Multi-Locus Integration of Antimicrobial Peptide Api137 in Saccharomyces cerevisiae Based on Ty Transposons:...

Antimicrobial peptides (AMPs) are promising alternatives to antibiotics for combating multidrug-resistant bacteria, yet their practical application is hindered by the low content of natural AMPs and the high cost of chemical synthesis. In this study, we developed a high-efficiency heterologous expression system for the proline-rich cationic antimicrobial peptide Api137 in Saccharomyces cerevisiae CENPK2 by engineering the Ty retrotransposon system composed of multi-locus integration. Recombinant plasmids carrying Api137 encoding elements were constructed and integrated into the CENPK2 genome, generating the engineered strain CENPK2 + Ty1-2/2/3/4. The target fusion peptide (5.2 kDa) was successfully expressed and identified by Tris-tricine-SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The quantification of Api137 from fermentation broth was applied by high-performance liquid chromatography (HPLC) which showed that the yield of tandem peptide in the fermentation supernatant reached 20.5 mg/L and the intracellular retention rate was 33.0%. Comparative analysis of MIC and MBC values revealed that the biologically synthesized Api137 exhibited slightly superior antibacterial activity relative to the chemically synthesized Api137. In vitro functional assays demonstrated that the fermentation supernatant of the engineered strain exhibited broad-spectrum antibacterial activity against five pathogenic bacteria, with a maximum antibacterial rate of 92.9% against Aeromonas veronii. Hemolysis assays and cytotoxicity tests confirmed that the fermentation supernatant exhibited neither hemolytic activity nor cytotoxicity. Moreover, the expression of Api137 did not impose a metabolic burden on the host. This study establishes a Ty transposon-mediated strategy for the high-level expression of Api137 in S. cerevisiae, which significantly demonstrates the antibacterial activity of Api137 while ensuring excellent biosafety.