General and High-Throughput Small Molecule Screens and Selections for Metabolic Engineering

项目摘要 该提案的目的是创建通用的、高通量的模块化分析， 广泛的范围，以克服目前的瓶颈，在测试所需的巨大多样性， 解决代谢工程问题。如果成功，这些技术将使强大的 定向进化方法常规应用于天然产物的生物合成， 他们的类似物。代谢工程涉及的文库大小高达1020个， 超越了现在常规的蛋白质工程，因为多个基因不仅在生物合成中， 在宿主菌株背景中也必须经常协同地优化。然而今天 代谢工程主要是通过在一个特定的时间内引入一些遗传修饰来进行的。 时间，然后通过低通量气相和液相色谱法测定所得菌株 质谱分析方法。先前用于代谢的高通量测定 工程技术仅限于不寻常的分子，如发色团。因此，我们在这里应用 从蛋白质受体置换竞争分子以产生两个 用于代谢工程的一般测定：荧光偏振（FP）测定和酵母 三杂交（Y3 H）选择。FP测定将作为第一代培养基实施， 吞吐量筛选，作为一个垫脚石，以Y3 H这将有更高的吞吐量更大 比108。当在有性生殖条件下进行诱变时， 同源重组（HR），可以搜索大于1020的文库。合作 与唐实验室（加州大学洛杉矶分校）和斯奈德实验室（芝加哥大学），我们挑战我们的 该技术具有增加真菌的生产滴度的代谢工程使命， 脱水四环素TAN-1612和在S.酿酒厂 对抗抗生素耐药性和其他应用。

Project Summary The objective of this proposal is to create general, high-throughput assays that are modular and broad in scope to overcome the current bottleneck in testing the enormous diversity required for solving metabolic engineering problems. If successful, these technologies will enable powerful directed evolution approaches to be routinely applied to the biosynthesis of natural products and their analogs. Metabolic engineering involves library sizes of up to 1020, many orders of magnitude beyond now routine protein engineering, because multiple genes not only in the biosynthetic pathway but also in the host strain background must be optimized often synergistically. Yet, today metabolic engineering is primarily performed by introducing just a few genetic modifications at a time and then assaying the resulting strains by low throughput gas- and liquid-chromatography mass spectrometry methods. Previous high-throughput assays employed in metabolic engineering have been limited to unusual molecules, such as chromophores. Thus, here we apply the concept of displacement of a competitor molecule from a protein receptor to develop two general assays for metabolic engineering: the fluorescence polarization (FP) assay and the yeast three-hybrid (Y3H) selection. The FP assay would be implemented as a first-generation, medium throughput screen, as a step stone to the Y3H which would have higher throughput of greater than 108. When carried out under the conditions of sexual reproduction with mutagenesis via homologous recombination (HR), libraries of greater than 1020 can be searched. In collaboration with the Tang laboratory (UCLA) and the Snyder laboratory (UChicago), we challenge our technology with the metabolic engineering mission of increasing production titers of the fungal anhydrotetracycline TAN-1612 and generating biologically active analogs in S. cerevisiae for combating antibiotic resistance and applications beyond.