Enhanced brevicidine production in Brevibacillus laterosporus by integrated fermentation and ARTP mutagenesis: mechanistic insights into the high‑yield phenotype

Brevicidine is a promising non-ribosomal antimicrobial peptide with potent activity against Gram-negative pathogens, but its practical development is limited by low natural productivity and insufficient understanding of the physiological basis underlying high-yield phenotypes. In this study, an integrated strategy combining fermentation optimization, atmospheric and room-temperature plasma mutagenesis, and mechanistic analysis was established to improve brevicidine production in Brevibacillus laterosporus 811. Single-factor optimization increased the brevicidine titer to 257.32 mg/L, and subsequent Plackett–Burman and Box–Behnken designs further improved production to 326.05 mg/L. A temperature-shift strategy combined with Tween-80 supplementation further increased the titer to 605.1 mg/L in shake-flask fermentation. In parallel, mutagenesis generated a stable high-yield mutant designated as T3, which showed a 26.6% increase in production relative to the wild type. Comparative genomics identified a cis-regulatory mutation located 75 bp upstream of spoVB . Transcriptomic, quantitative reverse-transcription PCR, and transmission electron microscopy analysis consistently showed that downregulation of spoVB in the mutant was associated with impaired spore cortex formation, indicating attenuation of sporulation-related development. Fed-batch fermentation in a 5 L bioreactor, combined with temperature shifting, low-residual-sugar feeding, and pH regulation, further increased brevicidine production to 820 mg/L. This study established an integrated route for improving brevicidine production at both the process and strain levels and provided mechanistic evidence linking suppression of sporulation-related development to the high-yield phenotype. These findings not only strengthen the production basis of brevicidine, but also provide a useful framework for the development and optimization of other non-ribosomal antimicrobial peptides.