Molecular cloning and antimicrobial activity of enterolysin A and helveticin J of bacteriolysins from metagenome of Chinese traditional fermented foods

Expansion of food industry and international trade in foodstuffs, together with increasing worldwide attention to food safety heightened the need of exploring natural preservatives such as bacteriocins to prevent food contamination and spoilage. In this study, we uncovered a significant diversity of enterolysin A and helveticin J gene sequences belonging to bacteriolysins in popular Chinese traditional fermented foods using culture-independent metagenomic approach. The CODEHOPs targeting conserved motifs of the enterolysin A and helveticin J genes from Lactobacillales were designed to amplify respective gene sequences from six metagenomic DNA extracts by PCR. The amplicons were evaluated by sequence clone libraries and phylogenetic analyses. All translated enterolysin A sequences (77-80 amino acids) derived from four libraries were grouped into one cluster with 84-96% identity to homologs from Lactobacillus in GenBank database. Deduced helveticin J sequences (169-196 amino acids) derived from five libraries were classified into two distinct clusters, which shared 58-100% identity with public homologs in the database. Based on the obtained sequences, one of intact enlA and hlvJ genes was amplified by genome walking TAIL-PCR, and cloned into the expression vector pET-28a in Escherichia coli BL21, respectively. The recombinant EnlA (DFE5) producing a 30.6-kDa protein displayed antimicrobial activity against three bacterial genera, including human pathogens Listeria monocytogenes and Staphylococcus aureus. The expressed HlvJ (RS9) encoding a 249-aa protein was observed to inhibit Lactobacillus helveticus. This study demonstrated the validity of the metagenomic approach used in this study for identifying natural variants directly from complex food samples, but also provided the evidence that Chinese traditional fermented foods could be a valuable bacteriocin gene source for further exploration in food industry. (c) 2012 Elsevier Ltd. All rights reserved.