Iron-Derepressed Robust Production of Fusarinine C Siderophore by <i>Aureobasidium melanogenum</i>

Siderophore biosynthesis is severely inhibited by iron sufficiency, limiting microbial production of siderophores on a large industrial scale. Herein, we report novel iron-derepressed and robust production of the fungal siderophore fusarinine C (FsC) in <i>Aureobasidium melanogenum</i>, achieved by metabolic reprogramming coupling Raman-based single-cell sorting (RACS). First, we deciphered the mechanisms of iron repression on siderophore biosynthesis in this fungus. Guided by this, we constructed an iron-depressed chassis. In this chassis, we reprogrammed the metabolic pathways of FsC involving the modules of l-ornithine, mevalonate, and siderophore biosynthesis, accomplishing iron-depressed, robust, and sole production of FsC with an extracellular titer of 763 mg L^-1 under iron-sufficient conditions in stainless steel fermenters. However, intrinsic heterogenicity occurred in FsC bioproduction. To overcome this, we developed an approach based on flow-mode RACS to select and acquire high FsC-producing single cells from each fermentation batch to be used as the seed for the next batch, thereby persistently maintaining the robustness of the FsC titer over 818 mg L^-1 in successive batch fermentation. This study showcases the principle for overcoming iron repression and heterogenicity of siderophore bioproduction to fit large-scale industrialization.