Engineering Increased Tolerance in S. Cerevisiae Through Efflux Pump Addition

Abstract

Biofuels are of particular interest in the quest to find a reliable source of renewable energy. Currently, bioethanol is the primary biofuel in use, but due to a number of significant drawbacks that come with using ethanol as a fuel source, methods to increase heavy alcohol production in microorganisms have been closely researched. Despite successes in the field, a substantial drawback comes from the toxicity of byproducts and the final products to the microorganisms that produce them. This study investigated a potential method of increasing tolerance to different groups of toxins in S. cerevisiae. The method investigated was the addition of efflux pumps that are known to have a role in increasing tolerance in C. albicans. After a number of qualitative screening assays, potential sets of pumps that may be effective against specific toxins were found. Further individualized testing was performed using the candidates found in the previous assays using quantitative growth rate assays. Although the quantitative analysis did not display an increase in tolerance in the engineered strains, there exists a great deal of potential in continuation of the work presented here. The qualitative results, the methods used to test and analyze the effectiveness of various pumps against toxins, and the background characterization of optimal testing conditions will all serve as useful tools to future endeavors into determining the potential role of these efflux pumps in conferring increased resistance in S. cerevisiae.