Optimized expression and antimicrobial activity of rainbow trout defensin 3 in Komagataella phaffii

Rainbow trout β-defensin 3 (rtDefb3) is identified as a small cationic antimicrobial peptide, but its recombinant expression and functional characterization have not been reported. In this study, the mature rtDefb3 Komagataella phaffii coding sequence was cloned from trout liver cDNA and heterologously expressed in GS115 using the pPIC9K with an α-factor secretion signal. Expression condition was optimized by adjusting methanol concentration (0.5-1.25%), induction temperature (26-30 °C), and induction duration (24-168 h). Optimized induction with 1.0% methanol at 30 ℃ for 96 h produced ~ 7 mg/mL of secreted peptide, which was purified to > 90% purity using one-step Ni-IDA affinity chromatography. The identity and purity of the recombinant rtDefb3 were confirmed by Tricine-SDS-PAGE, HPLC (~ 97% purity), and MALDI-TOF mass spectrometry. Functional assays revealed potent broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. These findings demonstrate, for the first time, the successful production of active rtDefb3 in Komagataella phaffii, establishing a scalable expression platform for fish β-defensins. The recombinant rtDefb3 peptide holds promise as a natural antimicrobial agent for aquaculture and potential therapeutic applications, addressing the urgent demand for alternatives to conventional antibiotics. KEY POINTS: • First successful recombinant production of rainbow trout β-defensin 3 in Komagataella phaffii • via optimized induction strategy. • High-yield secretory expression (~ 7 mg/mL) with > 90% purity confirmed by HPLC and biochemical analysis. • Recombinant rtDefb3 displayed potent broad-spectrum antibacterial activity, offering a repeatable and scalable system and functional validation.