GENERATION OF HYBRID BACTERIOCINS VARIANTS WITH ENHANCED ANTIMICROBIAL ACTIVITY

NAVARRO, S.A; FERNANDEZ DE ULLIVARRI, M.; LANZA, L; CHALON M.C.; BELLOMIO, A

Buenos Aires

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

Bacteriocins are bacterial antimicrobial peptides of ribosomal synthesis active on phylogenetically related microorganisms. The chimerical BACTERIOCIN-HINGE-MICROCIN named PedA1-GGG-ColV and Ent35-GGG-MccV are new hybrid peptides with broader antimicrobial spectrum than the parental molecules. Based on the hypothesis that changes in the hinge connecting the bacteriocins could improve the activity of the hybrids, the inter-peptide region was subjected to gene-based bioengineering to generate novel derivatives with enhanced bioactivity. In this work, we generate a bank of randomly mutated hybrid bacteriocin genes by saturation mutagenesis. The employed site-directed saturation mutagenesis method uses two primers containing a degenerate mixture of the four bases at the central codon of the three-amino-acids hinge-encoding region. These primers were added to starting plasmid template and thermal cycled to produce mutant DNA plasmids, which were subsequently transformed into competent Escherichia coli DH5α.The plasmid DNA was purified from all the obtained colonies and the preparation containing a plasmid mix was analyzed by DNA sequencing. Approximately equal quantities of the four bases were seen at each position of the mutated codon. The plasmid mixture was used to obtain an expression library in E. coli BL21. One hundred colonies were randomly selected and the expression of hybrid peptides was further induced with 0.01 mM IPTG. The antimicrobial 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 activity compared to the Ent35-GGG-MccV extract (parental hybrid). In particular, the extract obtained from the strain SN72 was four times more active on both E. coli and L. monocytogenes. The approach was highly successful to obtain mutants with improved bioactivity with respect to the original hybrids.