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)已成为一种重要的药物来源，并发挥了关键作用 作为治疗人类疾病的药物。真菌的快速扩张 基因组序列和生物信息学工具的发展使鉴定成为可能 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 发现新的真菌NPs的机会。然而，新的生物活性真菌NPs的发现 仍然具有挑战性，因为在确定真菌中的BGC和遗传操作的优先顺序方面存在困难。在……里面 这项建议，我们希望通过铺设管道，快速发现具有生物活性的天然新型真菌 可作为治疗人类疾病的下一代候选药物的产品 疾病；为了做到这一点，我们将应用CRISPR CAS基因组编辑技术并致力于 这些工具有助于真菌天然产物的生物合成。为了实现研究目标，我们首先 方向将侧重于鉴定和表征罕见的核糖体合成 和来自真菌来源的翻译后修饰多肽(RIPP)。由于Ripps的独特之处 生物合成机制、复杂的化学特性和重要的药理作用 细菌破碎机的特性引起了学术界和 制药业。然而，只有极少数的Ripps被从真菌中鉴定出来，甚至 尽管众所周知，真菌是NPs的高产生产者。通过表征新的生物合成 已知的Ripps和通过生物信息学分析鉴定的新真菌BGC的酶，我们预计 大大拓宽和加深我们对真菌碎屑的生物合成的理解，并扩展 新的真菌RIPP NPs谱系。我们的第二个方向将集中在扩大和 应用基于CRISPR的基因组工程工具包表征生物合成基因簇 来自丝状真菌。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。我们会 开发互补的CRISPR-CAS工具用于操纵真菌生物合成基因 在原生和异源表达宿主中都有簇。通过这样做，我们希望开发出一种 全套CRISPR基因编辑工具包，快速进行遗传操作，研究自然 丝状真菌中产物的生物合成。总而言之，这两个研究方向和合作 通过BGC表征、基因工具进步和新的研究努力 生物信息学算法开发将建立一条完整的管道，以显著增加 真菌NPs及其类似物，特别是真菌Ripps，使这些分子变得有价值 人类治疗学的候选药物。

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.