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

项目摘要 微生物天然产物是寻找药物候选物的丰富资源。下一个进展 世代测序已经指数地增加了可从放线菌获得的宏基因组数据， 已知其是生物活性次级代谢物的多产者。然而，许多天然产物 化学多样性尚未采用传统的、依赖于种植的战略加以探索。而且有 来自培养微生物来源基因簇的显著部分在标准发酵下保持沉默 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修饰的途径。 多样性来自未培养的生物体和神秘的途径。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，因为它经常非常困难。 耗时且昂贵，并且通常限于少量标准主机。 在第一阶段，将这个瓶颈，通过应用我们的无细胞表达技术，异源表达隐藏的， 放线菌基因簇。具有治疗作用的天然产物的隐蔽生物合成基因簇 价值可以以高通量和成本有效的方式来表征。我们建议实现这一目标 通过三个具体目标。目标1。多种放线菌无细胞系统平台开发。 我们预计，来自多个放线菌菌株的无细胞系统将提高隐蔽性的可能性。 簇异源表达。目标2.天然产物生物合成途径中隐生放线菌的鉴定 产品.我们将确定以前从未表达过的隐蔽放线菌生物合成途径，并基于过滤器 在我们的无细胞表达平台上的表达潜力。目标3：高通量体外无细胞 表情我们将进行高通量（384+）实验，然后表达和表征3个独特的隐蔽基因， 基因簇 如果成功的话，这个有价值的平台可以用来激活生物信息学上可用的神秘基因库。 生物合成基因簇用于发现高活性放线菌新天然产物 药理价值第二阶段的工作将侧重于平台的扩大以及确定和扩大 用于下游表征的潜在治疗候选物。

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.