Metabolic Engineering of Xylose-Fermenting Yeast: Isobutanol Production and an Investigation of Stability

Abstract

Biofuels have become an increasingly attractive alternative to traditional fossil fuels in the past few decades. Of particular interest are second generation biofuels as they are produced from renewable, cellulosic biomass rather than from feedstock as first generation biofuels were. One of the main components of lignocellulose is hemicellulose, which can consist of around 20% of the dry weight of lignocellulose. Composed of mainly xylose, hemicellulose is unable to be broken down by wild-type S. cerevisiae. The viability of using second generation biofuels in an industrial setting depends on the efficient breakdown of lignocellulose during fermentation reactions. Through the metabolic engineering of S. cerevisiae and introduction of enzymes for xylose assimilation, strains have been designed that are able to incorporate xylose. An important aspect of xylose assimilation investigated is the genomic stability of such strains as they are exposed to glucose rather than only xylose. Their subsequent growth rate and fermentation profiles after being inoculated in glucose are an important factor in deciding whether their use can be applied to an industrial setting. The main product currently produced during biofuel production is ethanol. The effective utilization of ethanol, however, is limited as it can only be blended into the current petroleum infrastructure due to its low energy density comparative to gasoline. In addition to investigating the genomic stability of xylose-assimiliating strains, the introduction of isobutanol pathways within these strains was explored. The upregulation of genes within the valine biosynthetic pathway has been shown to drastically improve isobutanol production within S. cerevisiae, which could then be integrated directly into the current petroleum infrastructure without need for costly modifications.