Development of a Bacterial Host for Natural Product Discovery and Production

项目摘要 从生命系统中分离出来的分子一直是药物发现的先导。然而，其中两个 从天然产物中发现药物的主要挑战是已知化合物的高再发现率 使用传统技术和低产量。基因组测序与合成生物学的最新进展 在私人和学术界激起了对天然产物发现的新兴趣。比如说， 对未充分探索的分类群的基因组挖掘增加了发现新化合物的机会。此外，鲁棒 宿主生物可以通过克服常见的低产障碍来促进发现工作。的细菌 变形菌门的伯克霍尔德菌目是天然产物的新兴来源。我们先前已经 在伯克霍尔德氏菌中实现了自体聚酮-非核糖体肽剪接体抑制素的高产率生产 物种我们建议了解，开发和应用这种菌株作为宿主，以发现和生产天然的 来自伯克霍尔德菌目（Burkholderiales）和潜在的其它变形菌门（Proteobacteria）的产物。虽然大肠杆菌长期以来 作为一种模式细菌宿主，合成生物学界正在远离“一个宿主适合”的想法， 所有”，而不是有主机定制的生物合成基因簇的来源。我们假设 所讨论的伯克霍尔德氏菌可以作为宿主，通过产生 异源天然产物的高产量。我们的初步数据支持这一假设为异源 编码套索肽capistruin的模型基因簇在该菌株中的表达导致capistruin的产生 产量至少是E.杆菌在目标1中，我们建议 通过a）根据伯克霍尔德菌目内的源属测试宿主的呼吸 和其他的β-变形菌，同时发现套索肽;和B）研究自体的调节， 剪接抑制素生物合成的最终目标是获得用于异源途径的调节部分 建设在目标2中，我们将开发宿主作为天然产物发现的工具， 表征启动子文库和产生最小化的基因组。在目标3中，我们将通过以下方式应用主机 从新建的伯克霍尔德菌环境收集中发现天然产品。该项目预计 提供工具，将简化和加速天然产品的发现，从一个有前途的，但未充分探索 源头所获得的工具和知识将被应用于发现新的，生物活性的天然产品，通过 伯克霍尔德菌属菌株收集的基因组挖掘。

Project Summary Molecules isolated from living systems have consistently served as leads in drug discovery. However, two of the main challenges of drug discovery from natural products are the high rate of rediscovery of known compounds using traditional technologies and low yields. Recent advances in genome sequencing and synthetic biology have spurred a renewed interest in natural product discovery in the private and academic sectors. For instance, genome mining of underexplored taxa increases the chances of novel compound discovery. Moreover, robust host organisms can facilitate discovery efforts by overcoming the common low-yield hurdle. Bacteria belonging to the Burkholderiales order of -Proteobacteria are an emerging source of natural products. We have previously achieved high yield production of autologous polyketide-nonribosomal peptide spliceostatins in a Burkholderia species. We propose to understand, develop, and apply this strain as a host to discover and produce natural products from Burkholderiales and potentially other -Proteobacteria. Although Escherichia coli has long been used as a model bacterial host, the synthetic biology community is moving away from the idea that “one host fits all” to instead have hosts tailored to the source of biosynthetic gene clusters. We hypothesize that the Burkholderia sp. in question can be used as a host to streamline the discovery process by producing heterologous natural products in high yields. Our preliminary data supports this hypothesis as heterologous expression of a model gene cluster encoding the lasso peptide capistruin in this strain led to capistruin production in yields that are at least 65-fold, and up to 580-fold higher than with E. coli. In Aim 1 we propose to test/understand the host by a) testing the breath of the host in terms of source genera within the Burkholderiales and other -Proteobacteria while discovering lasso peptides; and b) investigating the regulation of autologous spliceostatin biosynthesis with the ultimate goal of deriving regulatory parts for heterologous pathway construction. In Aim 2, we will develop the host as a tool for natural product discovery by discovering and characterizing a promoter library and by generating a minimized genome. In Aim 3 we will apply the host by discovering natural products from a newly built Burkholderiales environmental collection. This project is expected to provide tools that will streamline and accelerate natural product discovery from a promising yet underexplored source. The tools and knowledge obtained will be applied to discover novel, bioactive natural products via genome mining of a Burkholderiales strain collection.