title: Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin.
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.
authors.affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.
authors.forename: Stephanie P
authors.initials: SP
authors.lastname: Cartwright
authors.affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.|Thistle Scientific Ltd, Glasgow, G71 6NZ, UK.
authors.forename: Richard A J
authors.initials: RA
authors.lastname: Darby
authors.affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.
authors.forename: Debasmita
authors.initials: D
authors.lastname: Sarkar
authors.affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.|Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, 41296, Sweden.
authors.forename: Nicklas
authors.initials: N
authors.lastname: Bonander
authors.affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.
authors.forename: Stephane R
authors.initials: SR
authors.lastname: Gross
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, UK.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Ashe
authors.affiliation: School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK. r.m.bill@aston.ac.uk.
authors.forename: Roslyn M
authors.identifier: http://orcid.org/0000-0003-1331-0852
authors.initials: RM
authors.lastname: Bill
mesh_terms: D020121:5' Untranslated Regions; D051398:Aquaporin 1 / Q000096:biosynthesis* / Q000235:genetics*; D018388:Codon, Terminator; D004318:Doxycycline / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins / Q000235:genetics; D016366:Open Reading Frames; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011132:Polyribosomes; D043705:Receptor, Adenosine A2A / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D018388:Codon, Terminator; D043705:Receptor, Adenosine A2A; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D051398:Aquaporin 1; D049452:Green Fluorescent Proteins; D004318:Doxycycline
keywords: Aquaglyceroporin; Fps1 regulation; Leaky scanning; Recombinant protein; Saccharomyces cerevisiae; Translation initiation; Upstream open reading frame