CONICET | Buscador de Institutos y Recursos Humanos

Bacteriocins are bacterial antimicrobial peptides of ribosomal syn-thesis active on phylogenetically related microorganisms. The chi-merical BACTERIOCIN-HINGE-MICROCIN named PedA1-GGG-ColV and Ent35-GGG-MccV are new hybrid peptides with broaderantimicrobial spectrum than the parental molecules. Based on thehypothesis that changes in the hinge connecting the bacteriocinscould improve the activity of the hybrids, the inter-peptide regionwas subjected to gene-based bioengineering to generate novelderivatives with enhanced bioactivity. In this work, we generate abank of randomly mutated hybrid bacteriocin genes by saturationmutagenesis. The employed site-directed saturation mutagenesismethod uses two primers containing a degenerate mixture of thefour bases at the central codon of the three-amino-acids hinge-en-coding region. These primers were added to starting plasmid tem-plate and thermal cycled to produce mutant DNA plasmids, whichwere subsequently transformed into competent Escherichia coliDH5a. The plasmid DNA was purified from all the obtained coloniesand the preparation containing a plasmid mix was analyzed by DNA99sequencing. Approximately equal quantities of the four bases wereseen at each position of the mutated codon. The plasmid mixturewas used to obtain an expression library in E. coli BL21. One hun-dred colonies were randomly selected and the expression of hybridpeptides was further induced with 0.01 mM IPTG. The antimicrobi-al activity of cellular extracts was determined against the Gram (+)and Gram (-) indicator strains (L. monocytogenes FBUNT and E.coli MC4100). Some of the extracts showed higher antibiotic activ-ity compared to the Ent35-GGG-MccV extract (parental hybrid). Inparticular, the extract obtained from the strain SN35 was four timesmore active on both E. coli and L. monocytogenes. The approachwas highly successful to obtain mutants with improved bioactivitywith respect to the original hybrids.