School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia.
School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia.
Methods Enzymol. 2024;702:121-145. doi: 10.1016/bs.mie.2024.06.009. Epub 2024 Jul 23.
Siderophores are low-molecular-weight organic bacterial and fungal secondary metabolites that form high affinity complexes with Fe(III). These Fe(III)-siderophore complexes are part of the siderophore-mediated Fe(III) uptake mechanism, which is the most widespread strategy used by microbes to access sufficient iron for growth. Microbial competition for limited iron is met by biosynthetic gene clusters that encode for the biosynthesis of siderophores with variable molecular scaffolds and iron binding motifs. Some classes of siderophores have well understood biosynthetic pathways, which opens opportunities to further expand structural and property diversity using precursor-directed biosynthesis (PDB). PDB involves augmenting culture medium with non-native substrates to compete against native substrates during metabolite assembly. This chapter provides background information and technical details of conducting a PDB experiment towards producing a range of different analogues of the archetypal hydroxamic acid siderophore desferrioxamine B. This includes processes to semi-purify the culture supernatant and the use of liquid chromatography-tandem mass spectrometry for downstream analysis of analogues and groups of constitutional isomers.
铁载体是低分子量的细菌和真菌次生代谢物，能与 Fe(III)形成高亲和力的配合物。这些 Fe(III)-铁载体配合物是铁载体介导的 Fe(III)摄取机制的一部分，这是微生物获取生长所需足够铁的最广泛策略。微生物对有限铁的竞争是通过生物合成基因簇来满足的，这些基因簇编码具有可变分子支架和铁结合基序的铁载体。一些类别的铁载体具有明确的生物合成途径，这为使用前体定向生物合成 (PDB) 进一步扩大结构和性质多样性提供了机会。PDB 涉及在培养基中添加非天然底物，以在代谢物组装过程中与天然底物竞争。本章提供了进行 PDB 实验以生产典型的羟肟酸铁载体去铁胺 B 的一系列不同类似物的背景信息和技术细节。这包括半纯化培养上清液的过程以及使用液相色谱-串联质谱法对类似物和同系物组进行下游分析。
