Regulon engineering for rapid growth of Saccharomyces cerevisiae on non-native sugars
Endalur Gopinarayanan, Venkatesh.
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 ... read moreof substrate 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.
Keyword: Bioengineering.read less