Characterization of Variation in Natural Product Production Under Chemical Elicitation Using Parallel Stable Isotope Labeling

Most microorganisms produce far fewer secondary metabolites under laboratory culture conditions than would be expected based on the number of biosynthetic gene clusters (BGCs) present in their genomes. One strategy for inducing secondary metabolite production is to add chemical elicitors that disrupt bacterial metabolism. This one-strain-many-compounds (OSMAC) strategy has been used successfully to discover a broad range of natural products. However, traditional strategies for detecting changes in natural product production are not well suited to characterizing variations in the full secondary metabolome under elicitation conditions. One efficient tool to differentiate metabolites between experiments is IsoAnalyst, a parallel stable isotope labeling method that connects secondary metabolites to BGCs by determining the rates of incorporation for a set of isotopically labeled secondary metabolism building blocks. In this study three strains of Paraburkholderia were profiled under a range of OSMAC conditions and changes in secondary metabolism characterized using a combination of analytical tools including IsoAnalyst. Using these profiles, we assessed the degree of novel secondary metabolite production under different elicitation conditions. Prioritization of one compound class strongly induced in the presence of the antibiotic rifaximin led to the discovery of 2-hydroxyacyl putrescine compounds putrescinamides A (1) and B (2). The structures of these new metabolites were determined through a combination of multidimensional NMR experiments and total synthesis, which permitted the determination of their full absolute configurations. Together these stable isotope labeling experiments provide a unique perspective on system-wide variation in de novo secondary metabolite biosynthesis under elicitor conditions and highlight the impact of elicitor selection on metabolite induction in Burkholderiales strains.绝大多数微生物在实验室培养条件下产生的次级代谢产物，远低于基于其基因组中携带的生物合成基因簇（biosynthetic gene clusters, BGCs）数量所预期的水平。诱导次级代谢产物生成的策略之一，是添加可干扰细菌代谢的化学诱导子。这一“一菌多产物（one-strain-many-compounds, OSMAC）”策略已被成功用于发现多种天然产物。然而，传统的天然产物产量变化检测策略，并不适用于表征诱导条件下完整次级代谢组的变异情况。用于区分不同实验间代谢物的高效工具为IsoAnalyst——这是一种平行稳定同位素标记方法，可通过测定一组同位素标记的次级代谢前体的掺入速率，将次级代谢产物与生物合成基因簇（BGCs）相关联。本研究对3株帕拉伯克霍尔德菌在一系列OSMAC策略条件下进行了代谢组分析，并通过包括IsoAnalyst在内的多种分析工具组合，表征了其次级代谢的变化情况。基于这些代谢谱，我们评估了不同诱导条件下新型次级代谢产物的生成水平。针对抗生素利福昔明（rifaximin）存在下显著诱导产生的一类化合物进行优先筛选，最终发现了2-羟酰基腐胺类化合物——腐胺酰胺A（1）与B（2）。通过多维核磁共振（multidimensional NMR）实验与全合成（total synthesis）相结合的方法，我们解析了这些新型代谢物的结构，并确定了其完整的绝对构型。综上，这些稳定同位素标记实验为解析诱导条件下次级代谢产物从头生物合成的全系统变异提供了独特视角，并揭示了诱导子选择对伯克霍尔德菌目（Burkholderiales）菌株代谢物诱导产生的影响。