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线索。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，产量低。基因组测序和合成生物学的最新进展 激起了私营部门和学术界对天然产品发现的新兴趣。例如, 对未被开发的分类群进行基因组挖掘增加了发现新化合物的机会。此外，健壮 寄主生物可以通过克服常见的低产量障碍来促进发现工作。细菌所属 对于伯克霍尔德里亚目的-变形杆菌来说，它是一种新兴的天然产物来源。我们之前已经 在伯克霍尔德氏菌中实现自体聚酮-非核糖体肽剪接蛋白的高产生产 物种。我们建议了解、开发和应用这种菌株作为宿主来发现和生产天然的 来自伯克霍尔德雷亚斯和可能的其他变形杆菌的产品。尽管大肠杆菌长期以来一直是 被用作模型细菌宿主的合成生物界正在远离“一个宿主适合”的想法 所有“改为根据生物合成基因簇的来源量身定做宿主。我们假设 Burkholderia sp.可以用作主机，通过生成 产量高的异源天然产物。我们的初步数据支持这一假设，认为它是异源的 编码套索多肽辣椒素的模型基因簇在该菌株中的表达导致辣椒素的产生 其产量至少是大肠杆菌的65倍，最高可达580倍。在目标1中，我们建议 测试/了解寄主：a)根据伯克霍尔德雷亚目中的源属测试寄主的呼吸 和其他-变形杆菌，同时发现套索多肽；以及b)研究自体 剪接抑制素的生物合成，最终目标是获得异源途径的调节部分 建筑。在目标2中，我们将开发宿主作为发现天然产品的工具，通过发现和 表征启动子文库并生成最小化的基因组。在目标3中，我们将通过以下方式应用宿主 从新建的伯克霍尔德里亚斯环境收藏品中发现天然产品。这个项目是预期的 提供工具，以简化和加速天然产品的发现，从一个有希望但未被开发的 消息来源。获得的工具和知识将用于发现新的、具有生物活性的天然产品 伯克霍尔德雷亚斯菌株集合的基因组挖掘。

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.