Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica
Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica
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DOI：
10.1007/s00253-012-4421-5
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发表时间：
2013-04-01
影响因子：
5
通讯作者：
Alper, Hal S.
中科院分区：
文献类型：
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作者：
Blazeck, John;Reed, Ben;Alper, Hal S.
各种和强大的启动子对于任何宿主生物的代谢和途径工程应用都是必不可少的。为了实现这种能力，在这里我们展示了一种可推广的方法，用于从头构建强，合成的混合动力启动子库。具体而言，我们证明了如何利用启动子截断和碎片解剖分析来识别新型上游激活序列（UAS）和核心启动子 - 生成混合动力启动子所需的两个组件。作为基本情况，检查了Yarrowia脂溶液中的本机TEF启动子，以鉴定有假定的UAS元素，这些元素是模块化的合成转录激活剂。由新分离的230个核苷酸UAS（TEF）＃2元素激活的核心启动子区域的合成启动子，该区域被激活，显示了启动子强度的3至4.5倍。通过转录因子结合位点废除的进一步分析表明，对于完整的UAS（TEF）＃2功能，GCR1P结合位点是必需的。这些各种启动子在各种碳源中的功能进行了测试。最后，通过将不同的UAS元素（在这种情况下为UAS（TEF）和UAS1B）相结合，我们开发了一个高强度启动子，用于Y. lipolytica，其表达水平近7倍，高于强，组成型TEF启动子的表达水平。因此，此处描述的一般策略可以使合成启动子的从头构造有效地构建既增加天然表达能力，又可以产生可调基因表达的文库。
Both varied and strong promoters are essential for metabolic and pathway engineering applications in any host organism. To enable this capacity, here we demonstrate a generalizable method for the de novo construction of strong, synthetic hybrid promoter libraries. Specifically, we demonstrate how promoter truncation and fragment dissection analysis can be utilized to identify both novel upstream activating sequences (UAS) and core promoters-the two components required to generate hybrid promoters. As a base case, the native TEF promoter in Yarrowia lipolytica was examined to identify putative UAS elements that serve as modular synthetic transcriptional activators. Resulting synthetic promoters containing a core promoter region activated by between one and twelve tandem repeats of the newly isolated, 230 nucleotide UAS(TEF)#2 element showed promoter strengths 3- to 4.5-fold times the native TEF promoter. Further analysis through transcription factor binding site abrogation revealed the GCR1p binding site to be necessary for complete UAS(TEF)#2 function. These various promoters were tested for function in a variety of carbon sources. Finally, by combining disparate UAS elements (in this case, UAS(TEF) and UAS1B), we developed a high-strength promoter with for Y. lipolytica with an expression level of nearly sevenfold higher than that of the strong, constitutive TEF promoter. Thus, the general strategy described here enables the efficient, de novo construction of synthetic promoters to both increase native expression capacity and to produce libraries for tunable gene expression.