Metabolism Project

新陈代谢项目

• 批准号: 9073786
• 负责人: JOHN A GERLT
• 金额: $ 42.29万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9073786
• 关键词: Binding Proteins; Core Protein; Enzymatic Biochemistry; Enzymes; Fractionation; Genes; In Vitro; Individual; Knock-out; Lead; Ligands; Maps; Measures; Metabolic; Metabolic Pathway; Metabolism; Microbiology; Modeling; Pathway interactions; Phenotype; Production; Proteins; Research Infrastructure; Structure; Testing; Transcript; Update; base; genome-wide; in vitro activity; in vivo; metabolomics; microbial; novel; novel strategies; overexpression; protein purification; screening; tool; transcriptome sequencing; transcriptomics

PROJECT SUMMARY: METABOLISM PROJECT The Metabolism Project will provide the experimental infrastructure (enzymology, microbiology, transcriptomics, and metabolomics) for testing the EFI's sequence/structure-based tools for predicting in vitro enzymatic activities and in vivo metabolic functions. In using those tools, the Metabolism Project will take the lead in developing integrated strategies for discovering novel metabolic pathways and providing direction for enhancing the tools. The Specific Aims of the Metabolism Project are: 1) coordinate selection of targets for protein production by the Protein Core, protein purification and ligand screening by the Ligand Discovery Project, and integrative pathway mapping by the Modeling Project; 2) measure in vitro enzymatic activities of targets produced by the Protein Core to validate predictions from integrative pathway mapping by the Modeling Project; 3) construct null (knockout) strains of genes encoding enzymes for which in vitro activities are predicted by the Modeling Project; 4) perform targeted transcript analyses by qRT-PCR and, when appropriate, genome-wide transcriptomics by RNA-Seq to confirm predicted metabolic functions predicting by the Modeling Project; 5) analyze metabolites (metabolomics) predicted by the Modeling Project in wild type and knockout/overexpression strains under conditions in which phenotypes are observed, with the in vitro assigned activity facilitating both chromatographic fractionation of total metabolites and subsequent mass spectroscopic analysis; and 6) transfer in vitro enzymatic activities and in vivo metabolic functions to UniProt to update/correct the annotations for the individual binding proteins and enzymes in the metabolic pathways that are predicted and verified.

项目总结：新陈代谢项目 新陈代谢项目将提供实验基础设施(酶、微生物学、 转录学和代谢组学)，用于测试EFI基于序列/结构的体外预测工具 酶活性和体内代谢功能。在使用这些工具时，新陈代谢项目将采取 领导制定发现新代谢途径的综合战略，并为 增强工具。新陈代谢项目的具体目标是：1)协调选择目标 蛋白质核心蛋白生产、蛋白质纯化及配基筛选 2)体外酶活性测定 由蛋白质核心产生的靶点，用于通过建模验证来自整合路径图的预测 项目；3)构建编码在体外具有活性的酶的零(敲除)基因株 通过建模项目预测；4)通过qRT-PCR进行有针对性的转录本分析，并在适当的时候， RNA-Seq全基因组转录证实模型预测的代谢功能 项目；5)分析建模项目预测的野生型和 在观察到表型的条件下，基因敲除/过表达菌株，并在体外分配 促进总代谢物的层析分离和随后的质谱学的活性 分析；以及6)将体外酶活性和体内代谢功能转移到UniProt 更新/更正代谢途径中各个结合蛋白和酶的注释 都得到了预测和验证。