Optimization of the recombinant production of Hermetia illucens antimicrobial peptides in yeasts

Antimicrobial peptides (AMPs) play a key role in the innate immunity, the first line of defense against bacteria, fungi, and viruses. Their utilization can contribute to counteract the phenomenon of antibiotic resistance. AMPs are small molecules, ranging from 10 to 100 amino acid residues produced by all living organisms. Because of their wide biodiversity, insects are the richest and most innovative sources of AMPs. Hermetia illucens, a bioconverter insect, could be a great source of AMPs because of its extraordinary ability to live in harsh environments. In a previous study, H. illucens transcriptome was examined to identify all the sequences putatively encoding AMPs. These analyses allowed to identify 57 putative sequences suitable for subsequent experimental validation studies. Moreover, preliminary data showed that peptide fraction derived from H. illucens, whose AMPs production was induced by infection with Micrococcus flavus, showed antimicrobial activity against M. flavus and mild antimicrobial activity against Staphylococcus aureus and Escherichia coli. The aim of this work is the optimization of the AMPs production in Saccharomyces cerevisiae and Pichia pastoris expression systems, in which two putative AMP sequences of H. illucens were cloned. Yeasts have been selected as hosts in order to allow the correct eukaryotic post translational processing. Cloning experiments were performed using vectors with constitutive or inducible promoters suitable for gene expression of AMPs; these systems ensure AMP intracellular production in S. cerevisiae (YEp112T) and its secretion by P. pastoris (pPIC9K). AMP expression was first verified by real-time quantitative PCR, at different cell growth phases, and the most promising mutants were used to purify the peptides (using a polyhistidine-tag at the C terminus and a nickel-charged resin). Antimicrobial activity of the peptides was tested on E. coli, S. aureus and M. flavus strains using cell extracts or supernatants by the agar diffusion method and bioautography assay.