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

• 批准号: 10558961
• 负责人: VIRGINIA W CORNISH
• 金额: $ 6.37万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 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/10558961
• 关键词: Acids; Anabolism; Antibiotic Resistance; Antibiotics; Antibodies; Binding; 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

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 该提案的目标是创建通用的、高通量的分析，这些分析是模块化的 范围广泛，以克服目前在测试所需的巨大多样性方面的瓶颈 解决代谢工程问题。如果成功，这些技术将使强大的 定向进化方法将常规应用于天然产品的生物合成和 他们的类比。代谢工程涉及高达1020个数量级的文库 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 途径还必须在宿主菌株背景下进行优化，往往是协同作用的。然而，今天 代谢工程主要是通过在一年中只引入几个基因修改来执行的 时间，然后用低通量的气-液-色层析法分析产生的菌株 质谱学方法。以前用于代谢的高通量分析 工程学一直局限于不寻常的分子，如生色团。因此，我们在这里适用 将竞争分子从蛋白质受体置换成两个 代谢工程常用检测方法：荧光偏振法和酵母菌 三元杂交(Y3H)选择。FP检测将作为第一代介质实施 吞吐量筛，作为迈向Y3H机的一块踏脚石，它会有更高的吞吐量更大 而不是108。在有性繁殖条件下通过诱变进行 同源重组(HR)，可搜索大于1020的文库。在协作中 通过唐实验室(UCLA)和斯奈德实验室(UChicago)，我们挑战我们的 具有提高真菌产量效价的代谢工程任务的技术 脱水四环素Tan-1612及其在酿酒酵母中产生的生物活性类似物 与抗生素耐药性作斗争，并扩大应用范围。