The recombinant Salinispora tropica CNB-440 siderophore synthetase StDesD catalysed the assembly of pools of hydroxamic acid-containing chelators from a set of linear, non-native substrates containing internal polyethylene glycol (PEG)n spacer units. The internal (PEG)n units were capped at each end by an equivalent of the native StDesD substrate N-hydroxy-N-succinyl-cadaverine (HSC, 1) to give the length-modulated substrate set 1-(PEG)n-1-L (n = 1–6) (12.1–12.6). Chemoenzymatic reactions analysed by liquid chromatography-mass spectrometry showed StDesD catalysed end-to-end condensation reactions of 12.1–12.6 producing the cognate dihydroxamic acid macrocycles 1-(PEG)n-1-MC (13.1–13.6) as major products. Minor amounts of the macrocycle using a (1)2-L substrate with no PEG insert was observed, showing the flexible PEG linker in 12.1–12.6 improved positioning of the reactive termini in the StDesD active site to promote condensation. The major product using (1)2-L was instead the tetrahydroxamic acid macrocycle (1)4-MC desferrioxamine T1 (DFOT1, 7) formed from the sufficiently flexible precursor (1)4-L desferrioxamine S1 (DFOS1, 4). Reaction mixtures incubated with Ga(III) produced 1:1 Ga(III)-13.1–13.6 complexes. The work demonstrates chemoenzymatic synthesis as a facile approach to assemble structurally diverse and functional chelators alongside providing mechanistic insight of siderophore synthetases.