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

项目摘要/摘要 该项目的目标是利用地衣和非金属材料开发一个高度严谨的技术平台。 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。抗药性微生物的出现， 新型抗生素的供应不断减少，发现和使用抗生素的数量急剧减少 开发天然产物(NPs，也称为次生代谢物，SMS)和其他小分子 抗感染、抗增殖、抗病毒的细菌和真菌分子化合物 自20世纪60年代以来，炎症剂放大了一个严重的公共卫生问题，由 疾控中心和世卫组织。我们假设，大规模药物发现管道的复兴使用的是- 被开发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 一批新药引领和解决抗生素耐药性危机。真菌短信是 在其他广泛的临床应用中，也是抗癌化合物的重要来源。然而， 只有1%或更少的丝状真菌被测序，而高通量测序 研究表明，只有约10%的真菌SM生物合成基因簇(BGC)在 实验室条件。因此，迫切需要革命性的技术和工具来 发现并更有效地剖析真菌短信的生物合成，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，我们开发了一种 真菌人工染色体/质谱学(FAC-MS)新方法 一整套完整的SM-BGC的捕获、异源表达和化学分析 来自Clevenger等人所示的已测序真菌，NAT。化学。Biol，2017。我们还展示了 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，我们将通过以下方式开发一个创新的真菌技术平台 将FAC-MS与BAC/NGS测序和生物活性图谱相结合来证明这一概念 可从难生长的地衣真菌共生体(如地衣真菌共生体，I期)捕获新的生物活性SMS 和未测序真菌(II期)。这项技术应该会改进真菌SM的发现 100~1000倍，从地衣中发现至少5种新型抗菌药物先导化合物 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.