Using phosphoglucose isomerase-deficient (pgi1Δ) Saccharomyces cerevisiae to map the impact of sugar phosphate levels on d-glucose and d-xylose sensing

Abstract

Background Despite decades of engineering efforts, recombinant Saccharomyces cerevisiae are still less efficient at converting d-xylose sugar to ethanol compared to the preferred sugar d-glucose. Using GFP-based biosensors reporting for the three main sugar sensing routes, we recently demonstrated that the sensing response to high concentrations of d-xylose is similar to the response seen on low concentrations of d-glucose. The formation of glycolytic intermediates was hypothesized to be a potential cause of this sensing response. In order to investigate this, glycolysis was disrupted via the deletion of the phosphoglucose isomerase gene (PGI1) while intracellular sugar phosphate levels were monitored using a targeted metabolomic approach. Furthermore, the sugar sensing of the PGI1 deletants was compared to the PGI1-wildtype strains in the presence of various types and combinations of sugars. Results Metabolomic analysis revealed systemic changes in intracellular sugar phosphate levels after deletion of PGI1, with the expected accumulation of intermediates upstream of the Pgi1p reaction on d-glucose and downstream intermediates on d-xylose. Moreover, the analysis revealed a preferential formation of d-fructose-6-phosphate from d-xylose, as opposed to the accumulation of d-fructose-1,6-bisphosphate that is normally observed when PGI1 deletants are incubated on d-fructose. This may indicate a role of PFK27 in d-xylose sensing and utilization. Overall, the sensing response was different for the PGI1 deletants, and responses to sugars that enter the glycolysis upstream of Pgi1p (d-glucose and d-galactose) were more affected than the response to those entering downstream of the reaction (d-fructose and d-xylose). Furthermore, the simultaneous exposure to sugars that entered upstream and downstream of Pgi1p (d-glucose with d-fructose, or d-glucose with d-xylose) resulted in apparent synergetic activation and deactivation of the Snf3p/Rgt2p and cAMP/PKA pathways, respectively. Conclusions Overall, the sensing assays indicated that the previously observed d-xylose response stems from the formation of downstream metabolic intermediates. Furthermore, our results indicate that the metabolic node around Pgi1p and the level of d-fructose-6-phosphate could represent attractive engineering targets for improved d-xylose utilization.

Evaluation of Pyrophosphate-Driven Proton Pumps in Saccharomyces cerevisiae under Stress Conditions

“…All flow cytometry analyses for growth studies in bioreactors were carried out with a MACSQuant ® VYB flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) applying a flow rate of 50 µL·min −1 and a threshold of 0.83 on the forward scatter. The cell suspensions were diluted to an OD of 0.1 to 0.2 using PBS with 1.32 µg·mL −1 of propidium iodide [ 51 ]. The measurement of the fluorescence for the biosensors was started within 10 min of sampling.…”

Impact of xylose epimerase on sugar assimilation and sensing in recombinant Saccharomyces cerevisiae carrying different xylose-utilization pathways

“…Incorporation of the XR/XDH pathway allowed the cells to partially activate the sugar signaling pathways, evident from the induction of SUC2 p which is under the control of the SNF1/Mig1p nutrient sensing pathway [ 21 ]. Most likely, the intracellular metabolites responsible for this activation are glycolytic intermediates such as fructose-6-phosphate [ 22 ]. However, the potential redox imbalance in XR/XDH strains, caused by the promiscuous co-factor preference of XR, may also influence the SNF1/Mig1p sugar signaling pathway via crosstalk from redox-sensitive pathways.…”