McIsaac R Scott, Gibney Patrick A, Chandran Sunil S, Benjamin Kirsten R, Botstein David
The Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA, Amyris, Inc., Emeryville, CA 94608, USA, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA and Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Nucleic Acids Res. 2014 Apr;42(6):e48. doi: 10.1093/nar/gkt1402. Epub 2014 Jan 20.
A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input-output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
最近为酵母开发了一种快速起效、可调节且具有单基因特异性的条件基因表达系统。只要细胞表达人工转录因子Z3EV（Zif268 DNA结合结构域、人雌激素受体的配体结合结构域和病毒蛋白16的融合体），置于含有六个Zif268结合序列（单核苷酸间隔）的修饰GAL1启动子直接下游的基因在β-雌二醇存在下就会被选择性诱导。我们表明，可以使用简单的设计原则来修改Z3EV反应性启动子的强度。通过将Zif268结合位点移向转录起始位点，表达量可几乎翻倍。尽管报道雌激素受体二聚化是激素依赖性激活所必需的，但单个结合位点就足以激活靶基因。靶基因表达水平与启动子结合位点拷贝数相关，我们设计了一组具有不同输入-输出特性的可诱导启动子框架。最后，诱导剂特性与基因激活之间的偶联是灵活的：Z3EV的配体特异性可以重新编程以响应非激素小分子，只需在人雌激素受体结构域中进行五个氨基酸替换，这可能对工业应用有用。
