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

代谢工程的通用和高通量小分子筛选和选择

基本信息

批准号：
10797492
负责人：
VIRGINIA W CORNISH
金额：
$ 2.53万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10797492
关键词：
Academia Affinity Agar Air Amination Anabolism Antibiotic Resistance Antibiotics Award Bacterial Antibiotic Resistance Bacterial Infections Binding Biological Assay Catalysis Cellulases Centers for Disease Control and Prevention (U.S.)Cessation of life Chemicals Chemistry Collaborations Color Coupled Coupling DNA Binding Detection Dimerization Directed Molecular Evolution Drops Economics Educational process of instructing Engineering Enzymes FDA approved Fermentation Fluorescence Polarization Freedom Funding Future Gas Chromatography Gases Gene Deletion Generations Genes Genetic Genetic Recombination Genetic Transcription Gram-Negative Bacteria Growth Hand Heritability Hybrids In Vitro Incentives Industry Laboratories Lactamase Libraries Mass Fragmentography Mass Spectrum Analysis Measures Methodology Methods Methyltransferase Mining Minor Mission Modernization Modification Molecular Biology Molecular Conformation Mutagenesis Mutation Natural Products Nitrogen Oxidases Oxygenases Oxytetracycline Parents Pathway interactions Positioning Attribute Production Property Protein Engineering Proteins Public Health Publications Reducing Agents Reporter Genes Research Route Running Saccharomyces cerevisiae Sampling Science Secure Services Sexual Reproduction Solvents Specificity Streptomyces Structure System Technology Testing Tetracyclines Ticks Time Transaminases Transcriptional Regulation Transducers Tube United States Validation Variant Work World Health Organization Yeasts analog aptamer chromophore cost design drug discovery drug production experimental study flasks flexibility fluorophore functional group global health high throughput screening homologous recombination improved in vivo instrument interest liquid chromatography mass spectrometry member metabolic engineering novel novel strategies parent grant programs purge receptor reconstitution scaffold screening skills small molecule supply chain synthetic biology tool transcription factor

项目摘要

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.

项目概要家长奖的目标是创建模块化且广泛的通用、高通量检测方法。克服当前测试解决代谢问题所需的巨大多样性的瓶颈工程问题。如果成功，这些技术将实现强大的定向进化方法常规应用于天然产物及其类似物的生物合成。代谢工程涉及文库大小高达 1020，超出了现在常规蛋白质工程的多个数量级，因为多个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，今天的代谢工程主要是通过引入一些遗传基因来进行的。一次修饰，然后通过低通量气相色谱和液相色谱分析所得菌株质谱（GCMS 和 LCMS）方法。我们打算仅使用 LCMS 来确认我们的检测结果，显着减少与这些方法相关的成本和时间。以前使用的高通量测定代谢工程中的研究仅限于不寻常的分子，例如发色团。因此，我们在这里应用从蛋白质受体中置换竞争分子的概念，以开发两种通用检测方法代谢工程：荧光偏振（FP）测定和酵母三杂交（Y3H）选择。这 FP 测定将作为第一代、中等通量筛选来实施，作为 Y3H 的垫脚石这将具有大于108的更高吞吐量。当在性条件下进行时通过同源重组 (HR) 进行诱变繁殖，超过 1020 的文库可以搜索过。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）合作，我们挑战我们的技术具有提高真菌生产滴度的代谢工程使命脱水四环素 TAN-1612 并在酿酒酵母中生成生物活性类似物以对抗抗生素电阻和应用之外。