Identification of gene over-expression targets for the development of robust yeast strains for cellulosic ethanol production

Bioethanol production from cellulosic biomass is projected to significantly reduce the environmental impact over traditional methods of producing this biofuel. However, this technology is at its infancy, and several challenges, including biomass feedstock processing, need to be addressed to improve efficiency. For instance, acetate is present at growth inhibitory concentrations in biomass hydrolysates. Moreover, inhibition of growth is intensified by the presence of increasing concentrations of the produced bioethanol. Thus, engineering of resistance to growth inhibitors, including product inhibition, is essential to improve bioethanol yields while decreasing the need to detoxify the cellulosic feedstock. Here, we apply the Cytostat cell culture technique, developed in our lab, to screen for acetate and ethanol resistance phenotypes in Saccharomyces cerevisiae cells transformed with a genomic library, designed to investigate the effects of gene over-expression from native promoters. The main advantages of this approach are that, first, the Cytostat selects for inhibitor specific resistance because the continuous culture is maintained at very low cell densities, and thus the composition of the growth medium remains constant throughout the experiment. This circumvents the possibility of resistance arising from the metabolic consumption or modification of the inhibitor, such that the resistance phenotype must be established by a sustainable mechanism in response to the constant challenge. Second, identifying the genetic modification only requires the sequencing of a small genomic fragment from the library, not whole genome sequencing. Using this approach, acetic acid resistant transformants, with double the growth rate of the WT strain in minimal medium supplemented with 40 mM acetic acid, were rapidly enriched in a Cytostat from a pool of cells transformed with the library. Similarly, ethanol resistant transformants were isolated in minimal medium supplemented with 2.5% v/v ethanol, with nearly 40% improved growth rates over the WT. The roles of the over-expressed genes in the resistance phenotypes, including the kinetics of glucose consumption and ethanol production, cell size homeostasis, and viability will be presented.