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


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Article Info

DATE

2017-03-09

AUTHORS

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

ABSTRACT

BackgroundWe 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.ResultsPolysome 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.ConclusionsRegulation 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. More... »

PAGES

41

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s12934-017-0656-2

DOI

http://dx.doi.org/10.1186/s12934-017-0656-2

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https://app.dimensions.ai/details/publication/pub.1084251163

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/28279185


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30 schema:description BackgroundWe 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.ResultsPolysome 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.ConclusionsRegulation 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.
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37 BackgroundWe
38 FPS1 gene
39 Fps1
40 Further studies
41 GCN4
42 ORF
43 ORF upstream
44 Saccharomyces
45 ability
46 addition
47 additional strains
48 adenosine receptor 2A
49 aquaporins
50 base pairs
51 block
52 cell factories
53 cells
54 clear block
55 codon
56 conditions
57 constitutive phosphorylation
58 constructs
59 control
60 deletion
61 eIF2α
62 eukaryotic initiation factor
63 experiments
64 expression
65 expression constructs
66 expression plasmid
67 factors
68 factory
69 fluorescent protein
70 frame
71 genes
72 green fluorescent protein
73 high levels
74 high yields
75 horseradish peroxidase
76 implications
77 initiation
78 initiation block
79 initiation factors
80 investigation
81 leaky scanning
82 levels
83 low levels
84 mechanism
85 microbial cell factories
86 molecular mechanisms
87 normal conditions
88 pairs
89 part
90 peroxidase
91 phenotype
92 phosphorylation
93 plasmid
94 presence
95 primary ORF
96 production
97 protein
98 protein production
99 receptor 2A
100 recombinant protein production
101 recombinant yields
102 region
103 regulation
104 response
105 same plasmid
106 scanning
107 soluble green fluorescent protein
108 specific molecular mechanisms
109 state
110 stop codon
111 strains
112 strains of Saccharomyces
113 stress response
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115 stressed phenotype
116 study
117 sufficient yield
118 time
119 transcriptional activator GCN4
120 translation initiation
121 translational control
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123 translational state
124 uORF1
125 uORF2
126 uORFs
127 untranslated region
128 upstream
129 wild-type cells
130 yeast
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