Heterologous expression and optimization of the antimicrobial peptide acidocin 4356 in Komagataella phaffii to target Pseudomonas aeruginosa

Multidrug-resistant (MDR) pathogens, particularly Pseudomonas aeruginosa, pose a serious global health threat due to their increasing prevalence and limited therapeutic options. Antimicrobial peptides (AMPs) offer promising alternatives to traditional antibiotics, yet their large-scale application remains constrained by high production costs and technical challenges. This research sought to develop a yeast-based system for the cost-efficient synthesis of acidocin 4356 (ACD), an antimicrobial peptide proven effective against P. aeruginosa. A codon-optimized ACD gene was cloned into the pPICZα-A expression vector and integrated into the Komagataella phaffii (formerly Pichia pastoris) GS115 genome. Colony PCR confirmed successful integration, and specific transformants demonstrated expression of the 6 × His-ECS-rACD fusion protein, as verified by SDS-PAGE and dot blot analysis. After Ni-NTA chromatography and enterokinase digestion, rACD was found at ~ 20 kDa instead of 8.3 kDa, suggesting oligomerization or post-translational modifications. Response surface methodology determined the optimal temperature, pH, and methanol concentration for peptide synthesis. Under optimal circumstances (21 °C, pH 6.24, and 1.089% methanol), rACD synthesis increased by 34.12% over baseline conditions (30 °C, pH 6, 1% methanol). AlphaFold structural modeling identified three α-helices in high-confidence regions, implicated in bacterial membrane disruption. Antimicrobial assays demonstrated potent rACD activity against P. aeruginosa, yielding a 58.29% reduction in growth at 150 µg/mL and MIC50 and MIC90 values of 143.04 and 320.64 µg/mL, respectively. These findings underscore K. phaffii as a robust platform for AMP production and highlight rACD's therapeutic potential as an effective agent against MDR P. aeruginosa, warranting further investigation into its clinical and industrial applications. KEY POINTS: • Developing a novel K. phaffii strain for heterologous expression supports efficient rACD peptide production. • Optimized conditions boosted expression yield by 34.12% above the reference fermentation settings. • Recombinant acidocin suppressed Pseudomonas aeruginosa growth by 58%, indicating anti-MDR activity.