Abstract
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.
Highlights
The sugar sensing and carbon catabolite repression in Baker’s yeast Saccharomyces cerevisiae is gov‐ erned by three major signaling pathways that connect carbon source recognition with transcriptional regulation
Design and construction of the yeast biosensor strains A panel of eight S. cerevisiae sugar-responsive biosensor strains were constructed by coupling select promoters from the three main sugar sensing pathways in this yeast (Snf3p/Rgt2p, SNF1/Mig1p and cAMP/PKA; Fig. 1) with a green fluorescent protein (GFP) gene followed by genomic integration
The biosensor expression cassettes were produced by cloning of endogenous S. cerevisiae promoters circa 1 kb upstream from the HXT1/2/4, SUC2, CAT8, TPS1/2 and TEF4 genes in front of the yEGFP3 cassette [27] in the YIplac211 vector [50]
Summary
The sugar sensing and carbon catabolite repression in Baker’s yeast Saccharomyces cerevisiae is gov‐ erned by three major signaling pathways that connect carbon source recognition with transcriptional regulation. The recombinant strains, despite being successfully engineered to utilize xylose, do not seem to recognize this carbon source as a fermentable sugar, as has been implied in multiple studies [13,14,15,16,17,18,19]. Taken together, these advances suggest something is lacking in the sensing and signaling of xylose in S. cerevisiae, and that this is a plausible bottleneck that has to be overcome in order to improve productivity of e.g. ethanol from xylose
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