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.
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. Electronic supplementary material The online version of this article (10.1186/s12934-019-1141-x) contains supplementary material, which is available to authorized users.
BackgroundThe sugar sensing and carbon catabolite repression in Baker’s yeast Saccharomyces cerevisiae is governed by three major signaling pathways that connect carbon source recognition with transcriptional regulation. Here we present a screening method based on a non-invasive in vivo reporter system for real-time, single-cell screening of the sugar signaling state in S. cerevisiae in response to changing carbon conditions, with a main focus on the response to glucose and xylose.ResultsThe artificial reporter system was constructed by coupling a green fluorescent protein gene (yEGFP3) downstream of endogenous yeast promoters from the Snf3p/Rgt2p, SNF1/Mig1p and cAMP/PKA signaling pathways: HXT1p/2p/4p; SUC2p, CAT8p; TPS1p/2p and TEF4p respectively. A panel of eight biosensors strains was generated by single copy chromosomal integration of the different constructs in a W303-derived strain. The signaling biosensors were validated for their functionality with flow cytometry by comparing the fluorescence intensity (FI) response in the presence of high or nearly depleted glucose to the known induction/repression conditions of the eight different promoters. The FI signal correlated with the known patterns of the selected promoters while maintaining a non-invasive property on the cellular phenotype, as was demonstrated in terms of growth, metabolites and enzyme activity.ConclusionsOnce verified, the sensors were used to evaluate the signaling response to varying conditions of extracellular glucose, glycerol and xylose by screening in 96-well microtiter plates. We show that these yeast strains, which do not harbor any recombinant pathways for xylose utilization, are lacking a signaling response for extracellular xylose. However, for the HXT2p/4p sensors, a shift in the flow cytometry population dynamics indicated that internalized xylose does affect the signaling. These results suggest that the previously observed effects of this pentose on the S. cerevisiae physiology and gene regulation can be attributed to xylose and not only to a lack of glucose.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0580-x) contains supplementary material, which is available to authorized users.
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.
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