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

Autor: Richard A. J. Darby, Stephane R. Gross, Roslyn M. Bill, Debasmita Sarkar, Mark P. Ashe, Stephanie P. Cartwright, Nicklas Bonander
Jazyk: angličtina
Rok vydání: 2017
Předmět:
0301 basic medicine
Untranslated region
Saccharomyces cerevisiae Proteins
Recombinant protein
Receptor
Adenosine A2A
Genes
Fungal
Green Fluorescent Proteins
030106 microbiology
Saccharomyces cerevisiae
Bioengineering
Biology
Upstream open reading frame
Applied Microbiology and Biotechnology
Fps1 regulation
Open Reading Frames
03 medical and health sciences
Eukaryotic translation
Gene Expression Regulation
Fungal
Eukaryotic initiation factor
Translational regulation
Translation initiation
Peptide Chain Initiation
Translational
2. Zero hunger
Expression vector
Aquaporin 1
Research
Aquaglyceroporin
biology.organism_classification
Molecular biology
Recombinant Proteins
Open reading frame
030104 developmental biology
Doxycycline
Polyribosomes
Codon
Terminator
Leaky scanning
5' Untranslated Regions
Plasmids
Biotechnology
Zdroj: Cartwright, S P, Darby, R A J, Sarkar, D, Bonander, N, Gross, S R, Ashe, M P & Bill, R M 2017, ' Constitutively-stressed yeast strains are high-yielding for recombinant Fps1 : Implications for the translational regulation of an aquaporin ', Microbial Cell Factories, vol. 16, no. 1, 41 . https://doi.org/10.1186/s12934-017-0656-2
Microbial Cell Factories
ISSN: 1475-2859
DOI: 10.1186/s12934-017-0656-2
Popis: 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. 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.
Databáze: OpenAIRE
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