inoculated with cells from YPD overnight culture, washed twice in sterile deionised water, to achieve an initial optical density (OD) of 0.1. Three replicates of each strain were then cultured in 500 mL of YNB supplemented with 20g/l glucose and adjusted to either pH 5.5 or 2.5 with H2SO4. The pH was maintained at 5.5 and 2.5 using either 10 M NaOH or 1 M H3PO4 for pH adjustment. Temperature was maintained at 25°C, with agitation set at 300 rpm. For RNA extraction, 10-20 mL of cells with OD between 0.7 and 1.0 were harvested and promptly snap-frozen with liquid nitrogen.  Total RNA was extracted from 1x10^7 cells of three biological replicates harvested at the logarithmic growth phase (OD600 0.5-1.0) using the RNeasy Mini Kit (QIAGEN, Germany) following the manufacturer's protocol for enzymatic digestion of the cell wall followed by lysis of spheroplasts. The concentration of lyticase was optimized to 50U per 107 cells for efficient cell lysis. To eliminate genomic DNA contamination, an additional DNase treatment was performed using the RNase-free DNase set (QIAGEN, Germany) according to the manufacturer's instructions. The extracted RNA was quantified using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, United States).  Libraries were prepared from total RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc.) following the manufacturer's instructions. 