Redefining Fermentation Parameters in Natural Products Drug Discovery

项目摘要 从土壤微生物中发现抗生素被广泛认为是最重要的成就之一 在现代医学中，使许多重要的医疗程序，包括手术和癌症， 化疗然而，抗生素耐药性正在接近如此关键的水平，我们正面临着一个突出的问题。 公共卫生灾难，许多重大的医学进步可能不再可能。有一个 迫切需要开发和/或发现新型抗生素，特别是具有活性的抗生素 对抗高优先级的革兰氏阴性病原体天然产品已经成为了 我们目前的大多数抗生素，以及基因组学，宏基因组学和代谢组学的最新进展， 这表明细菌基因组中仍然有大量的生物合成潜力， 产生新的抗生素。不幸的是，在基因组中识别新的生物合成基因簇（BGC） 对它可能产生的天然产物的化学性质提供了非常少的信息。因此，在本发明中， 天然产物发现领域以及因此抗生素发现领域面临两个主要问题， 障碍：我们如何快速有效地鉴定有潜力产生所需新的 然后我们如何一致地诱导这些BGC的表达， 描述他们生产的化合物。产生抗生素的沉默BGC的诱导可能是 环境或社区依赖，特别是考虑到抗菌化合物的自我伤害作用。我们 先前的工作已经验证了一种用于鉴定产生抗微生物化合物的微生物的方法， 沉默的BGC，该提案描述了优化单一和混合培养物发酵的方法 条件来持续地产生这些化合物。在研究目标上，我们提出了两个互补的目标， 开发可重复和可扩展的发酵条件的方法， 抗生素生产，这通常是天然产物化学领域中的限速步骤。目标1将 扩大我们的观察，微生物增加生产抗菌化合物时，生长在 否则营养有限的培养基，其中复合多糖是它们的主要碳源。目标2将 在发酵之前使用共培养来操纵微生物生理学。我们希望我们优化的文化 条件将使我们能够获得含有足够的化合物的天然产品提取物， 分子网络（FBMN）分析，以在活性指导的 生物活性化合物的纯化和结构解析（目的3）。

PROJECT SUMMARY The discovery of antibiotics from soil microbes is widely regarded as one of the most significant achievements in modern medicine, enabling many important medical procedures including surgery and cancer chemotherapy. However, antibiotic resistance is approaching such a critical level that we are facing an eminent public health disaster where many significant medical advancements may no longer be possible. There is an urgent need to develop and/or discover novel classes of antibiotics, especially antibiotics that are active against high-priority Gram-negative pathogens. Natural products have served as the scaffold for the vast majority of our current antibiotics, and recent advances in genomics, metagenomics, and metabolomics clearly indicate that there is still a vast wealth of biosynthetic potential encoded in bacterial genomes that could produce novel antibiotics. Unfortunately, identifying a novel biosynthetic gene clusters (BGCs) in a genome provides us with very little information about the chemical nature of the natural product it might produce. Thus, the field of natural product discovery, and consequently the field of antibiotic discovery, faces two major obstacles: how do we quickly and efficiently identify strains that have the potential to produce desirable novel compounds from “silent” BGCs and then how do we consistently induce the expression of these BGCs to characterize the compounds they produce. The induction of silent BGCs that produce antibiotics is likely context or community dependent, especially given the self-harming effects of antimicrobial compounds. Our previous work has validated a method for identifying microbes that produce antimicrobial compounds from silent BGCs, and this proposal describes methods for optimizing single- and mixed-culture fermentation conditions to consistently produce these compounds. In the research aims, we propose two complementary approaches to develop reproducible and scalable fermentation conditions that can broadly stimulate silent antibiotic production, which is often a rate limiting step in the field of natural products chemistry. Aim 1 will expand on our observations that microbes increase the production of antimicrobial compounds when grown in otherwise nutrient-limited media where complex polysaccharides are their dominant carbon source. Aim 2 will use co-culture to manipulate microbial physiological prior to fermentation. We expect that our optimized culture conditions will enable us to obtain natural product extracts that contain sufficient compound for feature-based molecular networking (FBMN) analyses to dereplicate antimicrobial compounds prior to activity-guided purification and structural elucidation of bioactive compounds (Aim 3).