Dossani Zain Y, Reider Apel Amanda, Szmidt-Middleton Heather, Hillson Nathan J, Deutsch Samuel, Keasling Jay D, Mukhopadhyay Aindrila
DOE Joint Bioenergy Institute, Emeryville, California, USA.
Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
Yeast. 2018 Mar;35(3):273-280. doi: 10.1002/yea.3292. Epub 2017 Dec 7.
Despite the need for inducible promoters in strain development efforts, the majority of engineering in Saccharomyces cerevisiae continues to rely on a few constitutively active or inducible promoters. Building on advances that use the modular nature of both transcription factors and promoter regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein. Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes, and the hybrid promoters can be induced using estradiol, a compound with no detectable impact on S. cerevisiae physiology. Using combinations of one, two or three operator sequence repeats and a set of native S. cerevisiae promoters, we obtained a series of hybrid promoters that can be induced to different levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain.
尽管在菌株开发工作中需要诱导型启动子，但酿酒酵母中的大多数工程改造仍继续依赖少数组成型活性或诱导型启动子。基于利用转录因子和启动子区域的模块化性质所取得的进展，我们构建了一个由合成转录因子调控的杂交启动子文库。杂交启动子由天然酿酒酵母启动子组成，其中操纵子区域已被细菌LexA DNA结合蛋白识别的序列所取代。相应地，合成转录因子（TF）由LexA蛋白的DNA结合结构域组成，与人类雌激素结合结构域和病毒激活结构域VP16融合。所得具有细菌DNA结合结构域的系统避免了天然酿酒酵母基因的转录，并且杂交启动子可以使用雌二醇诱导，雌二醇是一种对酿酒酵母生理学无明显影响的化合物。通过使用一个、两个或三个操纵序列重复序列与一组天然酿酒酵母启动子的组合，我们获得了一系列杂交启动子，使用相同的合成TF和给定的雌二醇可以将其诱导到不同水平。这组启动子与我们的合成TF相结合，有可能在单个菌株中同时将众多基因或途径调控到多个所需水平。
