Production of antimicrobial peptides fused to a nanoparticle-forming polypeptide using Pichia pastoris

Abstract Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics, but their production remains challenging due to low recovery from natural sources, high costs associated with chemical synthesis, and toxicity-related limitations in bacterial expression systems. Yeast-based platforms provide a scalable and physiologically tolerant host for recombinant AMP production. Here, we used Pichia pastoris to produce AMPs fused to self-assembling polypeptide nanoparticles. Two constructs, aurein 1.2–CSB (Aur-CSB) and myxinidin–CSB (Myx-CSB), were designed by fusing the AMPs to a triblock polypeptide (CSB) that self-assembles into protein nanoparticles. Culture and induction parameters, including glycerol and methanol concentrations and pH, were systematically evaluated to enhance yield. Allowing extensive biomass accumulation prior to methanol induction, combined with permissive medium acidification to approximately pH 3, increased production yields. Under these conditions, Aur-CSB and Myx-CSB yields increased by up to 35.3-fold and 5.5-fold, respectively, reaching 35.95 ± 19.05 mg/L and 12.96 ± 5.75 mg/L. The purified polypeptides self-assembled into nanoparticles, as confirmed by atomic force microscopy and DLS. CSB formed rod-shaped nanoparticles with an average height of 3.55 ± 0.38 nm, whereas Aur-CSB and Myx-CSB formed globular nanoparticles with average heights of 9.79 ± 1.56 nm and 9.28 ± 0.99 nm, respectively. To provide a preliminary evaluation of biological functionality, the recombinant polypeptides were assessed against Escherichia coli using fluorescence assays. Membrane integrity was evaluated with a live/dead bacterial viability test, based on SYTO 9 and propidium iodide staining, while Hoechst was used to stain bacterial DNA. Both the culture supernatants containing the AMP-CSB expressed fusions and the purified nanoparticles displayed a preserved antimicrobial activity. Collectively, these findings establish key parameters for the efficient production of self-assembling AMP-based protein nanoparticles in Pichia pastoris and provide a scalable platform for their further development and functional characterization. Key points • High biomass before induction with methanol and a pH ~ 3 improve AMP-CSB production. • The purified AMP-CSB polypeptides retain the ability to self-assemble into nanoparticles. • The AMP-CSB nanoparticles show preliminary antimicrobial activity against E. coli.