Rapid discovery of thousands of intact biosynthetic gene pathways for bioactive natural product compounds from un-sequenced filamentous fungi using a novel FAC-NGS tool

使用新型 FAC-NGS 工具从未测序的丝状真菌中快速发现数千个完整的生物活性天然产物化合物的生物合成基因途径

• 批准号: 10348139
• 负责人: Jin Woo Bok
• 金额: $ 100万
• 依托单位: INTACT GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10348139
• 关键词: Address; Agriculture; Anabolism; Anti-Infective Agents; Antibiotic Resistance; Area; Artificial Chromosomes; Bacteria; Bacterial Artificial Chromosomes; Bar Codes; Biological Assay; Biology; Biomedical Research; Centers for Disease Control and Prevention (U.S.); Chemicals; Clinical; Collaborations; Collection; Communicable Diseases; Data; Development; Engineering; Gene Cluster; Genes; Genome; Genomics; Goals; Health; High Pressure Liquid Chromatography; High-Throughput Nucleotide Sequencing; Individual; Industry; Inflammation; Laboratories; Lead; Libraries; Lichen - organism; Link; Mass Spectrum Analysis; Medicine; Metagenomics; Methods; Microbe; Microbial Drug Resistance; Mining; Molds; Natural Products; Pathway interactions; Pharmacologic Substance; Phase; Ploidies; Process; Public Health; Research Project Grants; Source; Structure; Technology; Testing; Therapeutic; United States Department of Agriculture; anti-cancer; antimicrobial; antimicrobial drug; bioactive natural products; chronic infection; drug discovery; drug resistant microorganism; fungus; improved; indexing; innovation; next generation sequencing; novel; novel antibiotic class; novel therapeutics; pandemic preparedness; small molecule; symbiont; therapeutic lead compound; tool; virtual

Project Summary/Abstract The goal of this project is to develop a highly rigorous technology platform utilizing lichen and un- sequenced fungi to revive the discovery pipeline for fungal-derived therapeutics capable of treating chronic infections and health conditions. The emergence of drug resistant microbes, the diminishing supply of novel classes of antibiotics, and the dramatic reduction in discovery and development of natural products (NPs, also termed secondary metabolites, SMs) and other small molecule compounds from bacteria and fungi for anti-infective, anti-proliferation and anti- inflammation agents since 1960s have amplified a serious public health concern championed by the CDC and WHO. We posit that the revival of large-scale drug discovery pipelines using under- exploited microbes including lichen fungal symbionts and un-sequenced fungi will provide a new cadre of novel drug leads and solutions towards the antibiotic-resistance crisis. Fungal SMs are also important sources of anticancer compounds among other widespread clinical uses. However, only 1% or less of filamentous fungi have been sequenced and high throughput sequencing has shown that only about 10% of fungal SM-biosynthetic gene clusters (BGCs) are expressed under laboratory conditions. Therefore, revolutionary technologies and tools are urgently needed to discover and more effectively dissect the biosynthesis of fungal SMs in order to more efficiently access novel fungal metabolites as potential pharmaceutical agents. Recently, we developed a novel fungal artificial chromosome/mass spectrometry (FAC-MS) method that allows the direct capture, heterologous expression and chemical analysis of an entire set of large intact SM-BGCs from sequenced fungi as shown in Clevenger et al., Nat. Chem. Biol, 2017. We have also shown that shuttle bacterial artificial chromosome (BAC) technology combined with BAC pooling, indexing, and next-gen sequencing (NGS) can achieve 100kb-linked sequencing and assembly. Therefore, in this project, we will develop an innovative fungal technology platform by integrating FAC-MS with BAC/NGS sequencing and bioactivity profiling to prove the concept that novel bioactive SMs can be captured from hard to grow (e.g. lichen fungal symbionts, Phase I) and un-sequenced fungi (Phase II). This technology should improve fungal SM discovery 100~1000 fold and result in the discovery of at least 5 novel antimicrobial drug leads from lichen and un-sequenced fungi.

项目概要/摘要 该项目的目标是利用地衣和非生物技术开发一个高度严格的技术平台 对真菌进行测序，以重振真菌衍生疗法的发现管道 治疗慢性感染和健康状况。耐药微生物的出现， 新型抗生素的供应减少，发现和发现的抗生素数量急剧减少 天然产物（NP，也称为次级代谢物，SM）和其他小型产品的开发 来自细菌和真菌的分子化合物，用于抗感染、抗增殖和抗 自 20 世纪 60 年代以来的炎症因子加剧了严重的公共卫生问题 疾病预防控制中心和世界卫生组织。我们认为，大规模药物发现管道的复兴 被利用的微生物，包括地衣真菌共生体和未测序的真菌，将提供一种新的 应对抗生素耐药性危机的新药先导和解决方案的核心。真菌 SM 是 也是其他广泛临床用途中抗癌化合物的重要来源。然而， 只有 1% 或更少的丝状真菌已被测序，并且高通量测序已经完成 结果表明，只有约 10% 的真菌 SM 生物合成基因簇 (BGC) 在以下条件下表达： 实验室条件。因此，迫切需要革命性的技术和工具 发现并更有效地剖析真菌 SM 的生物合成，以便更有效地 获取新的真菌代谢物作为潜在的药物制剂。最近，我们开发了一个 新型真菌人工染色体/质谱（FAC-MS）方法，允许直接 对一整套大型完整 SM-BGC 进行捕获、异源表达和化学分析 来自测序的真菌，如 Clevenger 等人，Nat. 所示。化学。 Biol，2017。我们还展示了 将穿梭细菌人工染色体（BAC）技术与BAC池相结合， 索引和下一代测序（NGS）可以实现100kb的连锁测序和组装。 因此，在这个项目中，我们将开发一个创新的真菌技术平台 将 FAC-MS 与 BAC/NGS 测序和生物活性分析相结合，以证明以下概念： 新型生物活性 SM 可以从难以生长的物质中捕获（例如地衣真菌共生体，第一阶段） 和未测序的真菌（第二阶段）。这项技术应该可以改善真菌 SM 的发现 100~1000倍，并导致从地衣中发现至少5种新型抗菌药物先导物 和未测序的真菌。