The production of biofuels from cellulosic biomass is a promising technology for developing a renewable source of energy. Efforts to produce ethanol from cellulosic biomass using microbes, such as the yeast Saccharomyces cerevisiae, face major challenges, including the need for detoxification. Here, we apply a strategy to discover genetic alterations that lead to improved robustness of S. cerevisiae in the presence of acetate, which is present at toxic concentrations in hemicellulose hydrolysates. Acetate in its protonated form (acetic acid) enters the cell through passive diffusion and dissociates into a proton and acetate, acidifying the cytosol and inhibiting cell function, an effect that is exacerbated in the presence of sodium. Through flow cytometry analysis, implemented as part of a novel cell culture technique, the Cytostat, we characterized the deleterious effects of sodium acetate on growth and on cell size homeostasis. Further, using the Cytostat to screen a genome-wide, gene overexpression library, we identified that overexpressing the ENA2 gene, a P-type sodium pump ATPase, provides a significant growth improvement in the presence of sodium acetate. Together, our data support the proposed mechanism for the synergistic growth inhibition exerted by acetate and sodium, as well as the mechanism that develops resistance.
Bibliographical noteFunding Information:
Valuable discussions with Dr. A. Gilbert and Dr. G. Sitton are greatly appreciated. This study was supported by the Biotechnology Institute, University of Minnesota ; the Initiative for Renewable Energy and the Environment (IREE), University of Minnesota ; and Mascoma Corporation .
- Cell size
- Flow cytometry
- Overexpression library
- Sodium acetate