The studies described in this thesis were motivated by ongoing efforts to develop lignocellulosic biomass as an efficient and practical source of renewable energy. Enormous problems complicate these efforts to reduce reliance on greenhouse gas-generating fossil fuels. Simply obtaining the fermentable sugars available in the cellulose and hemicellulose components of lignocellulose to make ethanol or other biofuels generates compounds that inhibit the subsequent fermentation step itself. Acetic acid is one such inhibitor, formed by hydrolysis of acetylated hemicellulose and lignin during typical "pretreatments" of plant biomass performed to reduce the crystallinity of native cellulose (Klinke et al., 2004; Palmqvist and Hahn-Hagerdal, 2000).
The specific focus of this thesis is the problem of acetic acid-mediated growth inhibition of the yeast Saccharomyces cerevisiae. It describes experiments undertaken to obtain more resistant strains. S. cerevisiae has a long and rich history of use in food and beverage production, e.g., baking, winemaking, and brewing. Because of its simple growth requirements, high ethanol-producing capacity and robustness under process condition, S. cerevisiae remains the most widely used microorganism for large-scale bioethanol production. The development of strains with increased resistance to acetic acid will advance efforts to develop plant biomass as a practical source of renewable energy.
This dissertation starts with a review of the literature on acetic acid stress and response in the yeast S. cerevisiae (Chapter 1). The subsequent chapters describe experiments undertaken to obtain acetic acid-resistant yeast strains and to determine the basis for the resistance. Chapter 2 presents results from screening a yeast deletion library that demonstrates that the condition of nutritional auxotrophy itself increases yeast sensitivity to acetic acid. Chapter 3 describes a resistant mutant obtained from screening a library of overexpressed yeast genes in which an increase in vacuolar ATPase (V-ATPase) activity was found to correlate with increased tolerance for acetic and other acids. The study detailed in Chapter 4 tests the hypothesis that overexpression of acetyl-CoA synthetase can increase acetic acid tolerance by converting excess acetic acid into acetyl-CoA. In the final chapter (Chapter 5), the implications of these studies are discussed in the context of better understanding acid stress in yeast and for the application of this information for strain improvement.