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Robust and rapid xylose utilization is essential for developing sustainable bioprocesses capable of converting pentose-rich lignocellulosic biomass into renewable chemicals. Saccharomyces cerevisiae is a popular choice for industrial bioprocesses due to its high product yields, robust performance, and stress tolerance. Unfortunately, S. cerevisiae does not natively utilize xylose. Decades of research have led to the design of engineering strategies that enable and substantially improve the conversion of xylose, but further improvements are still needed to effectively compete with petroleum-based alternatives. Efforts have mostly targeted individual enzymes and proteins related to the central carbon metabolism, leading to a stepwise refinement over time. An alternative approach is to target the cellular regulatory network controlling the overall sugar utilization, enabling simultaneous modulation of dozens of downstream enzymes at once. This concept forms the central theme of my thesis. By investigating the response of S. cerevisiae regulatory networks to xylose, and subsequently engineering them to exhibit altered responses, I aimed to enhance xylose utilization for the production of sustainable biochemicals.

Three main pathways detect and respond to glucose and other carbon sources in S. cerevisiae: the Snf3p/Rgt2p pathway, the SNF1 pathway, and the PKA pathway. These pathways control sugar transport, alternative carbon utilization, and sugar feasting, respectively. Previous studies of these pathways have shown that xylose is not perceived as a rapidly fermentable sugar, as evidenced by the activation of the SNF1 pathway and lack of PKA activity. In my work, I found evidence that specific metabolic intermediates formed during xylose catabolism (fructose-6-phosphate in particular), and extracellular xylose itself for non-metabolizing cells, are likely behind this response. In addition, I showed that the response was independent from the xylose utilization pathway employed, and that the response occurred in both laboratory and industrial strains. As such, the response is a widespread phenomenon and represents an important target for future strain improvement.

In addition to this, I endeavored to design and implement xylose-specific receptors to co-stimulate all three pathways since this has been demonstrated to improve xylose utilization. Two separate approaches were pursued: 1) chimeric receptors were constructed by combining xylose-binding transporters with signaling domains to trigger the Snf3p/Rgt2p pathway, and 2) mutagenizing the PKA-activating G-protein coupled receptor Gpr1p. While the chimeric constructs initially showed promise, it was later revealed that the altered sugar signaling response was likely due to residual transport activity rather than signaling. In silico modelling of mutant Gpr1p candidates indicated potential for xylose binding, leading to the construction of a genetic library; however, screening of the library remains to be performed.

Overall, this thesis represents a step forward in the understanding of sugar signaling in S. cerevisiae on xylose and ways to alter it for improved pentose utilization.