Abstract: The rapid development of industries and decreasing fossil fuel reserves appeals for cleaner and sustainable methods for eco-friendly, industrial scale production of chemicals using biochemical methods with cheap, abundant and readily available feedstocks. However, not all industrially relevant microbes can metabolize these carbon sources, thereby requiring heterologous incorporation of s... read moreubstrate assimilation pathways. One such model organism, Saccharomyces cerevisiae (baker's yeast), has been widely used in diverse applications, from bread and wine making, to production of industrially relevant chemicals and high-value pharmaceuticals. However, it cannot metabolize pentoses, which make up a significant portion of lignocellulose. Substantial research years have been expended on engineering pentose metabolism in S. cerevisiae. To tackle the problem, researchers have taken a direct approach of constitutively overexpressing necessary catabolic enzymes to direct flux towards glycolysis. However, in stark contrast, native sugar metabolism is usually carefully regulated using sensing, signaling and metabolic components using regulatory systems referred to as regulons. In this work, I analyzed a well characterized natural sugar detection and assimilation system in yeast, the galactose (GAL) regulon, and compared it with engineering methods used for pentose metabolism. From literature review, we hypothesized that downstream genes of the GAL regulon might enhance growth on pentose. As the role of the downstream GAL regulon genes are uncharacterized, we uncoupled regulation from metabolism and demonstrated that these genes are essential for rapid growth of yeast on galactose. To make use of these genes for growth on xylose, we systematically re-engineered every component of GAL regulon resulting in high aerobic growth rates and cell densities on xylose. Transcriptomics analyses re-affirmed our hypothesis that downstream genes of GAL regulon required for growth, also get upregulated in the xylose regulon. We extended this approach for a second substrate, arabinose, and demonstrate the general applicability of this strategy. Finally, we re-designed the regulon engineering technique to construct a platform strain that obviates the need to re-engineer multiple components of the regulon for metabolizing a non-native substrate. Using this approach, we show enhanced growth in sugars, irrespective of whether they are detected by the regulon. Overall, this thesis deals with the need for new strategies to engineer non-native substrate utilization and provides a powerful and easy-to-implement 'Regulon Engineering' strategy in this yeast as a potential paradigm.
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: Nikhil Nair.
Committee: Kyongbum Lee, Joshua Kritzer, and Lee Lynd.