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

项目概要/摘要 该项目的目标是开发一个高度严格的技术平台，利用地衣和非地衣， 对真菌进行测序，以恢复真菌衍生疗法的发现管道， 治疗慢性感染和健康状况。抗药性微生物的出现， 新型抗生素的供应减少，发现和应用的急剧减少， 天然产物（NP，也称为次级代谢产物，SM）和其他小分子的开发 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，炎症因子加剧了严重的公共卫生问题， CDC和WHO。我们认为，使用以下技术的大规模药物发现管道的复兴 利用微生物，包括地衣真菌共生体和未测序的真菌，将提供一个新的 新型药物的干部领导和解决办法对抗药性危机。真菌SM是 也是抗癌化合物在其它广泛临床应用中的重要来源。然而，在这方面， 只有1%或更少的丝状真菌被测序，并且高通量测序已经 显示只有约10%的真菌SM-生物合成基因簇（BGC）表达于 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 获得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 新的真菌人工染色体/质谱（FAC-MS）方法，允许直接 捕获、异源表达和化学分析一整套大的完整SM-BGC 从Clevenger等人所示的测序真菌，自然化学生物学，2017年。我们还表明 穿梭细菌人工染色体（BAC）技术结合BAC池， 索引和下一代测序（NGS）可以实现100 kb连接的测序和组装。 因此，在本项目中，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 可以从难以生长的植物中捕获新的生物活性SM（例如地衣真菌共生体，I期） 和未测序的真菌（第二阶段）。这项技术应该改善真菌SM的发现 100~1000倍，至少发现了5个地衣类抗菌药物的新先导化合物 和未测序的真菌

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.