Optimisation and Scale-Up of the Heterologous Production of the Antifungal Protein PeAfpA From Penicillium expansum in Stirred-Tank Bioreactors.

Antifungal proteins from fungal origin (AFPs) are small, secreted, cationic, cysteine-rich proteins with potential as novel biofungicides. However, their application requires safe and cost-effective production platforms. PeAfpA is a highly active AFP naturally produced in large quantities by the phytopathogenic fungus Penicillium expansum and biotechnologically produced by the Generally Recognized As Safe fungus Penicillium chrysogenum although at lower yields. Here, the production of PeAfpA using the P. chrysogenum strain PCMG11552 was optimised in 0.5 L and scaled up to 5 L in stirred-tank bioreactors. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to evaluate the influence of process parameters on PeAfpA production, identifying medium pH and agitation rate as key factors, while dissolved oxygen proved critical during scale-up. The PeAfpA yields obtained were tenfold higher than those achieved previously in flask cultures, demonstrating the feasibility of scaling AFP production from laboratory to pilot scale for diverse applications. Finally, the P. chrysogenum marker-free PeAfpA-producing strain obtained via recyclable CRISPR/Cas9 system guarantees regulatory compliance and biosafety, making it suitable for its use in any commercial environment.