Heterotrophic fermentation of a robust human defensin in Chlamydomonas reinhardtii provides a stable and potent antibacterial

BACKGROUND: The growing threat of antibiotic resistance underscores the urgent need for novel therapeutic agents. Antimicrobial peptides (AMPs), such as human defensins, are promising candidates due to their broad-spectrum activities, but their widespread application is severely hindered by high production costs and limited stability. Among these, human neutrophil peptide-2 (HNP2), a member of the α-defensin family, is a particularly potent AMP that kills microorganisms by disrupting their membrane integrity. The eukaryotic microalga Chlamydomonas reinhardtii (C. reinhardtii) has emerged as a low-cost and efficient bioreactor for exogenous protein production, yet its potential for expressing human defensins remains largely unexplored. This study aimed to express a tandem trimer of the HNP2 mature peptide (3×HNP2) in C. reinhardtii and to characterize its stability, safety, and antibacterial functions. RESULTS: In this study, a gene encoding 3×HNP2 was successfully expressed in the C. reinhardtii strain CC-5325, yielding a fusion protein of approximately 22 kDa. The recombinant protein's expression remained stable for over five months of continuous subculturing. Obtained via fermentation technology and affinity purification, the purified 3×HNP2 demonstrated potent antibacterial activity against the Gram-negative pathogens Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The protein exhibited high thermal stability (up to 90 °C), broad pH tolerance (pH 2-10), and resistance to degradation by proteinase K and papain. Mechanistic investigations using propidium iodide (PI) staining and scanning electron microscopy (SEM) confirmed that 3×HNP2 acts by disrupting bacterial membrane integrity. Furthermore, qRT-PCR analysis revealed that 3×HNP2 significantly downregulated the expression of key virulence-associated genes (lecA, phzA2, csgA, and rpoS). Biosafety assays showed that the peptide had minimal hemolytic activity and low cytotoxicity against mammalian cell lines. CONCLUSIONS: This work establishes a green platform for producing a functional human defensin in C. reinhardtii. It demonstrates that algae-derived 3×HNP2 is a stable, safe, and effective antimicrobial agent, suggesting its potential as a candidate for further development in antimicrobial therapeutics or food safety applications.