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: 9918932
• 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 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918932
• 关键词: Amino Acids; Binding Proteins; Biological Assay; Carbohydrates; Communication; Core Protein; Development; Docking; Enzymes; Family; Firmicutes; 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 silico; in vivo; metabolomics; microbial; novel; novel strategies; operation; programs; protein expression; protein purification; protein transport; 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）：该计划项目的重点是在微生物物种中发现新的代谢途径的新型计算和实验策略的开发和应用，这些新代谢途径可为其提供完整的基因组序列。该计划项目涉及三个项目：1）用于选择目标选择的代谢项目以及对体外酶促活性和体内生理功能（途径）的预测验证； 2）通过差异扫描荧光法对两种溶质结合蛋白（SBP）的配体特异性进行大规模筛选的配体发现项目； 3）在计算机通路对接和集成途径映射中的建模项目以预测代谢途径。该计划项目涉及两个核心：1）管理核心，以协调“日常”操作和通信以及监督目标选择； 2）从GDNA，蛋白质表达和蛋白质纯化的高通量基因克隆的蛋白质核心提供SBPS和转录调节剂的样品，用于由配体发现项目和途径酶和途径酶进行配体筛选，用于由代谢项目提供体外酶试验的体外酶试剂。该计划项目的四个具体目标侧重于制定综合的一般策略，以发现途径，该途径有望广泛适用：1）对具有小分子配体库的SBP和转录调节器的大规模筛选，其目标是分配配体特异性并描述家族中的特定性/序列空间； 2）使用同源性建模的新代谢途径预测途径酶的结构（从SBPS的基因组邻域环境和转录调节剂中确定），在小分子库的硅配体中，以获取“ HIT”列表的“ hit hit”列表的硅酸盐配体，从SBP特异性，途径对接“命中列表”，化学反应库和相似性集成分析； 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途径； 4）转移注释向Uniprot转移，以扩展并用于改善新测序基因组的自动功能注释质量。该计划项目将说明这种策略，重点是在人类肠道微生物组中发现的碳水化合物和氨基酸分解代谢途径发现。

项目成果

Identification of a novel tRNA wobble uridine modifying activity in the biosynthesis of 5-methoxyuridine.

• DOI: 10.1093/nar/gky592
• 发表时间: 2018-09-28
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Ryu H;Grove TL;Almo SC;Kim J
• 通讯作者: Kim J

Characterization of an l-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations.

具有前所未有的酶促转化的 L-抗坏血酸分解代谢途径的表征。

• DOI: 10.1021/jacs.9b09863
• 发表时间: 2020
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Stack,TylerMM;Morrison,KatelynN;Dettmer,ThomasM;Wille,Brendan;Kim,Chan;Joyce,Ryan;Jermain,Madison;Naing,YadanarThan;Bhatti,Khadija;Francisco,BrianSan;Carter,MichaelS;Gerlt,JohnA
• 通讯作者: Gerlt,JohnA

Evolution of Enzyme Function and the Development of Catalytic Efficiency: Triosephosphate Isomerase, Jeremy R. Knowles, and W. John Albery.

• DOI: 10.1021/acs.biochem.1c00211
• 发表时间: 2021-11-23
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Gerlt, John A.

Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides.

• DOI: 10.1021/jacs.7b01283
• 发表时间: 2017-08-30
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Grove TL;Himes PM;Hwang S;Yumerefendi H;Bonanno JB;Kuhlman B;Almo SC;Bowers AA
• 通讯作者: Bowers AA

Discovery of novel pathways for carbohydrate metabolism.

• DOI: 10.1016/j.cbpa.2020.09.005
• 发表时间: 2021-04
• 期刊: Current opinion in chemical biology
• 影响因子: 7.8
• 作者: Stack TMM;Gerlt JA
• 通讯作者: Gerlt JA