P10: Comparative proteomic analysis of Pichia stipitis CBS 6054 adapted to a dilute acid pretreated waste stream for improved ethanol yield

Adaptation of yeast to dilute acid pretreated (DAP) hydrolysate has been shown to be a viable method for overcoming inhibition as well as increasing ethanol yields; however, the actual effects of adaptation at the molecular level are not well characterized. The aim of this work was to adapt Pichia stipitis CBS 6054 to a DAP lignocellulose-containing waste stream and perform comparative proteomic analysis to evaluate the biochemical and molecular mechanisms of adaptation. Adaptation was carried out over a period of three – four weeks using repeated sub-culturing in increasing concentrations of hydrolysate (up to 100%)  The fermentation capabilities of both the adapted and the unadapted strain were evaluated in order to verify the adaptation resulted in improved product yields, substrate utilization, and biomass production. The adapted strain produced >70% of the theoretical ethanol yield within 72 hours, using close to 100% of the available sugars. In contrast, the unadapted strain utilized < 1% of the available sugars and produced no ethanol over 72 hours. Comparative proteomic analysis using ¹⁶O/¹⁸O-labeling and LC-MS/MS was performed to determine which proteins are up/down regulated in the adapted vs. the wild types yeast, specifically in the xylose utilization, glycolytic, and pentose phosphate pathways. Regulation of the proteins involved in the stress response to changes in pH and detoxification of furfural and 5-hydroxymethyl furfural were also investigated. Determining the molecular and biochemical mechanisms involved in adaptation will benefit biofuels research by providing target proteins/pathways for bio-prospecting and in the design of novel genetically modified organisms.