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Abstract <p>Multidrug-resistant (MDR) pathogens, particularly <i>Pseudomonas aeruginosa</i>, 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 <i>P. aeruginosa</i>. A codon-optimized ACD gene was cloned into the pPICZα-A expression vector and integrated into the <i>Komagataella phaffii</i> (formerly <i>Pichia pastoris</i>) 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&#xa0;kDa instead of 8.3&#xa0;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&#xa0;°C, pH 6.24, and 1.089% methanol), rACD synthesis increased by 34.12% over baseline conditions (30&#xa0;°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 <i>P. aeruginosa</i>, yielding a 58.29% reduction in growth at 150&#xa0;µg/mL and MIC50 and MIC90 values of 143.04 and 320.64&#xa0;µg/mL, respectively. These findings underscore <i>K. phaffii</i> as a robust platform for AMP production and highlight rACD’s therapeutic potential as an effective agent against MDR <i>P. aeruginosa</i>, warranting further investigation into its clinical and industrial applications.</p> Graphical Abstract <p></p> Key points <p>•&#xa0;<i>Developing a novel K. phaffii strain for heterologous expression supports efficient rACD peptide production.</i></p> <p>•&#xa0;<i>Optimized conditions boosted expression yield by 34.12% above the reference fermentation settings.</i></p> <p>•&#xa0;<i>Recombinant acidocin suppressed Pseudomonas aeruginosa growth by 58%, indicating anti-MDR activity.</i></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月18日

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  • Ali Akbari,
  • Mahbubeh Talaee,
  • Mohammad Mir-Derikvand,
  • Navid Dad,
  • Sima Modiri,
  • Fatemeh Saadati,
  • Negin Noorbakhsh,
  • Hossein Shahbani Zahiri,
  • Hojatollah Vali,
  • Kambiz Akbari Noghabi

摘要

Abstract

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.

Graphical Abstract

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.