Accessing the Silent Majority: Induction of Gene Expression in Fungal Artificial Chromosomes for Natural Product Discovery

项目总结/摘要 真菌天然产物中含有大量的生物合成基因，是药物开发的宝贵资源 簇（BGC），其产生具有治疗感染性疾病潜力的结构独特的化合物， 免疫系统紊乱和许多其他疾病。尽管真菌中含有大量的生物活性化学物质， 最近的研究表明，只有不到5%的可能的真菌次级代谢产物尚未被发现。 发现了虽然基因组测序的进展已经暴露了这一差距，但成功刺激基因组的表达， 来自这些沉默的BGC的未知天然产物是阻碍药物发现的主要瓶颈 努力为了克服这一挑战，有针对性的方法，旨在了解和操纵 需要影响天然产物生物合成的遗传和环境线索。最近 在异源基因表达和比较代谢组学方面的创新导致了 真菌人工染色体代谢物评分（FAC-MS）管道，这使得插入真菌 将基因组DNA导入真菌宿主，鉴定异源表达的代谢物，并阐明其 生物合成途径尽管许多FACs表达了其他隐藏的代谢物，但许多BGC表达了 还没有用这种方法表达出来。本文中的提案详细说明了激活神秘的FAC-encoded BGC，能够对控制基因表达的因素进行靶向分析。在目标1中，我们将探讨 通过用表观遗传修饰剂培养FACs和通过 将FACs插入具有改变的重要调节蛋白水平的遗传染色质突变体中。对于Aim 2， 我们将通过在存在以下物质的情况下培养FACs来刺激BGC编码的防御化合物的表达： 真菌和细菌信号分子以及复杂的微生物提取物。活化的FAC编码化合物 将作为分离和生物合成研究的目标，并将评价其抗微生物活性。此外 为了发现新的代谢物，这种方法有望提供对许多环境 和调节基因表达的表观遗传线索。该平台旨在改善FAC-MS管道， 通过提供真菌的未开发潜力，加速新药先导的发现。这些研究 还将提供真菌遗传学，化学生态学和分析代谢组学方面的关键培训， 为独立的研究生涯奠定基础。

PROJECT SUMMARY/ABSTRACT Fungal natural products are an invaluable resource for drug discovery, containing numerous biosynthetic gene clusters (BGCs) that produce structurally unique compounds with the potential to treat infectious diseases, immune disorders, and many other conditions. Despite the plethora of bioactive chemicals contained within fungi, recent studies have suggested that less than 5% of the possible fungal secondary metabolites have yet been discovered. While advances in genomic sequencing have exposed this gap, the successful stimulation of unknown natural products from these silent BGCs represents a major bottleneck stymieing drug discovery efforts. To overcome this challenge, targeted approaches designed to understand and manipulate the genetic and environmental cues influencing natural product biosynthesis are required. Recent innovations in heterologous gene expression and comparative metabolomics have led to the development of the fungal artificial chromosome-metabolite scoring (FAC-MS) pipeline, which enables the insertion of fungal genomic DNA into a fungal host, identification of heterologously expressed metabolites, and elucidation of their biosynthetic pathways. Although many FACs have expressed otherwise cryptic metabolites, many BGCs have not yet been expressed with this approach. The proposal herein details plans for activating cryptic FAC-encoded BGCs, enabling targeted analysis of the factors controlling gene expression. In Aim 1, we will explore the impact of chromatin organization on gene expression by culturing FACs with epigenetic modifying agents and by inserting FACs into genetic chromatin mutants with altered levels of important regulatory proteins. With Aim 2, we will stimulate expression of BGC-encoded defense compounds through culturing FACs in the presence of fungal and bacterial signaling molecules and complex microbial extracts. Activated FAC-encoded compounds will be targeted for isolation and biosynthesis studies and will be evaluated for antimicrobial activity. In addition to uncovering novel metabolites, this approach is expected to provide insight into the numerous environmental and epigenetic cues that regulate gene expression. This platform aims to improve the FAC-MS pipeline to accelerate the discovery of novel drug leads by providing access to the untapped potential of fungi. These studies will also provide critical training in fungal genetics, chemical ecology, and analytical metabolomics, providing a foundation for an independent research career.