Chemoenzymatic synthesis of novel siderophore scaffolds

铁是所有微生物生命不可或缺的辅助因子。协调和激活分子氧的能力，以及电子传递的最佳氧化还原特性，使其成为许多细胞过程的核心。因此，铁稳态的谨慎管理至关重要。虽然铁具有高的天然丰度，但它在有氧环境中主要以Fe 3+存在，并且倾向于形成不溶性氢氧化铁，使其无法被微生物接近。因此，生物体进化出了复杂的铁获取和储存策略。虽然已知有几种机制，但细菌和真菌采用的一种常见方法是产生称为铁载体的低分子量化合物，其用作高亲和力铁螯合剂。在真菌中，产生的大多数铁载体化合物属于异羟肟酸类。该官能团源自L-鸟氨酸，其被N5-羟基化，随后被N5-酰化以产生双齿配体。通常，铁载体具有三个异羟肟酸单元，产生六齿配体，其促进多面体Fe 3+络合物的形成，其结合常数在1022 - 1032范围内。虽然异羟肟酸盐铁载体在真菌中的生理功能是公认的，但其生物合成的分子细节仍然很差。已知编码大的非核糖体肽合成酶（NRPS）的基因负责肽基铁载体的组装。最近的工作SidD NRPS，负责铁载体fusarinine C的生物合成，揭示了一个非常不寻常的非线性行为，以构建缩肽铁载体结构。这些包括观察到的氨基酸的模块间加载和链延伸reactions.The生物合成基因簇在红青霉负责fusarinine C，共享非常相似的结构域架构的粪原。它也有一个类似的底物，脱水甲羟戊酸-羟基鸟氨酸，尽管粪原是由反式异构体而不是顺式异构体制成的。尽管结构域和底物相似，但形成了非常独特的产物结构，粪原。这个博士项目的目的是研究负责粪原生产的生物合成途径，阐明反式单体单元和完整缩肽生物合成的分子细节，并确定NRPS酶复合物的3D结构。

Iron is an indispensable cofactor for all microbial life. The ability to coordinate and activate molecular oxygen, in addition to optimal redox properties for electron transport, places it central to numerous cellular processes. It is therefore vital that iron homeostasis is carefully managed. Although iron has a high natural abundance, it exists predominantly as Fe3+ in aerobic environments and tends to form insoluble ferric hydroxides rendering it inaccessible to microorganisms. Organisms have therefore evolved complex strategies for iron acquisition and storage. Whilst several mechanisms are known, a common approach employed by bacteria and fungi is the production of low-molecular-weight compounds known as siderophores, which serve as high-affinity iron chelators. In fungi, the majority of siderophore compounds produced belong to the hydroxamate class. This functionality originates from L-ornithine, which is N5-hydroxylated and subsequently N5-acylated to yield a bidentate ligand. Typically, siderophores possess three hydroxamate units, producing a hexadentate ligand, which promotes formation of a polyhedral Fe3+ complex with binding constants in the 1022 - 1032 range. Whilst the physiological function of hydroxamate siderophores in fungi is well established, the molecular details of their biosynthesis remain poorly characterised. Genes encoding for large non-ribosomal peptide synthetase (NRPS) enzymes are known to be responsible for the assembly of peptidyl siderophores. Recent work on the SidD NRPS, responsible for the biosynthesis of the siderophore fusarinine C, revealed a highly unusual nonlinear behaviour to construct the depsipeptide siderophore structure. These included observations of inter-module loading of amino acids and an iterative cycle of chain extension reactions.The biosynthetic gene cluster in Penicillium rubens responsible for fusarinine C, shares very similar domain architecture to that of coprogen. It also shares a similar substrate, anhydromevalonyl-hydroxyornithine, although coprogen is made from the trans isomer rather than the cis. Despite the similarity of domains and substrate, a very distinct product structure, coprogen, is formed. This PhD projects aim is to characterise the biosynthetic pathway responsible for coprogen production, elucidate the molecular details in the biosynthesis of the trans monomeric unit and the full depsipeptide, and to determine the 3D structure of the NRPS enzyme complex.