Discovery of Gene Overexpression Targets for Biofuel Product Tolerance in Yeast

Production of biofuels such as ethanol induces a potent stress on the microorganisms that produce them. Thus, engineering product tolerance is essential to improve yields and process efficiency.  We demonstrate the discovery of gene overexpression targets to significantly improve ethanol tolerance in Saccharomyces cerevisiae.  Using the Cytostat continuous culture technique we screened for a sustainable ethanol tolerance mechanism in a pool of S. cerevisiae cells transformed with a genome-wide overexpression library.  We discovered that an overlapping sequence containing genes HAP4 and SLD2 represented 90% of selected clones from a starting pool of 1,588, and that co-overexpressing these genes is sufficient to confer an increase of nearly 50% in both the growth rate and ethanol productivity under ethanol stress.  In addition, overexpression of these genes prevents the drastic effects of ethanol stress on cell size and the cell cycle.  Co-overexpression of HAP4 and SLD2 leads to significantly improved kinetics of glucose consumption and ethanol productivity during fed-batch growth under ethanol stress.  As demonstrated for the case of ethanol production the approach presents a general strategy for the improvement of a wide range of biotechnology processes.