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

Project Summary/AbstractThe 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 oftreating chronic infections and health conditions. The emergence of drug resistant microbes, thediminishing supply of novel classes of antibiotics, and the dramatic reduction in discovery anddevelopment of natural products (NPs, also termed secondary metabolites, SMs) and other smallmolecule compounds from bacteria and fungi for anti-infective, anti-proliferation and anti-inflammation agents since 1960s have amplified a serious public health concern championed bythe 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 newcadre of novel drug leads and solutions towards the antibiotic-resistance crisis. Fungal SMs arealso 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 hasshown that only about 10% of fungal SM-biosynthetic gene clusters (BGCs) are expressed underlaboratory conditions. Therefore, revolutionary technologies and tools are urgently needed todiscover and more effectively dissect the biosynthesis of fungal SMs in order to more efficientlyaccess novel fungal metabolites as potential pharmaceutical agents. Recently, we developed anovel fungal artificial chromosome/mass spectrometry (FAC-MS) method that allows the directcapture, heterologous expression and chemical analysis of an entire set of large intact SM-BGCsfrom sequenced fungi as shown in Clevenger et al., Nat. Chem. Biol, 2017. We have also shownthat 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 byintegrating FAC-MS with BAC/NGS sequencing and bioactivity profiling to prove the concept thatnovel 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 discovery100~1000 fold and result in the discovery of at least 5 novel antimicrobial drug leads from lichenand un-sequenced fungi.