Illumina HiSeq 4000 paired end sequencing; Transcriptional profile of the industrial hybrid Saccharomyces pastorianus reveals temperature-dependent allele expression bias and preferential orthologous protein assemblies
Transcriptional profile of the industrial hybrid Saccharomyces pastorianus reveals temperature-dependent allele expression bias and preferential orthologous protein assemblies
To determine the genes affected by temperature stress in different culture media, the expression profiles of the S. pastorianus CBS 1513 (Group I strain) at 13 °C, 22 °C and 30 °C in SD media, wort (maltose rich medium), SD media with 6% ethanol, and SD media without leucine were analysed. These media were chosen given their relevance to S. pastorianus domesticated habitat, as wort is used during the beer fermentation, alcohol is produced during this process, and the leucine amino acid metabolism is responsible for important flavour compound production, such as the isoamyl-acetate production (fruity flavour). S. pastorianus CBS 1513 cells are incubated overnight in Yeast Peptone Dextrose (2% glucose) at 25 °C with shaking at 200 rpm. The pre-cultured yeast cells were inoculated in A. fresh industrial wort from Brouwland (according to Giannakou et al., 2021), B. synthetic dextrose (SD) with all amino acids +2% glucose (complete), C. SD without leucine, or D. SD with 6% ethanol media. Culture temparatures were at 13 °C, 22 °C or 30 °C until mid-phase of exponential growth. Liquid fitness assays have been performed for expression validation, three technical replicates for three biological replicates. Cells were grown at 30 °C from an optical density of 0.1 (measured at a wavelength of 600nm). Growth was measured every 5 minutes as previously described by Naseeb and Delneri 2012, and recorded by a BMG FLUOstar OPTIMA Microplate Reader (OD 595nm), for up to 55 hours incubation time. Growth parameters were calculated using “Growthcurver” R package.  Total RNA was isolated from three biological replicates of cells collected at mid-log phase (OD600 0.4-0.6) using the RNeasy Mini Kit (QIAGEN, Germany). The lysis was performed by enzymatic digestion of cell wall followed by lysis of spheroplasts. To eliminate genomic DNA contamination, an additional DNAse treatment was performed with RNAse-free DNase set (QIAGEN, Germany) following the manufacturer's protocol. Truseq stranded mRNA Assay. Paired end.