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，这些微生物在几乎不产生任何影响的条件下培养 3 与它们产生的环境的相似性。传统发现方法的失败 4 跟上对新药先导化合物的需求，再加上我们对分子的了解的加深 5 天然产物生物合成的遗传学，刺激了天然产物新方法的开发 6 发现。这些努力主要集中在基因组挖掘和孤儿基因操纵上 7 个生物合成基因簇，旨在绕过控制化合物的调控机制 8 生产。尽管已经取得了重要进展，但这些方法尚未产生大量新成果 9个化学支架。在这里，我们提出了一种采用复合优先方法的替代发现范式。 10 我们开发了小分子原位树脂捕获 (SMIRC) 技术来回收微生物天然产物 11 直接来自环境样本，从而绕过了培养的需要。我们已将这种方法应用于 12 个海洋沉积物，其中存在复杂的微生物群落，间隙水流有利于复合 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 此技术提供的潜力。在这里我们建议测试新的捕获和分析的应用 16 条直接从海洋沉积物中发现微生物天然产物的管道。通过引入抗生素筛查 17 在工作流程中，我们针对生物医学相关化合物进行分离和结构表征。一个 18 微分馏技术将用于分离复杂的混合物并指导活性、低浓度的分离 19种丰度化合物。我们利用“纳摩尔级”天然产物表征方面的创新， 20 集成微冷冻探针 NMR 和 MS 分析进行亚微分子级结构测定 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。我们进一步建议使用 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。这种方法无需建立适合培养的实验室条件 25 和基因簇表达（阻碍当前天然产物发现工作的两个主要瓶颈） 26 相反，让环境充当天然生物反应器。最终，使用发现的先导化合物 27 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 宏基因组学为种植提供信息。