Extension of the substrate range is among one of the metabolic engineering goals for microorganisms used in biotechnological processes because it enables the use of a wide range of raw materials as substrates. One of the most prominent examples is the engineering of baker’s yeast Saccharomyces cerevisiae for the utilization of d-xylose, a five-carbon sugar found in high abundance in lignocellulosic biomass and a key substrate to achieve good process economy in chemical production from renewable and non-edible plant feedstocks. Despite many excellent engineering strategies that have allowed recombinant S. cerevisiae to ferment d-xylose to ethanol at high yields, the consumption rate of d-xylose is still significantly lower than that of its preferred sugar d-glucose. In mixed d-glucose/d-xylose cultivations, d-xylose is only utilized after d-glucose depletion, which leads to prolonged process times and added costs. Due to this limitation, the response on d-xylose in the native sugar signaling pathways has emerged as a promising next-level engineering target. Here we review the current status of the knowledge of the response of S. cerevisiae signaling pathways to d-xylose. To do this, we first summarize the response of the native sensing and signaling pathways in S. cerevisiae to d-glucose (the preferred sugar of the yeast). Using the d-glucose case as a point of reference, we then proceed to discuss the known signaling response to d-xylose in S. cerevisiae and current attempts of improving the response by signaling engineering using native targets and synthetic (non-native) regulatory circuits.

In Saccharomyces cerevisiae, pH homeostasis is reliant on ATP due to the use of proton-translocating ATPase (H⁺-ATPase) which constitutes a major drain within cellular ATP supply. Here, an exogenous proton-translocating pyrophosphatase (H⁺-PPase) from Arabidopsis thaliana, which uses inorganic pyrophosphate (PP_i) rather than ATP, was evaluated for its effect on reducing the ATP burden. The H⁺-Ppase was localized to the vacuolar membrane or to the cell membrane, and their impact was studied under acetate stress at a low pH. Biosensors (pHluorin and mQueen-2m) were used to observe changes in intracellular pH (pH_i) and ATP levels during growth on either glucose or xylose. A significant improvement of 35% in the growth rate at a pH of 3.7 and 6 g·L⁻¹ acetic acid stress was observed in the vacuolar membrane H⁺-PPase strain compared to the parent strain. ATP levels were elevated in the same strain during anaerobic glucose and xylose fermentations. During anaerobic xylose fermentations, co-expression of pHluorin and a vacuolar membrane H⁺-PPase improved the growth characteristics by means of an improved growth rate (11.4%) and elongated logarithmic growth duration. Our study identified a potential method for improving productivity in the use of S. cerevisiae as a cell factory under the harsh conditions present in industry.

Unlike glucose, the sub-optimal xylose utilization in recombinant Saccharomyces cerevisiae strains may stem from an unusual signaling response that is not adapted to detecting xylose as a fermentable substrate. We hypothesize that the membrane receptor Snf3p, known for sensing extracellular low glucose levels, may contribute to xylose recognition. To test this, we explored the effect of SNF3 inactivation and overexpression by measuring the response of the HXT2p-GFP biosensor integrated into S. cerevisiae strains with heterogeneous xylose assimilation and metabolism capacities. We showed that the absence of SNF3 effectively reduced HXT2p induction, while its overexpression improved signaling in the presence of xylose, suggesting the involvement of the receptor in the extracellular detection of this sugar. Although we attempted to engineer a xylose sensing system based on a chimeric receptor, its integration did not lead to considerable improvements in signal activation, indicating the need for further investigation. Finally, we showed that triggering the Snf3p pathway impacted xylose metabolism, with altered receptor levels prompting shifts in both biomass production and metabolite accumulation. Our findings suggest that understanding xylose sensing and its metabolic connection is essential for promoting more efficient xylose utilization in S. cerevisiae, a key step toward optimizing industrial bioprocesses.