A toolkit for precise, multigene control in Saccharomyces cerevisiae

Systems that allow researchers to precisely control the expression of genes are fundamental to biological research, biotechnology, and synthetic biology. However, few inducible gene expression systems exist that can enable simultaneous, multi-gene control in the important model organism and chassis, Saccharomyces cerevisiae. Here, we repurposed ligand binding domains (LBDs) from mammalian Type I nuclear receptors to establish a family of up to five orthogonal synthetic gene expression systems in yeast. Our systems enable tight, independent, multi-gene control through the addition of inert hormones, and are capable of driving robust gene expression outputs. As a proof-of-principle, we placed expression of four enzymes from the violacein biosynthetic pathway under independent expression control to selectively route pathway flux. Our results establish a modular, versatile, and potentially expandable toolkit for multidimensional control of gene expression in yeast that can be used to constr..., These data were generated to support the development and characterization of a toolkit used to construct orthogonal, small molecule-inducible genetic programs in yeast. The toolkit is based on synthetic transcription factors derived from human type I nuclear receptors fused to zinc finger DNA binding domains and transcription activation domains. In the published work, we extensively characterized synthetic transcription factor performance and orthogonality. As a proof-of-concept, we modulated flux through the violacein biosynthetic pathway by selectively inducing expression of combinations of pathway enzymes. Our datasets, comprised of data gathered using flow cytometry and high-performance liquid chromatography, support the four major figures of our manuscript: The mechanism of action, features, and design of synthetic transcription factors for genetic circuit construction in yeast. The characterization of maximum induction, basal expression, and dose curve response for the toolkit e..., The raw datasets require FlowJo to open, and the processed data files require GraphPad Prism.能够让研究人员精准调控基因表达的系统，是生物学研究、生物技术与合成生物学的核心基础。然而，在重要的模式生物与底盘细胞（chassis）酿酒酵母（Saccharomyces cerevisiae）中，可实现多基因同步精准调控的诱导型基因表达系统却十分稀缺。本研究中，我们对哺乳动物I型核受体（Type I nuclear receptors）的配体结合域（ligand binding domains, LBDs）进行重编程，在酿酒酵母中构建了一套最多包含五种正交型人工基因表达系统的家族。我们开发的系统可通过添加惰性激素实现严谨且独立的多基因调控，并可驱动稳定可靠的基因表达输出。作为原理验证，我们将紫色杆菌素（violacein）生物合成途径中的四种酶的表达置于独立调控之下，以此选择性地调控途径代谢流。本研究成果构建了一套模块化、多功能且具备潜在扩展性的工具箱，可用于酿酒酵母中基因表达的多维调控，进而可用于…… 本数据集的生成旨在支持一套工具箱的开发与表征，该工具箱可用于在酿酒酵母中构建正交型小分子诱导遗传程序。该工具箱基于源自人类I型核受体的人工转录因子，这类因子与锌指DNA结合域及转录激活域融合表达。在已发表的研究中，我们对人工转录因子的性能与正交性进行了全面表征。作为概念验证，我们通过选择性诱导途径酶组合的表达，调控了紫色杆菌素生物合成途径的代谢流。 我们的数据集包含通过流式细胞术（flow cytometry）与高效液相色谱（high-performance liquid chromatography, HPLC）获取的实验数据，可支撑本论文的四张核心图表： 1. 用于酿酒酵母遗传回路构建的人工转录因子的作用机制、核心特征与设计方案； 2. 该工具箱的最大诱导水平、基础表达水平及剂量曲线响应特性等；原始数据集需使用FlowJo软件打开，处理后的数据文件则需使用GraphPad Prism软件查看。