Cell-free Expression of Cryptic Actinomycetes Gene Clusters for Natural Products Discovery

用于天然产物发现的隐性放线菌基因簇的无细胞表达

• 批准号: 9348513
• 负责人: Zachary Z. Sun
• 金额: $ 22.5万
• 依托单位: SYNVITROBIO, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2018-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9348513
• 关键词: Actinomyces Infections; Address; Alpha Cell; Anti-Infective Agents; Area; Bioinformatics; Cell-Free System; Cells; Chemicals; Chemistry; Data; Development; Enzymes; Family; Fermentation; Gene Cluster; Genes; Genome; Genomics; Health; Human; In Vitro; Individual; Metagenomics; Methods; Micromonospora; Mining; Modeling; Molecular; Natural Products; Organism; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Probability; Production; Resources; Rhodococcus; Running; Saccharopolyspora; Source; Streptomyces; System; Technology; Terpenes; Testing; Therapeutic; Time; Universities; Variant; base; bioactive natural products; cost effective; drug candidate; drug development; experimental study; feeding; improved; microbial; microorganism; next generation sequencing; novel; novel therapeutics; research and development; scale up; small molecule; theories; therapeutic candidate

Project Summary Microbial natural products are known to be rich resources for the search of drug candidates. Advances in next generation sequencing have exponentially increased the metagenomic data available from actinomycetes, which are known to be prolific producers of bioactive secondary metabolites. However, much of natural product chemical diversity has not been explored using traditional, cultivation-dependent strategies. Furthermore, a significant fraction of gene clusters from cultivated microbial sources remain silent under standard fermentation conditions. In theory, heterologous expression of natural product gene clusters can provide access to chemical diversity from both uncultivated organisms and cryptic pathways. However, current cell-based heterologous expression approach remains to be the biggest bottleneck for genomic mining as often times it can be very time consuming and expensive, and usually limited to a small number of standard hosts. In Phase I, will this bottleneck by applying our cell-free expression technology to heterologously express cryptic actinomycete gene clusters in vitro. Cryptic biosynthetic gene clusters of natural products with therapeutic value can be characterized in a high-throughput and cost-effective fashion. We propose to achieve this objective through three specific aims. Aim 1. Platform development of diverse actinomycetes cell-free systems. We anticipate that cell-free systems from multiple actinomycete strains will improve the probability of cryptic cluster heterologous expression. Aim 2. Identification of cryptic actinomycetes biosynthetic pathways of natural products. We will identify never before expressed cryptic actinomycetes biosynthetic pathways and filter based on expression potential in our cell-free expression platform. Aim 3. High throughput in vitro cell-free expression. We will run high-throughput (384+) experiments to then express and characterize 3 unique cryptic gene clusters. If successful, this valuable platform can be used to activate the pool of bioinformatically-available cryptic biosynthetic gene clusters for the discovery of new natural products from actinomycetes with high pharmacological value. Phase II efforts will focus on platform scale up and the identification and scaling of potential therapeutic candidates for downstream characterization.

项目摘要 众所周知，微生物天然产品是搜索候选药物的丰富资源。下一个进步 生成测序已成倍地增加了来自放线菌的宏基因组数据， 已知它们是生物活性次生代谢产物的产生者。但是，大部分天然产品 尚未使用传统的，依赖培养的策略来探索化学多样性。此外， 在标准发酵中，来自培养微生物源的基因簇的显着比例保持沉默 状况。从理论上讲，天然产物基因簇的异源表达可以提供化学的访问 未经培养的生物和神秘途径的多样性。但是，当前基于细胞的异源 表达方法仍然是基因组采矿的最大瓶颈 耗时且昂贵，通常仅限于少数标准主机。 在第一阶段，将通过应用我们的无细胞表达技术来异源表达神秘性来通过这种瓶颈 放线菌基因簇在体外。具有治疗的天然产物的神秘生物合成基因簇 价值可以以高通量和具有成本效益的方式来表征。我们建议实现这一目标 通过三个特定目标的目标。 AIM 1。无细胞动力菌的平台开发。 我们预计来自多种放线菌菌株的无细胞系统将提高隐性的可能性 簇异源表达。目标2。鉴定自然的神秘放线菌生物合成途径 产品。我们将在表达的神秘放线菌生物合成途径和基于滤波器的基于滤波器的表达中永远不会确定 关于我们无细胞表达平台的表达潜力。 AIM 3。无体外细胞的高通量 表达。我们将运行高通量（384+）实验，然后表达和表征3个独特的隐性 基因簇。 如果成功，这个有价值的平台可用于激活生物信息可用的隐秘池 生物合成基因簇，用于从较高的放线菌发现新天然产物 药理价值。第二阶段的努力将集中在平台规模上，并识别和缩放 潜在的治疗候选者，用于下游特征。