Efficient production and characterization of the novel and highly active antifungal protein AfpB from Penicillium...

Area of Science:

• Mycology
• Protein biochemistry
• Biotechnology

Background:

• Filamentous fungi produce antifungal proteins (AFPs) with therapeutic potential.
• Penicillium digitatum has a single AFP gene (afpB), but its protein (AfpB) was previously undetectable.
• Fungi are typically immune to their own AFPs, like Penicillium chrysogenum's PAF.

Purpose of the Study:

• To achieve biotechnological production and secretion of AfpB from Penicillium digitatum.
• To characterize the antifungal activity and properties of the produced AfpB.
• To evaluate AfpB as a candidate for controlling pathogenic fungi.

Main Methods:

• Utilized a P. chrysogenum-based expression cassette for AfpB production in P. digitatum.
• Employed the Pichia pastoris yeast system with an α-factor signal peptide for AfpB secretion.
• Confirmed AfpB absence in wild-type strains via immunodetection.
• Assessed antifungal activity, protease/thermal stability, and haemolytic activity of purified AfpB.

Main Results:

• Successful biotechnological production and secretion of functional AfpB in both P. digitatum and P. pastoris.
• AfpB exhibited higher antifungal activity than P. chrysogenum PAF against various fungi, including P. digitatum.
• Spectroscopic data indicated native folding is not essential for AfpB activity.
• AfpB demonstrated stability against proteases and heat, with no observed haemolytic activity.

Conclusions:

• AfpB can be efficiently produced and secreted using heterologous expression systems.
• AfpB is a potent antifungal agent, effective even against its producing species, P. digitatum.
• AfpB's stability and lack of haemolytic activity make it a promising candidate for antifungal drug development.