Redefining Fermentation Parameters in Natural Products Drug Discovery

项目总结 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成为可能，包括外科手术和癌症 化疗。然而，抗生素耐药性正接近如此关键的水平，以至于我们面临着一个杰出的 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/或发现新类别的抗生素，特别是活性抗生素 对抗高优先级的革兰氏阴性病原体。天然产品已经成为广大人民的脚手架 我们目前的大多数抗生素，以及基因组学、元基因组学和代谢组学的最新进展 表明在细菌基因组中仍有大量的生物合成潜力，可以 生产新的抗生素。不幸的是，在基因组中鉴定一个新的生物合成基因簇(BGC) 为我们提供了很少的关于它可能产生的天然产品的化学性质的信息。因此， 天然产物发现领域，以及随后的抗生素发现领域，面临着两大 障碍：我们如何快速有效地识别有可能产生理想小说的菌株 来自“沉默的”BGC的化合物，那么我们如何持续地诱导这些BGC的表达 描述它们所产生的化合物的特征。诱导沉默的BGC产生抗生素的可能性很大 依赖于环境或社区，特别是考虑到抗菌化合物的自我伤害效应。我们的 以前的工作已经验证了一种方法，用于识别从 无声BGC，本提案描述了优化单一和混合培养发酵的方法 持续生产这些化合物的条件。在研究目标上，我们提出了两个互补的研究目标 开发可广泛刺激沉默的可重复和可扩展的发酵条件的方法 抗生素生产，这通常是天然产品化学领域的限速步骤。目标1将 扩展我们的观察，即微生物在生长时会增加抗菌化合物的产生 否则营养有限，复杂的多糖是他们的主要碳源。目标2将 在发酵前使用共培养来操纵微生物生理。我们希望我们优化的文化 条件将使我们能够获得天然产品提取物，含有足够的化合物用于基于特征的 抗菌化合物活性导向前的分子网络(FBMN)分析 生物活性化合物的纯化和结构鉴定(目标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).