Heterologous Expression of Thanatin Using Dual Transposon System in Saccharomyces cerevisiae.

Thanatin is a small cationic antimicrobial peptide with broad-spectrum antibacterial activity, considered a promising candidate to combat the global antibiotic resistance crisis. Microbial biosynthesis represents an attractive strategy for sustainable production of antimicrobial peptides. In this study, we constructed a heterologous expression system for thanatin in Saccharomyces cerevisiae CENPK2 using both PiggyBac (PB) and Ty2 retrotransposon systems, demonstrating the utility of yeast as a host for peptide production and transposon-based genetic engineering. By designing transposon-based expression plasmids incorporating the CL1 selection tag, we achieved efficient expression of thanatin. The expression of thanatin-derived peptides was confirmed by Tris-tricine-SDS-PAGE and liquid chromatography-mass spectrometry (LC-MS). In vitro antibacterial assays demonstrated that the fermentation supernatant of engineered yeast significantly inhibited the growth of Escherichia coli O157: H7, Salmonella typhimurium, Aeromonas veronii (JL-2), and Acinetobacter baumannii. Notably, strains harboring both PB and Ty2 systems exhibited stronger antibacterial activity than those with a single system. Scanning electron microscopy further revealed that thanatin caused damage to bacterial cell membranes and led to cell lysis. Hemolysis tests proved that the physiological saline-replaced fermentation supernatant possessed low hemolytic activity. This study successfully established a transposon-based platform for the heterologous expression of thanatin.