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

项目摘要 家长奖的目标是创建通用的、高通量的分析，这些分析是模块化的和广泛的 在测试解决新陈代谢所需的巨大多样性方面克服目前的瓶颈 工程问题。如果成功，这些技术将使强大的定向进化方法能够 常规应用于天然产物及其类似物的生物合成。代谢工程涉及到 文库规模高达1020个数量级，比现在常规的蛋白质工程高出许多数量级，因为 不仅生物合成途径中的基因，而且宿主菌株背景中的基因也必须经常优化 协同作用。然而，今天的新陈代谢工程主要是通过引入一些基因来实现的 一次修改，然后用低通量气相和液色谱分析所产生的菌株 质谱学(GCMS和LC MS)方法。我们只打算使用LC MS来确认我们的检测结果， 显著降低与这些方法相关的成本和时间。以前使用的高通量分析 代谢工程方面的研究一直局限于不寻常的分子，如生色团。因此，我们在这里适用 从蛋白质受体上置换竞争分子的概念，以开发两种通用的分析方法 代谢工程：荧光偏振(FP)试验和酵母三杂交(Y3H)选择。这个 FP检测将作为第一代中等吞吐量筛查实施，作为迈向Y3H的踏脚石 这将具有大于108的更高吞吐量。当在性行为条件下进行时 通过同源重组(HR)的突变繁殖，大于1020的文库可以 搜查过了。在与唐实验室(加州大学洛杉矶分校)和斯奈德实验室(芝加哥大学)的合作下，我们挑战 我们的技术具有提高真菌生产效价的代谢工程任务 脱水四环素Tan-1612及其在酿酒酵母中产生抗抗生素活性类似物的研究 阻力和应用范围以外的。

Project Summary The objective of the parent award 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 (GCMS and LCMS) methods. We intend to only use LCMS to confirm our assay results, significantly reducing cost and time associated with these 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.