Department of Chemical Engineering, The University of Texas at Austin, 1 University Station, Austin, TX 78712, USA.
Biotechnol Bioeng. 2012 Nov;109(11):2884-95. doi: 10.1002/bit.24552. Epub 2012 May 17.
A dynamic range of well-controlled constitutive and tunable promoters are essential for metabolic engineering and synthetic biology applications in all host organisms. Here, we apply a synthetic hybrid promoter approach for the creation of strong promoter libraries in the model yeast, Saccharomyces cerevisiae. Synthetic hybrid promoters are composed of two modular components-the enhancer element, consisting of tandem repeats or combinations of upstream activation sequences (UAS), and the core promoter element. We demonstrate the utility of this approach with three main case studies. First, we establish a dynamic range of constitutive promoters and in doing so expand transcriptional capacity of the strongest constitutive yeast promoter, P(GPD) , by 2.5-fold in terms of mRNA levels. Second, we demonstrate the capacity to impart synthetic regulation through a hybrid promoter approach by adding galactose activation and removing glucose repression. Third, we establish a collection of galactose-inducible hybrid promoters that span a nearly 50-fold dynamic range of galactose-induced expression levels and increase the transcriptional capacity of the Gal1 promoter by 15%. These results demonstrate that promoters in S. cerevisiae, and potentially all yeast, are enhancer limited and a synthetic hybrid promoter approach can expand, enhance, and control promoter activity.
在所有宿主生物中，代谢工程和合成生物学应用都需要具有良好控制的组成型和可调启动子的动态范围。在这里，我们应用一种合成杂交启动子方法在模型酵母酿酒酵母中创建强启动子文库。合成杂交启动子由两个模块化组件组成 - 增强元件，由串联重复或上游激活序列（UAS）的组合组成，以及核心启动子元件。我们通过三个主要案例研究证明了这种方法的实用性。首先，我们建立了组成型启动子的动态范围，从而在 mRNA 水平上将最强的组成型酵母启动子 P（GPD）的转录能力扩展了 2.5 倍。其次，我们通过添加半乳糖激活和去除葡萄糖抑制来证明通过杂交启动子方法赋予合成调节的能力。第三，我们建立了一个半乳糖诱导的杂交启动子集合，其半乳糖诱导表达水平的动态范围跨越近 50 倍，并将 Gal1 启动子的转录能力提高了 15%。这些结果表明，酿酒酵母中的启动子，以及可能所有酵母中的启动子，都受到增强子限制，并且合成杂交启动子方法可以扩展、增强和控制启动子活性。
