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

摘要 真菌天然产物（NPs）是一种卓越的药物来源， 用于治疗人类疾病的药物。真菌基因组序列的快速扩增 生物信息学工具的发展已经能够鉴定数千种真菌NP 生物合成基因簇（BGC），从而为发现新的真菌提供了前所未有的机会 NP。然而，新的生物活性真菌纳米颗粒的发现仍然具有挑战性，这是由于在以下方面的困难： 优先考虑BGC和真菌的遗传操作。在这份提案中，我们希望建造管道， 快速发现可作为下一代药物的新型生物活性真菌天然产物， 用于治疗人类疾病的候选人;为了做到这一点，我们将应用CRISPR Cas基因组编辑 技术和专用这些工具的真菌天然产物的生物合成。实现 研究目标，我们的第一个方向将集中在识别和表征很少发现 核糖体合成和后修饰的肽（RIPPs）来自真菌来源。由于 RIPPs独特的生物合成机制、复杂的化学特性和重要的药理作用， 细菌RIPP的特性引起了学术界和制药界的强烈兴趣。 行业然而，只有少数RIPP已被确定从真菌，即使真菌是已知的， 成为NP的主要生产商。通过表征已知RIPP的新型生物合成酶和新的RIPP的生物合成酶， 通过生物信息学分析确定的真菌BGC，我们期望大大拓宽和深化我们的 了解真菌RiPP的生物合成，并扩大新型真菌RiPP NP的库。 我们的第二个方向将专注于扩展和应用基于CRISPR的基因组工程工具包， 表征来自丝状真菌的生物合成基因簇。CRISPR-Cas工具已经成功地 已证明在真菌物种中是可行的，但很少用于真菌NP的调查 生物合成我们将开发互补的CRISPR-Cas工具集，用于操纵真菌 在天然和异源表达宿主中的生物合成基因簇。通过这样做，我们希望 开发一套完整的CRISPR基因编辑工具包，以快速进行基因操作， 丝状真菌中天然产物的生物合成。这两个研究方向和 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RIPP的库，使这些分子成为有价值的药物 用于人类治疗的候选物。

Abstract 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 profilic 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.