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：
9974122
负责人：
VIRGINIA W CORNISH
金额：
$ 54.83万
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 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9974122
关键词：
Acids Anabolism Antibiotic Resistance Antibiotics Antibodies Binding Biological Biological Assay Chemicals Chemistry Collaborations Data Detection Dimerization Directed Molecular Evolution Engineering FDA approved FK506 Family Fluorescein Fluorescence Polarization Foundations Gas-Liquid Chromatography Generations Genes Genetic Genetic Transcription Hand Hybrids In Vitro Laboratories Libraries Manuals Methodology Methods Minocycline Minor Mission Modification Molecular Structure Mutagenesis Natural Products Nobel Prize Organism Pathway interactions Positioning Attribute Production Property Protein Engineering Proteins Publications Publishing Reporter Genes Reporting Repressor Proteins Reproduction Resistance Saccharomyces cerevisiae Signal Transduction Structure Technology Testing Tetracyclines Therapeutic Time Yeasts analog base chromophore combat design high throughput screening homologous recombination improved in vivo liquid chromatography mass spectrometry mathematical model metabolic engineering mutant predictive test professor protein metabolite receptor resistant strain screening small molecule synthetic biology tool transcription factor virtual virtual library

项目摘要

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.

项目概要该提案的目标是创建通用的、模块化的、高通量的检测方法。范围广泛，以克服当前测试所需的巨大多样性的瓶颈解决代谢工程问题。如果成功的话，这些技术将带来强大的定向进化方法通常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，多个数量级超越现在常规的蛋白质工程，因为多个基因不仅在生物合成中途径而且在宿主菌株背景下也必须经常协同优化。然而，今天代谢工程主要是通过在一定范围内引入一些基因修饰来进行的时间，然后通过低通量气相色谱和液相色谱分析所得菌株质谱方法。以前用于代谢的高通量测定工程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以开发两种的概念代谢工程的一般检测：荧光偏振 (FP) 检测和酵母三杂交（Y3H）选择。 FP 测定将作为第一代介质进行实施吞吐量屏幕，作为 Y3H 的垫脚石，Y3H 将具有更高的吞吐量超过108。当在通过诱变进行有性繁殖的条件下进行时同源重组（HR），可搜索大于1020的文库。合作中与唐实验室（加州大学洛杉矶分校）和斯奈德实验室（芝加哥大学）一起，我们挑战我们的具有提高真菌生产滴度的代谢工程使命的技术脱水四环素 TAN-1612 并在酿酒酵母中生成生物活性类似物对抗抗生素耐药性及其应用。