As a result, it is very difficult to avoid biased assessment for the complex interactions of ethanol tolerance in yeast. Table 1 Recent studies on gene expression response and genes related to ethanol tolerance for Saccharomyces cerevisiae Method Strain Growth condition Cell growth stage Ethanol challenge concentration (%, v/v) Sampling time-points Reference qRT-PCR Array NRRL Y-50316 YM, 30°C CP673451 supplier OD600 = 0.15 8 0, 1, 6, 24, 48 h This work   NRRL Y-50049           Microarray S288c YPD, 28°C OD660 = 0.8 7 0, 0.5 h [11] Microarray PMY 1.1 YNB, 30°C OD620 = 1.0 5 0, 1, 3 h [12]   FY834           Microarray S288c IFO2347 YPD, 30°C OD660 = 1.0 5 0, 0.25, 0.5, 1, 2, 3 [13]

Microarray FY834 A1 YPD, 30°C Initial 10 log phase [15] Microarray Vin13 Grape juice, 30°C None 0 Varied ethanol concentrations [16]   K7             K11           Microarray K701 SR4-3 YPAD, 20°C None 0 log phase [17] Microarray EC1118 Synthetic must, 24°C None 0 SBE-��-CD mw fermentation stages1 to 6 [18]   K-9           Microarray X2180-1A YPD, 30°C None 0 log phase [19] SAGE EC1118 Synthetic must, 28°C None 0 0, 20, 48, 96 h [20] Microarray Kyokai no. 701 Sake mash, 15°C None 0 2, 3, 4, 5, 6, 8, 11, 14, 17 day [21] Yeast tolerance to ethanol is complex involving multiple genes and multiple quantitative trait loci [31]. Development of

ethanol-tolerant strains has been hindered by using conventional genetic engineering methods. On the other hand, yeast is adaptable to stress conditions under directed evolutionary engineering [2, 32–34]. Adaptation LY411575 nmr and evolutionary engineering have been successfully applied in obtaining ethanol tolerant strains at varied levels [26, 27, 35, 36]. Previously, we developed tolerant ethanologenic

yeast S. cerevisiae NRRL Y-50049 that is able to withstand and in situ detoxify numerous fermentation inhibitors that are derived from lignocellulose-to-ethanol conversion such as furfural and 5-hydroxymethylfurfural (HMF) [33, 37, 38]. Building upon the inhibitor-tolerant yeast, we recently developed ethanol-tolerant yeast NRRL Y-50316 using an adaptation evolutionary engineering method under laboratory settings. The qRT-PCR is an accurate assay platform and considered as an assay of choice for quantitative gene expression analysis. Oxalosuccinic acid It is commonly used to confirm high throughput expression data obtained by microarray which has higher levels of variations from multiple sources. For absolute quantitative gene expression analysis, due to the necessary wells required for the construction of standard curves, very limited number of wells are available for target gene assays [37, 39]. Recently, a significant advance has been made to safeguard data accuracy and reproducibility with two new components, a robust mRNA serving as PCR cycle threshold reference and a master equation of standard curves [37, 40, 41].