Changing Paradigms in Natural Product Discovery: A Molecule to Microbe Approach

改变天然产品发现范式：从分子到微生物的方法

• 批准号: 9808022
• 负责人: PAUL R JENSEN
• 金额: $ 23.63万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808022
• 关键词: Address; Anabolism; Antibiotics; Applications Grants; Bioinformatics; Biological; Biological Assay; Bioreactors; Bypass; Chemicals; Circular Dichroism; Communicable Diseases; Complex; Complex Mixtures; Coupled; Crude Extracts; Cues; Data; Data Set; Detection; Development; Environment; Failure; Fractionation; Future; Gene Cluster; Gene Expression; Genes; Genetic; Genome; Goals; High Pressure Liquid Chromatography; Home environment; In Situ; Individual; Ions; Knowledge; Laboratories; Lead; Link; Marine Sediment; Mass Spectrum Analysis; Medicine; Metagenomics; Methods; Microbe; Mining; Molecular; Natural Products; Nutrient; Oceans; Organism; Orphan; Outcome; Parents; Pilot Projects; Plant Resins; Production; Research; Sampling; Site; Source; Structure; Taxonomy; Techniques; Testing; Ursidae Family; Work; base; case-by-case basis; chemical synthesis; drug discovery; frontier; genetic manipulation; improved; innovation; insight; interest; interstitial; metabolome; metabolomics; microbial; microbial community; novel; novel strategies; novel therapeutics; practical application; programs; scaffold; scale up; small molecule; social; success; synthetic biology; tool; water flow

1 Microbial natural products represent one of our most important sources of medicines. Traditionally, these 2 compounds have been discovered from microbes cultured in the laboratory using conditions that bear little 3 resemblance to the environments from which they were derived. The failure of traditional discovery approaches 4 to keep pace with the need for new drug leads, coupled with our improved understanding of the molecular 5 genetics of natural product biosynthesis, spurred the development of new approaches for natural product 6 discovery. These efforts have largely focused on genome mining and the genetic manipulation of orphan 7 biosynthetic gene clusters with the aim of bypassing the regulatory mechanisms that control compound 8 production. While important progress has been made, these approaches have yet to yield a wealth of new 9 chemical scaffolds. Here we propose an alternative discovery paradigm that takes a compound first approach. 10 We developed a Small Molecule In situ Resin Capture (SMIRC) technique to recover microbial natural products 11 directly from environmental samples thus by-passing the need for cultivation. We have applied this approach to 12 ocean sediments, where complex microbial communities occur and interstitial water flow facilitates compound 13 capture. Coupled with mass-spectrometry based metabolomics, we demonstrate that SMIRC can capture known 14 microbial metabolites and thousands of compounds that cannot be readily identified, thus hinting at the discovery 15 potential afforded by this technique. Here we propose to test the applications of a new capture and analysis 16 pipeline to discover microbial natural products directly from marine sediments. By introducing an antibiotic screen 17 into the workflow, we target biomedically relevant compounds for isolation and structural characterization. A 18 micro-fractionation technique will be used to separate complex mixtures and guide the isolation of active, low 19 abundance compounds. We capitalize on innovations in 'nanomole-scale' natural product characterization and 20 the integration of microcryoprobe NMR and MS analyses for sub-micromole level structure determination 21 (including stereostructures). Our ability to prioritize leads based on MS de-replication and biological activity 22 provide opportunities to tease out deep chemical diversity from complex extracts. We further propose to use 23 metagenomics to link compounds to their biogenic sources and thus gain taxonomic perspective on unrealized 24 biosynthetic potential. This approach eliminates the need to establish laboratory conditions suitable for cultivation 25 and gene cluster expression (two of the major bottlenecks hampering current natural product discovery efforts) 26 and instead allows the environment to act as a natural bioreactor. Ultimately, lead compounds discovered using 27 this approach can be produced via chemical synthesis, bulk environmental extraction, synthetic biology, or 28 metagenomics informed cultivation.

1微生物天然产物是我们最重要的药物来源之一。传统上，这些 从实验室培养的微生物中发现了2种化合物， 3与它们所来自的环境的相似性。传统发现方法的失败 4跟上新药先导的需求，再加上我们对分子生物学的理解， 5天然产物生物合成的遗传学，促进了天然产物生物合成新途径的发展 6发现这些努力主要集中在基因组挖掘和孤儿的遗传操作上。 7个生物合成基因簇，目的是绕过控制化合物的调节机制 8生产。虽然取得了重要进展，但这些办法尚未产生大量新的成果， 9化学品在这里，我们提出了一个替代的发现范式，采取复合第一的方法。 我们开发了一种小分子原位树脂捕获（SMIRC）技术，用于回收微生物天然产物 11直接来自环境样品，从而绕过了培养的需要。我们将这种方法应用于 12海洋沉积物，其中存在复杂的微生物群落，间隙水流促进化合物 13捕捉结合基于质谱的代谢组学，我们证明SMIRC可以捕获已知的 14种微生物代谢产物和数千种无法轻易识别的化合物，从而暗示了这一发现。 15、这种技术的潜力。在这里，我们提出了一个新的捕获和分析测试的应用程序 16管道发现微生物天然产物直接从海洋沉积物。通过引入抗生素筛选 17进入工作流程，我们针对生物医学相关化合物进行分离和结构表征。一 18微分馏技术将用于分离复杂的混合物，并指导分离活性，低 19种丰度化合物。我们利用“纳米级”天然产物表征的创新， 20用于亚微摩尔级结构测定的微型冷冻探针NMR和MS分析的集成 21（包括立体结构）。我们能够根据MS去复制和生物活性对线索进行优先排序 22提供了从复杂提取物中梳理出深层化学多样性的机会。我们还建议使用 23宏基因组学将化合物与其生物来源联系起来，从而获得未实现的分类学视角 24生物合成潜力。这种方法消除了建立适合培养的实验室条件的需要 25和基因簇表达（阻碍当前天然产物发现努力的两个主要瓶颈） 26，而是允许环境充当自然生物反应器。最终，使用 这种方法可以通过化学合成、大量环境提取、合成生物学或 28宏基因组学告知种植。