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

摘要 真菌天然产物(NPs)一直是一种重要的药物来源，并发挥着关键作用 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使数以千计的真菌NP得以识别 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 NPS。然而，新的生物活性真菌NPs的发现仍然具有挑战性，因为 在真菌中优先考虑BGC和遗传操作。在这项提案中，我们预计将修建管道，以 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 治疗人类疾病的候选者；为此，我们将应用CRISPR Cas基因组编辑 并将这些工具专门用于真菌天然产品的生物合成。要实现 研究目标，我们的第一个方向将集中在识别和表征罕见的发现 来自真菌来源的核糖体合成和翻译后修饰的多肽(RIPP)。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 细菌裂解蛋白的特性引起了学术界和医药界的浓厚兴趣 工业。然而，尽管真菌是已知的，但从真菌中鉴定出的Ripps却很少。 成为一名形象鲜明的NPS制作人。通过对已知的Ripps和新的Ripps的新的生物合成酶进行表征 通过生物信息学分析鉴定的真菌BGC，我们预计将极大地拓宽和深化我们的 了解真菌RIPP的生物合成，并扩大新的真菌RIPP NPs的库。 我们的第二个方向将集中在扩展和应用基于CRISPR的基因组工程工具包 丝状真菌生物合成基因簇的特征。CRISPR-CAS工具已经成功 已证明在真菌物种中是可行的，但很少应用于真菌NP的研究 生物合成。我们将开发一套互补的CRISPR-CAS工具来操纵真菌 在自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 开发全套CRISPR基因编辑工具包，快速进行遗传操作研究 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、遗传工具进步和新的 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，使这些分子成为有价值的药物 人类治疗学的候选人。

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.