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

项目摘要 微生物天然产物被认为是寻找候选药物的丰富资源。下一步的进展 世代测序已经指数级地增加了放线菌可用的元基因组数据， 它们被认为是生物活性次生代谢物的高产生产者。然而，许多天然产品 化学多样性还没有使用传统的、依赖于栽培的策略来探索。此外，a 在标准发酵下，来自培养微生物来源的相当大一部分基因簇保持沉默 条件。从理论上讲，天然产物基因簇的异源表达可以提供化学物质 来自未培养生物和神秘路径的多样性。然而，目前基于细胞的异源 表达方法仍然是基因组挖掘的最大瓶颈，因为它经常非常 既耗时又昂贵，通常仅限于少量标准主机。 在第一阶段，通过应用我们的无细胞表达技术来异源表达会不会成为瓶颈 放线菌基因簇的体外实验。天然产物的隐蔽生物合成基因簇与治疗 价值可以以高吞吐量和高成本效益的方式来表征。我们建议实现这一目标 目的通过三个具体目标。目的1.多种放线菌无细胞体系的平台开发。 我们预计，来自多个放线菌菌株的无细胞系统将提高隐匿性 簇状异源表达。目的2.天然放线菌生物合成途径的鉴定 产品。我们将鉴定以前从未表达过的隐蔽放线菌生物合成途径和基于过滤器的 在我们的无细胞表达平台中的表达潜力。目标3.无细胞体外高通量 表情。我们将运行高通量(384+)实验来表达和表征3种独特的密码 基因簇。 如果成功，这个有价值的平台可以用来激活生物信息可用密码池 生物合成基因簇在发现高活性放线菌新天然产物中的应用 药理价值。第二阶段的工作将侧重于平台扩展以及确定和扩展 下游表征的潜在治疗候选者。

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.