Engineering Bacillus Subtilis and Fermentation Process Optimization for High-Level Production of Pulcherriminic Acid

Pulcherriminic acid (PA) is a typical microbial natural cyclic dipeptide and secondary metabolite. It serves as the biosynthetic precursor of pulcherrimin, an iron-chelating siderophore with promising potential for agricultural biocontrol. Low inherent biosynthesis severely limits its practical exploitation as a bioactive natural product. Here, we screened five Bacillus subtilis strains and selected 164T7P as the metabolic engineering chassis. Replacing the native promoter of the yvmC-cypX cluster with the xylose-inducible T7 promoter increased PA titer by 7.18-fold to 0.201 g/L. Knockout of abrB or spoIVB enhanced PA formation, whereas codY deletion suppressed its biosynthesis. Further optimization of carbon source, l-leucine supplementation and FeCl₃ concentration raised the titer to 1.284 g/L at 0.6 g/L FeCl₃. With glycerol as the primary feedstock supplemented with l-leucine and FeCl₃, fed-batch fermentation in a 5-L bioreactor achieved a final PA titer of 3.91 g/L, the highest reported titer of this natural product to date. This study establishes an efficient biosynthetic platform for high-level production of PA and facilitates further development of this bioactive microbial natural product.