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

推进 CRISPR-Cas 技术用于新型真菌天然产物的发现和表征

• 批准号: 10029379
• 负责人: Xue Gao
• 金额: $ 37.73万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10029379
• 关键词: Academia; Anabolism; Bioinformatics; CRISPR/Cas technology; Characteristics; Chemical Structure; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Development; Drug Industry; Drug resistance; Enzymes; Fungal Genome; Gene Cluster; Genes; Genetic; Genome engineering; Goals; Human; Investigation; Medicine; Molds; Natural Products; Pathway interactions; Peptides; Pharmacologic Substance; Pharmacology; Play; Property; Research; Ribosomes; Role; Source; Technology; Therapeutic; algorithm development; analog; base; bioinformatics tool; drug candidate; drug discovery; fungus; genetic manipulation; genome editing; human disease; interest; next generation; novel; novel therapeutics; tool

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.

抽象的 真菌天然产物（NP）一直是卓越的药物来源，并在以下领域发挥着关键作用： 用于治疗人类疾病的药物 真菌基因组序列的快速扩展。 生物信息学工具的发展使得数千种真菌NP的鉴定成为可能 生物合成基因簇（BGC），从而为发现新真菌提供了前所未有的机会 然而，由于困难，新的生物活性真菌纳米粒子的发现仍然具有挑战性。 在本提案中，我们希望优先考虑 BGC 和真菌基因操作。 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药物；为此，我们将应用 CRISPR Cas 基因组编辑 技术并将这些工具致力于真菌天然产物的生物合成，以实现这一目标。 研究目标，我们的第一个方向将侧重于识别和表征罕见发现 来自真菌的核糖体合成和翻译后修饰肽（RiPP）。 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浓厚兴趣 然而，尽管真菌是已知的，但仅从真菌中鉴定出少数 RiPP。 通过表征已知 RiPP 和新的新型生物合成酶，成为 NP 的专业生产商。 通过生物信息学分析鉴定真菌 BGC，我们期望大大拓宽和深化我们的研究范围 了解真菌 RiPP 的生物合成并扩展新型真菌 RiPP NP 的库。 我们的第二个方向将侧重于扩展和应用基于 CRISPR 的基因组工程工具包 CRISPR-Cas 工具已成功表征丝状真菌的生物合成基因簇。 真菌物种，但很少被证明应用于真菌 NP 的研究 我们将开发用于操纵真菌的互补 CRISPR-Cas 工具集。 通过这样做，我们期望能够在天然和异源表达宿主中生物合成基因簇。 开发全套CRISPR基因编辑工具包，快速进行基因操作研究 丝状真菌中的天然产物生物合成。这两个研究方向和 通过 BGC 表征、遗传工具进步和新方法进行合作研究 生物信息学算法开发将构建完整的管道，显着提高 真菌 NP 和类似物的全部成分，尤其是真菌 RiPP，使这些分子成为有价值的药物 人类疗法的候选者。