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.

项目摘要：新陈代谢项目 新陈代谢项目将提供实验基础设施（酶学、微生物学、 转录组学和代谢组学），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 领导开发综合策略以发现新的代谢途径并为 增强工具。代谢项目的具体目标是：1）协调选择目标 通过 Protein Core 进行蛋白质生产，通过 Ligand Discovery 进行蛋白质纯化和配体筛选 项目和建模项目的综合路径图； 2) 测量体外酶活性 蛋白质核心产生的目标，以验证建模的综合路径图谱的预测 项目; 3) 构建编码酶基因的无效（敲除）菌株，其体外活性为 由建模项目预测； 4) 通过 qRT-PCR 进行靶向转录本分析，并在适当时， 通过 RNA-Seq 进行全基因组转录组学，以确认通过建模预测的代谢功能 项目; 5) 分析模型项目预测的野生型代谢物（代谢组学） 在观察到表型的条件下敲除/过表达菌株，并在体外分配 活性促进总代谢物的色谱分离和随后的质谱分析 分析; 6) 将体外酶活性和体内代谢功能转移至UniProt 更新/更正代谢途径中各个结合蛋白和酶的注释 被预测和验证。