(41f) Ethanol Tolerance in Yeast Is Determined By Membrane Gradients

Ethanol made from the yeast Saccharomyces cerevisiae will continue to be the dominant mass-market biofuel in the near to intermediate term.  One of the primary bottlenecks to higher bioethanol production is the toxicity of ethanol itself, with cell death occurring at concentrations greater than 15-20% (v/v).  Increasing ethanol tolerance could improve both product yield and the viability of yeast for reuse in subsequent fermentations.  However, its biological basis remains elusive: whole genome studies have shown that ethanol tolerance is influenced by many genes, and thus far, none have produced significant improvement when manipulated alone.  An alternative approach, which would not necessitate any genetic manipulation of strains, is to uncover extracellular conditions that would desensitize cells to high ethanol concentrations.  To this end, we have identified several micronutrients which, when added to fermentation culture, can increase ethanol production in S. cerevisiae by >50% in the very high sugar and high cell density conditions that mimic industrial fermentation.  Moreover, we find that these additives confer increased resistance to propanol, collectively suggesting the augmentation of general cellular tolerance to solvents.  Dissecting the component effects of these additives led us to uncover a novel mechanism of ethanol toxicity based on the perturbation of membrane gradients.  Indeed, by using yeast mutants that specifically strengthen or weaken these gradients, we can elicit corresponding increases or decreases to ethanol performance.  The strengthening of membrane gradients by these culture additives is not unique to special strains of S. cerevisiae: this effect raises ethanol yields (to nearly 100%) universally among a sampling of genetically divergent industrial and laboratory strains.  Moreover, it is not specific to glucose: these media supplements also raise ethanol yields in a strain engineered to ferment xylose.  Taken together, these results point to an unanticipated method for increasing the economic potential of cellulosic biomass and reveal the fundamental role that membrane gradients play in determining ethanol tolerance.