A 6His-2Flag dual-tag strategy enables high-yield production of antimicrobial peptide N6 in Pichia pastoris to combat multidrug-resistant Klebsiella pneumonia.

In response to the global threat posed by multidrug-resistant Klebsiella pneumoniae, this study established an efficient heterologous expression system for the antimicrobial peptide N6 in Pichia pastoris X-33. By employing an innovative 6His-2Flag dual-tag fusion strategy coupled with formic acid-mediated cleavage, we achieved a high-yield production of N6, with a final titer of 1.02 g/L-significantly surpassing previous reports and meeting the threshold for scalable industrial production. The expressed N6 exhibited potent and broad-spectrum antibacterial activity against clinical isolates of K. pneumoniae, with MIC values ranging from 2 to 8 μg/mL. Mechanistic studies revealed that N6 exerted rapid bactericidal and biofilm-eradicating effects through a multimodal action involving membrane disruption, metabolic interference, and induction of oxidative stress. In a murine systemic infection model, the N6 treatment significantly improved survival rates of mice to 70% and reduced bacterial burdens in key organs. This work not only identifies N6 as a promising therapeutic candidate against drug-resistant bacterial infections, but also provides a streamlined and cost-effective synthetic biology platform for the high-level production of antimicrobial peptides, thereby facilitating their clinical translation and industrial application.