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：
10295418
负责人：
VIRGINIA W CORNISH
金额：
$ 2.12万
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/10295418
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 Resistance Saccharomyces cerevisiae Sexual Reproduction 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.

项目摘要该提案的目的是创建模块化和范围广泛，以克服当前的瓶颈测试所需的巨大多样性解决代谢工程问题。如果成功，这些技术将使强大定义的进化方法通常适用于天然产物的生物合成和他们的类似物。代谢工程涉及的图书馆尺寸最高为1020，许多数量级超越现在的常规蛋白质工程，因为多个基因不仅在生物合成中途径，但在宿主应变背景中也必须经常协同优化。但是，今天代谢工程主要是通过仅在一个遗传修饰中引入一些遗传修饰来进行的时间，然后通过低吞吐气和液态染料来测定所得的菌株质谱法。代谢的先前高通量测定工程仅限于异常分子，例如发色团。因此，我们在这里申请竞争者分子从蛋白质受体中取代的概念以发展两个代谢工程的一般测定：荧光极化（FP）测定和酵母三杂交（Y3H）选择。 FP测定将作为第一代，媒介实施吞吐量屏幕，作为Y3H的阶梯石，将具有更大的吞吐量大于108。可以搜索同源重组（HR），可以搜索大于1020的库。合作在唐实验室（UCLA）和Snyder实验室（Uchicago）的情况下，我们挑战我们具有代谢工程任务的技术，即增加真菌的生产效率赤铁环素tan-1612，并在酿酒酵母中生成生物活性类似物的生物类似物打击抗生素耐药性和范围之外的应用。