The production of siderophore analogues using precursor-directed biosynthesis.

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.

Acetyl-CoA-Mediated Post-Biosynthetic Modification of Desferrioxamine B Generates N- and N-O-Acetylated Isomers Controlled by a pH Switch.

Biosynthesis of the hydroxamic acid siderophore desferrioxamine D1 (DFOD1, 6), which is the N-acetylated analogue of desferrioxamine B (DFOB, 5), has been delineated. Enzyme-independent Ac-CoA-mediated N-acetylation of 5 produced 6, in addition to three constitutional isomers containing an N-O-acetyl group installed at either one of the three hydroxamic acid groups of 5. The formation of N-Ac-DFOB (DFOD1, 6) and the composite of N-O-acetylated isomers N-O-Ac-DFOB[001] (6a), N-O-Ac-DFOB[010] (6b), and N-O-Ac-DFOB[100] (6c) (defined as the N-O-Ac motif positioned within the terminal amine, internal, or N-acetylated region of 5, respectively), was pH-dependent, with 6a-6c dominant at pH < 8.5 and 6 dominant at pH > 8.5. The trend in the pH dependence was consistent with the pKa values of the NH3+ (pKa ∼ 10) and N-OH (pKa ∼ 8.5-9) groups in 5. The N- and N-O-acetyl motifs can be conceived as a post-biosynthetic modification (PBM) of a nonproteinaceous secondary metabolite, akin to a post-translational modification (PTM) of a protein. The pH-labile N-O-acetyl group could act as a reversible switch to modulate the properties and functions of secondary metabolites, including hydroxamic acid siderophores. An alternative (most likely minor) biosynthetic pathway for 6 showed that the nonribosomal peptide synthetase-independent siderophore synthetase DesD was competent in condensing N'-acetyl-N-succinyl-N-hydroxy-1,5-diaminopentane (N'-Ac-SHDP, 7) with the dimeric hydroxamic acid precursor (AHDP-SHDP, 4) native to 5 biosynthesis to generate 6. The strategy of diversifying protein structure and function using PTMs could be paralleled in secondary metabolites with the use of PBMs.

Analogues of desferrioxamine B (DFOB) with new properties and new functions generated using precursor-directed biosynthesis.

Desferrioxamine B (DFOB) is a siderophore native to Streptomyces pilosus biosynthesised by the DesABCD enzyme cluster as a high affinity Fe(III) chelator. Although DFOB has a long clinical history for the treatment of chronic iron overload, limitations encourage the development of new analogues. This review describes a recent body of work that has used precursor-directed biosynthesis (PDB) to access new DFOB analogues. PDB exploits the native biosynthetic machinery of a producing organism in culture medium augmented with non-native substrates that compete against native substrates during metabolite assembly. The method allows access to analogues of natural products using benign methods, compared to multistep organic synthesis. The disadvantages of PDB are the production of metabolites in low yield and the need to purify complex mixtures. Streptomyces pilosus medium was supplemented with different types of non-native diamine substrates to compete against native 1,5-diaminopentane to generate DFOB analogues containing alkene bonds, fluorine atoms, ether or thioether functional groups, or a disulfide bond. All analogues retained function as Fe(III) chelators and have properties that could broaden the utility of DFOB. These PDB studies have also added knowledge to the understanding of DFOB biosynthesis.