This method and other similar approaches
have been rigorously examined with demonstrated advantages of reliability and MRT67307 price reproducibility over housekeeping genes [37, 40–45]. In the present study, we compared cell growth, cell viability, ethanol production and gene expression under the ethanol stress between two very closely related strains, the lignocellulosic inhibitor-tolerant Y-50049 and its ethanol-tolerant derivative Y-50316 retaining the inhibitor-tolerance characteristics. Using the recently developed pathway-based qRT-PCR array assays, we investigated transcription dynamics of over 170 selected genes based on previous reports and our preliminary screening in response to ethanol challenge using a time-course study. The
objective of this study was to identify candidate and key genes responsible for ethanol tolerance to support complete ethanol fermentation. Our results uncovered previously unreported genes accountable for ethanol tolerance and identified legitimate candidate genes of ethanol tolerance based on the ethanol-tolerant Y-50316. Results of this study will aid dissection of ethanol tolerance mechanisms in yeast and metabolic engineering efforts for more tolerant strain development. selleck products Results Tolerance and viability On a solid medium of 2% glucose containing 8% ethanol, ethanol-tolerant strain Y-50316 showed cell growth from reduced cell concentrations at 10- to 100-fold Phenylethanolamine N-methyltransferase dilutions (Figure 1A). In contrast, cells of Y-50049 failed to grow at any reduced cell concentration levels. Strain Y-50316, an ethanol-tolerant derivative from its parental Y-50049, maintained the inhibitor-tolerance and able to in situ detoxify furfural and HMF derived from pretreatment of lignocellulosic biomass. On a medium of 2% glucose containing furfural and HMF at 10 mM each, both strains showed similar growth patterns
against the inhibitors at all cell dilution levels from 10- to 1000-fold (Figure 1B). On a liquid YM of 2% glucose containing furfural and HMF, both strains showed similar growth pattern and reached stationary phase in 30 h (data not shown) Figure 1 Cell growth response to ethanol and inhibitors. Comparison of cell growth and colony appearance for ethanol-tolerant and inhibitor-tolerant mutant Saccharomyces cerevisiae NRRL Y-50316 and its parental inhibitor-tolerant strain NRRL Y-50049 on YM plate of 2% glucose containing 8% (v/v) ethanol (A) or amended with inhibitors of furfural and 5-hydroxymethylfurfural each at 10 mM (B). The cultures initially applied were estimated with viable cell account of approximately 1.0 × 107 per ml as measured by colony forming units. A Selleckchem Apoptosis Compound Library serial of 10-fold culture dilutions in water were spotted onto a medium plate containing ethanol or inhibitors and cell growth examined 7 days after incubation at 30°C.