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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 yTHC BMS1  (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 (A 2a R) 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 yTHC BMS1  (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 yTHC BMS1  (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 A 2a R, 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.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-017-0656-2) contains supplementary material, which is available to authorized users.

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