Chemoenzymatic synthesis of novel siderophore scaffolds

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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.