A bacillomycin D lipopeptide controls Aspergillus flavus through membrane disruption and an L-homoserine-linked redox imbalance.

Abstract

Aspergillus flavus and its aflatoxins threaten global food safety, creating an urgent need for microbial biocontrol agents. Here, we isolated Bacillus velezensis 1-11-2 from soil and identified its primary antifungal metabolite as a bacillomycin D-type cyclic lipopeptide, bacillomycin D2. Bacillomycin D2 inhibited A. flavus with a minimum inhibitory concentration (MIC) of 32 μg/mL and a mycelial-growth IC50 of 12.08 μg/mL, and dose-dependently suppressed spore production, biomass, and aflatoxin accumulation. It remained active across a wide temperature and pH range and resisted metal ions and proteases. Mechanistically, bacillomycin D2 acted via two complementary routes. As the primary route, it bound phosphatidylethanolamine in the cell membrane, reducing membrane fluidity, dissipating membrane potential, and increasing permeability. As an auxiliary route, it triggered excessive reactive oxygen species (ROS) accumulation by impairing mitochondrial function and disturbing antioxidant homeostasis. Integrated transcriptomic and metabolomic profiling linked the observed redox imbalance to the cysteine/methionine metabolic pathway and identified L-homoserine as a contributing intracellular metabolic node. Isothermal titration calorimetry and molecular docking indicated an interaction between bacillomycin D2 and L-homoserine, suggesting that this interaction may interfere with O-acetyl-L-homoserine formation and consequently affect downstream cysteine and glutathione biosynthesis. Exogenous L-homoserine fully reversed antifungal activity at low, but not high, lipopeptide concentrations, suggesting that membrane disruption contributes substantially to the antifungal effect. These findings support an integrated, multi-target antifungal mode of action and provide candidate agents for controlling A. flavus contamination.