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Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin

Overview of attention for article published in Microbial Cell Factories, March 2017
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  • Above-average Attention Score compared to outputs of the same age and source (64th percentile)

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Title
Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin
Published in
Microbial Cell Factories, March 2017
DOI 10.1186/s12934-017-0656-2
Pubmed ID
Authors

Stephanie P. Cartwright, Richard A. J. Darby, Debasmita Sarkar, Nicklas Bonander, Stephane R. Gross, Mark P. Ashe, Roslyn M. Bill

Abstract

We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5' untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10-80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A2aR) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described. Polysome profiling experiments were used to analyze the translational state of spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); all but gcn5Δ were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31aΔ, rpl22aΔ, ssf1Δ and nop1Δ) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2α. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5'UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3Δ or wild-type cells, suggesting that high-yielding strains are able to bypass 5'uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories. Regulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 13 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
China 1 8%
Austria 1 8%
Unknown 11 85%

Demographic breakdown

Readers by professional status Count As %
Researcher 5 38%
Student > Ph. D. Student 3 23%
Lecturer > Senior Lecturer 1 8%
Professor 1 8%
Student > Master 1 8%
Other 2 15%
Readers by discipline Count As %
Agricultural and Biological Sciences 6 46%
Biochemistry, Genetics and Molecular Biology 3 23%
Medicine and Dentistry 2 15%
Unknown 2 15%

Attention Score in Context

This research output has an Altmetric Attention Score of 2. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 21 April 2017.
All research outputs
#4,787,649
of 9,719,571 outputs
Outputs from Microbial Cell Factories
#392
of 807 outputs
Outputs of similar age
#119,658
of 255,649 outputs
Outputs of similar age from Microbial Cell Factories
#6
of 17 outputs
Altmetric has tracked 9,719,571 research outputs across all sources so far. This one is in the 49th percentile – i.e., 49% of other outputs scored the same or lower than it.
So far Altmetric has tracked 807 research outputs from this source. They receive a mean Attention Score of 3.6. This one has gotten more attention than average, scoring higher than 50% of its peers.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 255,649 tracked outputs that were published within six weeks on either side of this one in any source. This one has gotten more attention than average, scoring higher than 51% of its contemporaries.
We're also able to compare this research output to 17 others from the same source and published within six weeks on either side of this one. This one has gotten more attention than average, scoring higher than 64% of its contemporaries.