School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK.
Thistle Scientific Ltd, Glasgow, G71 6NZ, UK.
Microb Cell Fact. 2017 Mar 9;16(1):41. doi: 10.1186/s12934-017-0656-2.
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 (AR) 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 AR, 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.
我们之前选择了四种酿酒酵母菌株，因为它们能够以足够的产量生产水通道蛋白 Fps1，以便进一步研究。与表达质粒相比，spt3Δ、srb5Δ、gcn5Δ 和 yTHCBMS1（添加 0.5μg/mL 强力霉素）的酵母菌株的产量增加了 10-80 倍，该质粒除了主要的 FPS1 开放阅读框（ORF）之外，还包含 249 个碱基对的 5'非翻译区（UTR）。其中一种菌株提高了 G 蛋白偶联受体腺苷受体 2a（AR）和可溶性绿色荧光蛋白（GFP）的重组产量。高产量 Fps1 表型的具体分子机制仍未完全描述。
多核糖体谱分析实验用于分析 spt3Δ、srb5Δ、gcn5Δ 和 yTHCBMS1（添加 0.5μg/mL 强力霉素）的翻译状态；除了 gcn5Δ 之外，所有菌株都发现翻译起始明显受阻。另外四个具有已知起始阻断的菌株（rpl31aΔ、rpl22aΔ、ssf1Δ 和 nop1Δ）与野生型相比，也提高了重组 Fps1 的产量。与野生型相比，spt3Δ、srb5Δ、gcn5Δ 和 yTHCBMS1（添加 0.5μg/mL 强力霉素）中真核转录激活因子 GCN4 的表达增加；这四种菌株还表现出真核起始因子 eIF2α的组成性磷酸化。这两种反应都表明存在一种持续的应激表型。在表达构建体中对 FPS1 的 5'UTR 进行研究，发现了在主要 ORF 上游的两个未翻译的 ORF（uORF1 和 uORF2）。删除任一个 uORF1 或 uORF1 和 uORF2 都进一步提高了我们四种菌株的重组产量；uORF 缺失的最高产量是从野生型细胞获得的。使天然 uORF（uORF2）的终止密码子发生移码，从而使其延伸到 FPS1 ORF 中，并没有显著改变 spt3Δ 或野生型细胞中 Fps1 的产量，这表明高产菌株能够通过渗漏扫描绕过 FPS1 基因中的 5'uORF，这是一种已知的应激反应机制。在一个或多个酵母菌株中，重组 AR、GFP 和辣根过氧化物酶的产量可以得到提高，这表明应激表型在高产细胞工厂中也可能很重要。
在酵母中，Fps1 水平的翻译控制可能具有重要的功能意义；天然 uORF（uORF2）的存在可能是在正常条件下维持低水平 Fps1 所必需的，但作为应激反应的一部分，需要更高水平的 Fps1。持续受到应激的酵母菌株可能是用于重组蛋白生产的高产微生物细胞工厂的有用工具。
