Applied Microbiology, Faculty of Biology and Biotechnology, Ruhr University Bochum, Bochum, Germany.
Organic Chemistry II, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany.
Microbiol Spectr. 2024 Jul 2;12(7):e0042324. doi: 10.1128/spectrum.00423-24. Epub 2024 Jun 12.
Clorobiocin is a well-known, highly effective inhibitor of DNA gyrase belonging to the aminocoumarin antibiotics. To identify potentially novel derivatives of this natural product, we conducted an untargeted investigation of clorobiocin biosynthesis in the known producer DS 12.976 using LC-MS, molecular networking, and analysis of fragmentation spectra. Previously undescribed clorobiocin derivatives uncovered in this study include bromobiocin, a variant halogenated with bromine instead of chlorine, hydroxylated clorobiocin, carrying an additional hydroxyl group on its 5-methyl-pyrrole 2-carboxyl moiety, and two other derivatives with modifications on their 3-dimethylallyl 4-hydroxybenzoate moieties. Furthermore, we identified several compounds not previously considered clorobiocin pathway products, which provide new insights into the clorobiocin biosynthetic pathway. By supplementing the medium with different concentrations of potassium bromide, we confirmed that the clorobiocin halogenase can utilize bromine instead of chlorine. The reaction, however, is impeded such that non-halogenated clorobiocin derivatives accumulate. Preliminary assays indicate that the antibacterial activity of bromobioin against and efflux-impaired matches that of clorobiocin. Our findings emphasize that yet unexplored compounds can be discovered from established strains and biosynthetic gene clusters by means of metabolomics analysis and highlight the utility of LC-MS-based methods to contribute to unraveling natural product biosynthetic pathways.
The aminocoumarin clorobiocin is a well-known gyrase inhibitor produced by the gram-positive bacterium DS 12.976. To gain a deeper understanding of the biosynthetic pathway of this complex composite of three chemically distinct entities and the product spectrum, we chose a metabolite-centric approach. Employing high-resolution LC-MS analysis, we investigated the pathway products in extracted culture supernatants of the natural producer. Novel pathway products were identified that expand our understanding of three aspects of the biosynthetic pathway, namely the modification of the noviose, transfer and methylation of the pyrrole 2-carboxyl moiety, and halogenation. For the first time, brominated products were detected. Their levels and the levels of non-halogenated products increased in medium supplemented with KBr. Based on the presented data, we propose that the enzyme promiscuity contributes to a broad product spectrum.
氯比醇是一种众所周知的、高效的 DNA 回旋酶抑制剂，属于氨基香豆素抗生素。为了鉴定这种天然产物的潜在新型衍生物，我们使用 LC-MS、分子网络和碎片光谱分析，对已知产DS 12.976 的氯比醇生物合成进行了非靶向调查。本研究中发现的以前未描述的氯比醇衍生物包括溴比醇，一种用溴而不是氯取代的卤代变体，羟基化氯比醇，在其 5-甲基-吡咯 2-羧酸部分上带有额外的羟基，以及另外两个在其 3-二甲基烯丙基 4-羟基苯甲酸酯部分上有修饰的衍生物。此外，我们还鉴定了几种以前不被认为是氯比醇途径产物的化合物，这为氯比醇生物合成途径提供了新的见解。通过在培养基中补充不同浓度的溴化钾，我们证实氯比醇卤化酶可以利用溴而不是氯。然而，该反应受到阻碍，导致非卤代氯比醇衍生物积累。初步测定表明，溴比醇对 和外排受损 的抗菌活性与氯比醇相当。我们的研究结果强调，可以通过代谢组学分析从已建立的菌株和生物合成基因簇中发现尚未探索的化合物，并突出了基于 LC-MS 的方法在揭示天然产物生物合成途径方面的效用。
氨基香豆素氯比醇是一种由革兰氏阳性菌 DS 12.976 产生的著名的拓扑异构酶抑制剂。为了更深入地了解这个由三种化学上截然不同的实体组成的复杂混合物的生物合成途径和产物谱，我们选择了一种以代谢物为中心的方法。我们采用高分辨率 LC-MS 分析，研究了天然产生菌提取培养上清液中的途径产物。鉴定出了新的途径产物，这些产物扩展了我们对生物合成途径三个方面的理解，即新戊糖的修饰、吡咯 2-羧酸部分的转移和甲基化以及卤化。首次检测到溴化产物。在补充 KBr 的培养基中，它们的水平和非卤化产物的水平增加。基于所呈现的数据，我们提出酶的混杂性有助于产生广泛的产物谱。
