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Real-time monitoring of the sugar sensing in Saccharomyces cerevisiae indicates endogenous mechanisms for xylose signaling

Brink, Daniel P LU ; Borgström, Celina LU ; Tueros, Felipe G LU and Gorwa-Grauslund, Marie F LU (2016) In Microbial Cell Factories 15(183).
Abstract
The 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.
Results
The 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... (More)
The 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.
Results
The 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.
Conclusions
Once 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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Saccharomyces cerevisiae , Biosensor, Sugar sensing, Signaling, Xylose, GFP, cAMP/PKA, Snf3p/Rgt2p, SNF1/ Mig1p, Flow cytometry
in
Microbial Cell Factories
volume
15
issue
183
pages
17 pages
publisher
BioMed Central
external identifiers
  • wos:000385987100001
  • scopus:84992336030
ISSN
1475-2859
DOI
10.1186/s12934-016-0580-x
language
English
LU publication?
yes
id
5c5c9df3-caad-417c-8afb-8657ef5093b8
date added to LUP
2016-10-28 11:24:33
date last changed
2017-10-22 05:21:09
@article{5c5c9df3-caad-417c-8afb-8657ef5093b8,
  abstract     = {The 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.<br/>Results<br/>The 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.<br/>Conclusions<br/>Once 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.},
  author       = {Brink, Daniel P and Borgström, Celina and Tueros, Felipe G and Gorwa-Grauslund, Marie F},
  issn         = {1475-2859},
  keyword      = {Saccharomyces cerevisiae ,Biosensor,Sugar sensing,Signaling,Xylose,GFP,cAMP/PKA, Snf3p/Rgt2p,SNF1/ Mig1p,Flow cytometry},
  language     = {eng},
  month        = {10},
  number       = {183},
  pages        = {17},
  publisher    = {BioMed Central},
  series       = {Microbial Cell Factories},
  title        = {Real-time monitoring of the sugar sensing in Saccharomyces cerevisiae indicates endogenous mechanisms for xylose signaling},
  url          = {http://dx.doi.org/10.1186/s12934-016-0580-x},
  volume       = {15},
  year         = {2016},
}