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

接触沉默的大多数：诱导真菌人工染色体中的基因表达以发现天然产品

• 批准号: 10214637
• 负责人: Lindsay Kate Caesar
• 金额: $ 6.64万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214637
• 关键词: Academia; Affect; Anabolism; Artificial Chromosomes; Aspergillus; Behavior; Bioinformatics; Biological; Biology; Cell Communication; Cells; Chemicals; Chemistry; Chromatin; Communicable Diseases; Communication; Complex; Cues; DNA; DNA Packaging; DNA Sequence; Data Set; Development; Ecology; Environment; Enzymes; Epigenetic Process; Event; Evolution; Factor Analysis; Foundations; Future; Gene Cluster; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomic DNA; Genomics; Goals; Histone Deacetylase; Histone Deacetylase Inhibitor; Human; Immune System Diseases; Improve Access; Laboratories; Length; Location; Medical; Medicine; Metabolic; Molds; Natural Products; Nature; Organism; Pathway interactions; Phenotype; Phylogenetic Analysis; Plants; Population; Postdoctoral Fellow; Ralstonia solanacearum; Regulation; Regulatory Element; Research; Research Personnel; Role; Signaling Molecule; Soil; Stimulus; Structure; System; Technical Expertise; Training; Translations; Work; antimicrobial; base; bioinformatics tool; career; chemical genetics; chromatin modification; comparative; design; drug discovery; drug resource; fungal genetics; fungus; gene induction; gene repression; genetic architecture; genetic regulatory protein; histone methylation; histone modification; improved; innovation; insight; metabolome; metabolomics; method development; microbial; microbial community; microorganism; mutant; novel; novel therapeutics; pathogenic bacteria; quorum sensing; stressor; tool; training opportunity

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.

项目摘要/摘要 真菌天然产品是药物发现的宝贵资源，包含许多生物合成基因 簇（BGC）产生结构独特的化合物，具有治疗传染病的潜力， 免疫疾病和许多其他疾病。尽管真菌中包含过多的生物活性化学物质，但 最近的研究表明，不到5％的真菌二级代谢产物尚未 发现。尽管基因组测序的进步已经揭示了这一差距，但成功刺激了 这些无声BGC的未知天然产品代表了一个主要的瓶颈阻塞药物发现 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提示。最近的 异源基因表达和比较代谢组学的创新导致了 真菌人造染色体 - 金代谢物评分（FAC-MS）管道，可以插入真菌 基因组DNA进入真菌宿主，异源表达的代谢产物的鉴定，并阐明其 生物合成途径。尽管许多FACS表达了其他代谢物，但许多BGC都有 尚未用这种方法表达。此处的提案详细介绍了激活神秘FAC编码的计划 BGC，实现了控制基因表达的因素的针对性分析。在AIM 1中，我们将探索影响 通过用表观遗传修饰剂培养FAC的染色质组织，并通过 将FACS插入具有重要调节蛋白水平的遗传染色质突变体中。与AIM 2一起 我们将在存在下通过培养FACS刺激BGC编码的防御化合物的表达 真菌和细菌信号分子和复杂的微生物提取物。活化的FAC编码化合物 将针对分离和生物合成研究，并将评估抗菌活性。此外 要揭示新的代谢产物，这种方法有望提供对众多环境的见解 和调节基因表达的表观遗传提示。该平台旨在改善FAC-MS管道 通过提供尚未开发的真菌潜力，加速了新型药物铅的发现。这些研究 还将提供对真菌遗传学，化学生态学和分析代谢组学的重要培训，提供了一种 独立研究职业的基础。