Pamamycin Disrupts the Cell Envelope and Mitochondrial Potential to Inhibit Aspergillus flavus and Aflatoxin Production in a Peanut Kernel Model

<i>Aspergillus flavus</i> contaminates food commodities and produces carcinogenic aflatoxins. Pamamycin, a macrodiolide antibiotic from <i>Streptomyces alboflavus</i> TD-1, shows potent antifungal activity, yet its action against <i>A. flavus</i> and efficacy in complex food matrices largely remains unknown. Here, pamamycin was purified and evaluated using in vitro assays together with a peanut kernel model. Pamamycin reduced colony formation of <i>A. flavus</i> on PDA in a concentration-dependent manner, with near-complete inhibition at 4.0 mg/L on surface-treated PDA plates. Microscopy revealed progressive deformation and collapse of conidia and hyphae. Pamamycin increased membrane permeability, as indicated by elevated extracellular nucleic acid leakage, and impaired cell envelope integrity, as reflected by alkaline phosphatase release. In addition, pamamycin reduced Rh123-associated fluorescence, indicating an apparent dissipation of mitochondrial membrane potential under the tested conditions. Notably, at pamamycin concentrations of ≥0.5 mg/L, AFB1 accumulation was markedly reduced and fell below the limit of detection (LOD). This suppression was accompanied by distinct transcriptional changes in the aflatoxin regulatory network. RT-qPCR showed concentration-dependent repression of the pathway-specific regulators <i>aflR</i> and <i>aflS</i>, whereas the global regulator <i>veA</i> displayed a biphasic response with transient upregulation at lower concentrations. Notably, at 0.5 mg/L, multiple structural genes (<i>aflC</i>, <i>aflD</i>, <i>aflK</i>, <i>aflP</i>, and <i>aflQ</i>) were reduced to near-background transcript levels, coinciding with the loss of detectable AFB1. In inoculated peanut kernels incubated under high-humidity conditions, pamamycin significantly reduced fungal colonization and decreased AFB1 accumulation by >99%. Transcriptomic analysis of cultures treated with 0.5 mg/L pamamycin further revealed extensive transcriptional reprogramming, with enrichment of pathways related to branched-chain amino acid biosynthesis, central carbon metabolism, and ABC transporters. Collectively, pamamycin inhibits <i>A. flavus</i> through combined disruption of cell envelope integrity, apparent mitochondrial potential collapse, and broad suppression of the aflatoxin biosynthetic pathway, supporting its potential utility for mitigating aflatoxin contamination in peanut kernels, pending further safety evaluation.