Novel Strategies for the Discovery of Microbial Metabolic Pathways

发现微生物代谢途径的新策略

• 批准号: 9557783
• 负责人: JOHN A GERLT
• 金额: $ 11.68万
• 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/9557783
• 关键词: Amino Acids; Binding Proteins; Biological Assay; Carbohydrates; Carrier Proteins; Communication; Computer Simulation; Core Protein; Development; Docking; Enzymes; Family; Genetic; Genetic Transcription; Genome; Genomic DNA; Goals; Growth; Homology Modeling; Human; In Vitro; Libraries; Ligands; Metabolic Pathway; Metabolism; Modeling; Neighborhoods; Pathway interactions; Phenotype; Physiological; Sampling; Scanning; Specificity; Structure; System; chemical reaction; enzyme pathway; gene cloning; gut microbiome; improved; in vivo; metabolomics; microbial; novel; novel strategies; operation; programs; protein expression; protein purification; public health relevance; screening; small molecule; small molecule libraries; solute; transcriptomics

DESCRIPTION (provided by applicant): This Program Project focuses on the development and application of novel computational and experimental strategies for the discovery of novel metabolic pathways in microbial species for which complete genome sequences are available. The Program Project involves three Projects: 1) Metabolism Project for target selection and experimental verification of predicted in vitro enzymatic activities and in vivo physiological functions (pathways); 2) Ligand Discovery Project for large-scale screening of ligand specificities of both solute binding proteins (SBPs) for transport systems and transcriptional regulators by differential scanning fluorimetry; and 3) Modeling Project for in silico pathway docking and integrative pathway mapping to predict metabolic pathways. The Program Project involves two Cores: 1) Administrative Core to coordinate "day-to-day" operations and communications as well as oversee target selection; and 2) Protein Core for high-throughput gene cloning from gDNAs, protein expression, and protein purification to provide samples of SBPs and transcriptional regulators for ligand screening by the Ligand Discovery Project and of pathway enzymes for in vitro enzymatic assays by the Metabolism Project. The Program Project has four Specific Aims focused on developing an integrated general strategy for the discovery of pathways that is expected to be broadly applicable: 1) large-scale screening of families of SBPs and transcriptional regulators with small molecule ligand libraries, with the goal of assigning ligand specificities and describing specificity/sequence space in the families; 2) prediction of novel metabolic pathways using homology modeling to obtain structures for pathway enzymes (identified from genome neighborhood context of the SBPs and transcriptional regulators), in silico ligand docking of small molecule libraries to obtain "hit" lists of substrates for all enzyms in the pathway ("pathway docking"), and integrative pathway mapping to identify an "optimized" pathway using clues from SBP specificity, pathway docking "hit lists", a library of chemical reactions, and similarity ensemble analysis; 3) verification of the predicted pathways using in vitro enzymatic activities, growth phenotypes, genetics, transcriptomics, and metabolomics; and 4) transfer of annotations to UniProt for dissemination and use in improving the quality of automatic functional annotations for newly sequenced genomes. The Program Project will illustrate this strategy with a focus on discovery of carbohydrate and amino acid catabolic pathways in Firmicute species found in the human gut microbiome.

描述（由申请人提供）：该计划项目侧重于新型计算和实验策略的开发和应用，以发现可获得完整基因组序列的微生物物种中的新型代谢途径。该计划项目包括三个项目：1）代谢项目，用于预测体外酶活性和体内生理功能（途径）的靶点选择和实验验证； 2) 配体发现项目，通过差示扫描荧光法大规模筛选运输系统和转录调节剂的溶质结合蛋白（SBP）的配体特异性； 3) 用于预测代谢途径的计算机途径对接和综合途径绘图的建模项目。该计划项目涉及两个核心：1）行政核心，协调“日常”运营和沟通，并监督目标选择； 2) 蛋白质核心，用于从 gDNA 进行高通量基因克隆、蛋白质表达和蛋白质纯化，为配体发现项目的配体筛选提供 SBP 和转录调节因子样品，为代谢项目的体外酶测定提供途径酶样品。该计划项目有四个具体目标，重点是开发一个综合的总体策略，用于发现预计广泛适用的途径：1）利用小分子配体文库大规模筛选 SBP 和转录调节因子家族，目标是分配配体特异性并描述家族中的特异性/序列空间； 2) 使用同源模型预测新的代谢途径，以获得途径酶的结构（从 SBP 和转录调节因子的基因组邻域背景中识别），小分子库的计算机配体对接以获得途径中所有酶的“命中”底物列表（“途径对接”），以及综合途径作图以使用以下线索识别“优化”途径 SBP特异性、通路对接“命中列表”、化学反应库和相似性集成分析； 3）利用体外酶活性、生长表型、遗传学、转录组学和代谢组学验证预测的途径； 4) 将注释转移到 UniProt 进行传播和使用，以提高新测序基因组的自动功能注释的质量。该计划项目将阐明这一策略，重点是发现人类肠道微生物组中厚壁菌门物种中的碳水化合物和氨基酸分解代谢途径。