A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering.
A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering.
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DOI：
10.1002/yea.3292
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发表时间：
2018-03
期刊：
影响因子：
--
通讯作者：
Mukhopadhyay A
中科院分区：
文献类型：
--
作者：
Dossani ZY;Reider Apel A;Szmidt-Middleton H;Hillson NJ;Deutsch S;Keasling JD;Mukhopadhyay A
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仍然依赖于一些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的进展，我们建立了一个由合成转录因子调节的混合启动子库。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替换。相应地，合成转录因子 (TF) 由 LexA 蛋白的 DNA 结合结构域、与人雌激素结合结构域和病毒激活结构域 VP16 融合组成。由此产生的具有细菌 DNA 结合结构域的系统避免了天然酿酒酵母基因的转录，并且可以使用雌二醇诱导杂合启动子，雌二醇是一种对酿酒酵母生理学没有可检测到的影响的化合物。使用一个、两个或三个操纵子序列重复和一组天然酿酒酵母启动子的组合，我们获得了一系列杂合启动子，可以使用相同的合成TF和给定的雌二醇将其诱导至不同水平。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调节多个基因或途径，达到多个所需水平。
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.
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