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

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.

Exploring the xylose paradox in Saccharomyces cerevisiae through in vivo sugar signalomics of targeted deletants.

BACKGROUND: There have been many successful strategies to implement xylose metabolism in Saccharomyces cerevisiae, but no effort has so far enabled xylose utilization at rates comparable to that of glucose (the preferred sugar of this yeast). Many studies have pointed towards the engineered yeast not sensing that xylose is a fermentable carbon source despite growing and fermenting on it, which is paradoxical. We have previously used fluorescent biosensor strains to in vivo monitor the sugar signalome in yeast engineered with xylose reductase and xylitol dehydrogenase (XR/XDH) and have established that S. cerevisiae senses high concentrations of xylose with the same signal as low concentration of glucose, which may explain the poor utilization. RESULTS: In the present study, we evaluated the effects of three deletions (ira2∆, isu1∆ and hog1∆) that have recently been shown to display epistatic effects on a xylose isomerase (XI) strain. Through aerobic and anaerobic characterization, we showed that the proposed effects in XI strains were for the most part also applicable in the XR/XDH background. The ira2∆isu1∆ double deletion led to strains with the highest specific xylose consumption- and ethanol production rates but also the lowest biomass titre. The signalling response revealed that ira2∆isu1∆ changed the low glucose-signal in the background strain to a simultaneous signalling of high and low glucose, suggesting that engineering of the signalome can improve xylose utilization. CONCLUSIONS: The study was able to correlate the previously proposed beneficial effects of ira2∆, isu1∆ and hog1∆ on S. cerevisiae xylose uptake, with a change in the sugar signalome. This is in line with our previous hypothesis that the key to resolve the xylose paradox lies in the sugar sensing and signalling networks. These results indicate that the future engineering targets for improved xylose utilization should probably be sought not in the metabolic networks, but in the signalling ones.

Assessing the effect of d-xylose on the sugar signaling pathways of Saccharomyces cerevisiae in strains engineered for xylose transport and assimilation.

One of the challenges of establishing an industrially competitive process to ferment lignocellulose to value-added products using Saccharomyces cerevisiae is to get efficient mixed sugar fermentations. Despite successful metabolic engineering strategies, the xylose assimilation rates of recombinant S. cerevisiae remain significantly lower than for the preferred carbon source, glucose. Previously, we established a panel of in vivo biosensor strains (TMB371X) where different promoters (HXT1/2/4p; SUC2p, CAT8p; TPS1p/2p, TEF4p) from the main sugar signaling pathways were coupled with the yEGFP3 gene, and observed that wild-type S. cerevisiae cannot sense extracellular xylose. Here, we expand upon these strains by adding a mutated galactose transporter (GAL2-N376F) with improved xylose affinity (TMB372X), and both the transporter and an oxidoreductase xylose pathway (TMB375X). On xylose, the TMB372X strains displayed population heterogeneities, which disappeared when carbon starvation was relieved by the addition of the xylose assimilation pathway (TMB375X). Furthermore, the signal in the TMB375X strains on high xylose (50 g/L) was very similar to the signal recorded on low glucose (≤5 g/L). This suggests that intracellular xylose triggers a similar signal to carbon limitation in cells that are actively metabolizing xylose, in turn causing the low assimilation rates.

Characterization of Clostridium thermocellum (B8) secretome and purified cellulosomes for lignocellulosic biomass degradation.

The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC-MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren't detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70°C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70°C and PC showed remarkable thermostability at 50 and 60°C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50°C.