Supplement to Advancing CRISPR-Cas Technologies for Discovery and Characterization of Novel Fungal Natural Products

项目摘要/摘要（30行文本） 真菌天然产物（NPs）是一种重要的药物来源， 作为治疗人类疾病的药物的作用。真菌的快速扩张 基因组序列和生物信息学工具的发展使识别 数以千计的真菌NP生物合成基因簇（BGC），从而提供了前所未有的 有机会发现新的真菌纳米粒子。然而，新的生物活性真菌纳米颗粒的发现， 由于在优先考虑BGC和真菌中的遗传操作方面存在困难，因此仍然具有挑战性。在 这一建议，我们希望建立管道，以快速发现新的生物活性真菌天然 产品，可以作为下一代候选药物治疗人类 为此，我们将应用CRISPR Cas基因组编辑技术， 这些工具的生物合成真菌天然产物。为了实现研究目标，我们首先 方向将集中在识别和表征很少发现的核糖体合成 和来自真菌来源的后修饰肽（RIPPs）。由于RIP的独特性， 生物合成机制，复杂的化学特性，以及重要的药理作用， 细菌RIPP的特性引起了学术界和 医药行业。然而，只有少数RIPP已被确定从真菌，甚至 尽管已知真菌是NP的多产者。通过表征新的生物合成 通过生物信息学分析确定了已知的RIPPs和新的真菌BGC的酶，我们预计 大大拓宽和加深我们对真菌RIPPs生物合成的理解， 新的真菌RiPP NP的库。我们的第二个方向将专注于扩大和 应用基于CRISPR的基因组工程工具包来表征生物合成基因簇 从丝状真菌。CRISPR-Cas工具已被成功证明是可行的 但很少应用于真菌NP生物合成的研究。我们将 开发用于操纵真菌生物合成基因的互补CRISPR-Cas工具集 在天然和异源表达宿主中的簇。通过这样做，我们希望开发一个 全套CRISPR基因编辑工具包，可快速进行基因操作， 丝状真菌中的产物生物合成。这两个研究方向和合作 通过BGC表征，遗传工具的进步，以及新的 生物信息学算法开发将建立一个完整的管道，以显着增加 真菌NP和类似物，特别是真菌RIPPs的库，使这些分子有价值 用于人类治疗的候选药物。

Project Summary/Abstract (30 lines of text) Fungal natural products (NPs) have been a preeminent source of medicine and played pivotal roles as pharmaceuticals for the treatment of human diseases. The rapid expansion of fungal genome sequences and the development of bioinformatics tools have enabled the identification of thousands of fungal NP biosynthetic gene clusters (BGCs), thus providing an unprecedented opportunity to discover new fungal NPs. However, the discovery of new bioactive fungal NPs remains challenging, due to difficulties in prioritizing BGCs and genetic manipulations in fungi. In this proposal, we expect to build pipelines to rapidly discover novel bioactive fungal natural products that can serve as the next generation of drug candidates for the treatment of human diseases; to do this, we will apply the CRISPR Cas genome editing technologies and dedicate these tools to the biosynthesis of fungal natural products. To achieve the research goal, our first direction will focus on identifying and characterizing rarely discovered ribosomally synthesized and post-translationally modified peptides (RiPPs) from fungal origins. Due to RiPPs’ unique biosynthetic machinery, complex chemical characteristics, and important pharmacological properties, bacterial RiPPs have drawn strong interest from both academia and the pharmaceutical industry. However, only a handful of RiPPs have been identified from fungi, even though fungi is known to be a prolific producer of NPs. By characterizing novel biosynthetic enzymes of known RiPPs and new fungal BGCs identified by bioinformatics analysis, we expect to greatly broaden and deepen our understanding of the biosynthesis of fungal RiPPs and expand the repertoire of novel fungal RiPP NPs. Our second direction will focus on expanding and applying CRISPR-based genome engineering toolkits to characterize biosynthetic gene clusters from filamentous fungi. CRISPR-Cas tools have been successfully demonstrated to be feasible in fungal species but are rarely applied in the investigation of fungal NP biosynthesis. We will develop complementary sets of CRISPR-Cas tools for manipulating fungal biosynthetic gene clusters in both native and heterologous expression hosts. By doing so, we expect to develop a full set of CRISPR gene-editing toolkits to rapidly carry out genetic manipulations to study natural product biosynthesis in filamentous fungi. Together, the two research directions and collaborative research endeavors through BGC characterization, genetic tool advancement, and new bioinformatics algorithm development will build a complete pipeline to significantly increase the repertoire of fungal NPs and analogs, especially fungal RiPPs, making these molecules valuable drug candidates for human therapeutics.