Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
Joint BioEnergy Institute, Emeryville, CA 94608, USA.
Nucleic Acids Res. 2018 Nov 30;46(21):e127. doi: 10.1093/nar/gky734.
Functional characterization of regulatory DNA elements in broad genetic contexts is a prerequisite for forward engineering of biological systems. Translation initiation site (TIS) sequences are attractive to use for regulating gene activity and metabolic pathway fluxes because the genetic changes are minimal. However, limited knowledge is available on tuning gene outputs by varying TISs in different genetic and environmental contexts. Here, we created TIS hexamer libraries in baker's yeast Saccharomyces cerevisiae directly 5' end of a reporter gene in various promoter contexts and measured gene activity distributions for each library. Next, selected TIS sequences, resulted in almost 10-fold changes in reporter outputs, were experimentally characterized in various environmental and genetic contexts in both yeast and mammalian cells. From our analyses, we observed strong linear correlations (R2 = 0.75-0.98) between all pairwise combinations of TIS order and gene activity. Finally, our analysis enabled the identification of a TIS with almost 50% stronger output than a commonly used TIS for protein expression in mammalian cells, and selected TISs were also used to tune gene activities in yeast at a metabolic branch point in order to prototype fitness and carotenoid production landscapes. Taken together, the characterized TISs support reliable context-independent forward engineering of translation initiation in eukaryotes.
在广泛的遗传背景下对调控 DNA 元件进行功能表征是对生物系统进行正向工程设计的前提。翻译起始位点（TIS）序列因其遗传变化最小，因此是调节基因活性和代谢途径通量的理想选择。然而，在不同的遗传和环境背景下通过改变 TIS 来调整基因输出的知识有限。在这里，我们在酿酒酵母（Saccharomyces cerevisiae）中直接在报告基因的 5' 端的各种启动子背景下创建了 TIS 六聚体文库，并测量了每个文库的基因活性分布。接下来，我们选择了 TIS 序列，这些序列导致报告基因输出几乎发生了 10 倍的变化，并在酵母和哺乳动物细胞中的各种环境和遗传背景下进行了实验表征。从我们的分析中，我们观察到 TIS 顺序和基因活性之间的所有两两组合之间存在很强的线性相关性（R2 = 0.75-0.98）。最后，我们的分析确定了一个 TIS，其输出比哺乳动物细胞中常用的用于蛋白质表达的 TIS 强近 50%，并选择了 TIS 来调整酵母中代谢分支点的基因活性，以原型适应度和类胡萝卜素生产景观。总之，所鉴定的 TIS 支持真核生物中翻译起始的可靠、独立于上下文的正向工程设计。
