Novel Strategies for the Discovery of Microbial Metabolic Pathways

描述（由申请人提供）：该计划项目的重点是开发和应用新的计算和实验策略，以发现微生物物种中的新代谢途径，并提供完整的基因组序列。该项目包括三个项目：1）代谢项目，用于靶标选择和体外酶活性和体内生理功能预测的实验验证2）通过差示扫描荧光法大规模筛选溶质结合蛋白（SBP）对转运系统和转录调节因子的配体特异性的配体发现项目;以及3）用于计算机途径对接和整合途径作图以预测代谢途径的建模项目。该计划项目涉及两个核心：1）行政核心，以协调“日常”的运作和沟通，以及监督目标的选择;和2）用于从gDNA高通量基因克隆，蛋白表达，和蛋白质纯化，以提供SBP和转录调节因子的样品，用于配体发现项目的配体筛选，以及代谢研究所的体外酶促测定的途径酶的样品项目该计划项目有四个具体目标，重点是制定一个综合的一般战略，发现途径，预计将广泛适用：1）大规模筛选家庭的SBPs和转录调控因子的小分子配体库，与目标分配配体特异性和描述特异性/序列空间的家庭; 2）使用同源建模预测新的代谢途径以获得途径酶的结构（从SBP和转录调节子的基因组邻近环境中鉴定），小分子文库的计算机配体对接以获得途径中所有酶的底物的“命中”列表（“途径对接”），和整合途径作图，以使用来自SBP特异性、途径对接“命中列表”、化学反应库和相似性系综分析的线索鉴定“优化”途径; 3）使用体外酶活性、生长表型、遗传学、转录组学和代谢组学验证预测的途径;以及4）将注释转移到UniProt以用于传播和用于改进新测序的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，重点是在人类肠道微生物组中发现的厚壁菌属物种中发现碳水化合物和氨基酸分解代谢途径。

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.