Cartwright, Stephanie P., Darby, Richard A.J., Sarkar, Debasmita, Bonander, Nicklas, Gross, Stephane R., Ashe, Mark P. and Bill, Roslyn M. (2017). Constitutively-stressed yeast strains are high-yielding for recombinant Fps1:implications for the translational regulation of an aquaporin. Microbial Cell Factories, 16 (1),
Abstract
Background: 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. Results: 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. Conclusions: 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.
Publication DOI: | https://doi.org/10.1186/s12934-017-0656-2 |
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Dataset DOI: | https://doi.org/10.17036/researchdata.aston.ac.uk.00000176 |
Divisions: | College of Health & Life Sciences College of Health & Life Sciences > School of Biosciences College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine |
Additional Information: | © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
Uncontrolled Keywords: | aquaglyceroporin,Fps1 regulation,leaky scanning,recombinant protein,saccharomyces cerevisiae,translation initiation,upstream open reading frame,Biotechnology,Bioengineering,Applied Microbiology and Biotechnology |
Publication ISSN: | 1475-2859 |
Last Modified: | 02 Oct 2024 07:16 |
Date Deposited: | 15 Mar 2017 13:55 |
Full Text Link: | |
Related URLs: |
http://www.scop ... tnerID=8YFLogxK
(Scopus URL) |
PURE Output Type: | Article |
Published Date: | 2017-03-09 |
Accepted Date: | 2017-03-09 |
Submitted Date: | 2016-10-04 |
Authors: |
Cartwright, Stephanie P.
Darby, Richard A.J. Sarkar, Debasmita Bonander, Nicklas Gross, Stephane R. ( 0000-0002-0867-8866) Ashe, Mark P. Bill, Roslyn M. ( 0000-0003-1331-0852) |