Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin.
Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin.
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DOI：
10.1186/s12934-017-0656-2
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发表时间：
2017-03-09
影响因子：
6.4
通讯作者：
Bill RM
中科院分区：
文献类型：
--
作者：
Cartwright SP;Darby RA;Sarkar D;Bonander N;Gross SR;Ashe MP;Bill RM
我们先前选择了四株酿酒酵母的能力，生产水通道蛋白Fps 1在足够的产量为进一步研究。用含有249个碱基对的5′非翻译区（UTR）和一级FPS 1开放阅读框（ORF）的表达质粒转化的酵母菌株spt 3 Δ、srb 5 Δ、gcn 5 Δ和yTHCBMS 1（补充有0.5 μg/mL强力霉素）的产量比表达相同质粒的野生型细胞的产量高10-80倍。其中一种菌株增加了G蛋白偶联受体腺苷受体2a（A2 aR）和可溶性绿色荧光蛋白（GFP）的重组产量。支持高产Fps 1表型的特定分子机制仍不完全描述。使用多核糖体分析实验来分析spt 3 Δ、srb 5 Δ、gcn 5 Δ和yTHCBMS 1（补充有0.5 μg/mL多西环素）的翻译状态;发现除了gcn 5 Δ之外的所有蛋白在翻译起始中表现出明显的阻断。与野生型相比，具有已知起始阻断的四种另外的菌株（rpl 31 a Δ、rpl 22 a Δ、ssf 1 Δ和nop 1 Δ）也提高了重组Fps 1的产量。真核转录激活因子GCN 4的表达在spt 3 Δ、srb 5 Δ、gcn 5 Δ和yTHCBMS 1（补充0.5 μg/mL多西环素）中增加;这四种菌株还表现出真核起始因子eIF 2 α的组成性磷酸化。这两种反应都指示组成性应激表型。对表达载体中FPS 1的5′UTR的研究显示，在原始ORF上游有两个未翻译的ORF（uORF 1和uORF 2）。删除uORF 1或uORF 1和uORF 2进一步提高了我们的四个菌株的重组产量;从野生型细胞中获得的uORF删除的产量最高。将天然uORF（uORF 2）的终止密码子移码，使其延伸到FPS 1 ORF中，并没有显著改变spt 3 Δ或野生型细胞中的Fps 1产量，这表明高产菌株能够通过泄漏扫描绕过FPS 1基因中的5′ uORF，这是一种已知的应激反应机制。重组A2 aR，GFP和辣根过氧化物酶的产量可以在一种或多种酵母菌株中得到改善，这表明应激表型在高产细胞工厂中也可能是重要的。通过翻译控制调节酵母中的Fps 1水平可能在功能上是重要的;在正常条件下可能需要天然uORF（uORF 2）的存在来维持低水平的Fps 1，但作为应激反应的一部分，可能需要更高的水平。组成性应激酵母菌株可能是用于重组蛋白生产的有用的高产微生物细胞工厂。本文的在线版本（doi：10.1186/s12934-017-0656-2）包含补充材料，可供授权用户使用。
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. 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. 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. The online version of this article (doi:10.1186/s12934-017-0656-2) contains supplementary material, which is available to authorized users.