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: 9297333
• 负责人: JOHN A GERLT
• 金额: $ 232.88万
• 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/9297333
• 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)：该计划项目专注于开发和应用新的计算和实验策略，以发现可获得完整基因组序列的微生物物种中的新代谢途径。该计划项目包括三个项目：1)代谢项目，用于体外酶活性和体内生理功能预测的目标选择和实验验证；2)配体发现项目，用于通过差示扫描荧光法大规模筛选运输系统的溶质结合蛋白(SBPs)和转录调控蛋白的配体特异性；以及3)建模项目，用于电子途径对接和整合路径绘制，以预测代谢途径。该计划项目涉及两个核心：1)行政核心，协调“日常”操作和沟通，并监督目标选择；2)高通量gDNA基因克隆、蛋白质表达和蛋白质纯化的蛋白质核心，为配基发现计划的配体筛选提供SBPs和转录调节因子样本，并为新陈代谢计划的体外酶分析提供途径酶。该计划项目有四个具体目标，重点是开发一种综合的一般策略，以发现有望广泛适用的途径：1)大规模筛选SBP家族和具有小分子配基文库的转录调控因子，目标是指定配体特异性并描述家族中的特异性/序列空间；2)使用同源建模来预测新的代谢途径以获得途径酶的结构(从SBPs和转录调节因子的基因组邻域上下文中识别)，在小分子文库的硅胶配基对接中获得途径中所有酶的底物的“命中”列表(“途径对接”)，以及使用来自SBP特异性的线索、途径对接“命中列表”、化学反应文库和相似性集成分析来识别“优化的”途径的综合途径图；3)使用体外酶活性、生长表型、遗传学、转录组和代谢组学来验证预测的途径；4)将注释传输到UniProt以供分发和用于提高新测序基因组的自动功能注释的质量。该计划项目将说明这一战略，重点是发现在人类肠道微生物组中发现的非米库特物种中的碳水化合物和氨基酸分解代谢途径。