Heterologous Expression of Bacteriocins from the Metagenome Mining of Cotija Cheese

Bacteriocins are a heterologous group of ribosomal peptides with antibacterial activity. They are of interest to the pharmaceutical and food industries due to their potential to fight antibiotic-resistant pathogens and improve microbial food safety, respectively. Metagenomic data mining for antibacterial activity is valuable for the information it provides from unstudied genomic sequences. Furthermore, the higher biosynthetic yield obtained by the heterologous expression of putative bacteriocins allows their subsequent purification and characterization. This work aimed to express antilisterial bacteriocins in Escherichia coli after obtaining their gene sequences by in silico mining the bacterial metagenome of Cotija cheese. This artisanal Mexican cheese is manufactured with unpasteurized milk and ripens for at least 3 months. Analyzing the Cotija cheese bacterial shotgun metagenome allowed us to select two sequences (QC1 and QC2) encoding novel Class IId bacteriocins belonging to the lactococcin family. These genes were expressed as (His)6-fusion proteins in E. coli BL21 (DE3) and showed high antimicrobial activity against Listeria monocytogenes, with a minimum inhibitory concentration of 78 µg/mL. QC1 and QC2 were tested against several pathogenic bacteria and showed activity exclusively against L. monocytogenes. QC2 has a novel sequence that showed no matches against the UniProt database. It was purified by Ni²⁺ affinity chromatography and retained its activity after heating at 70 °C for 30 min. As the sequences were obtained by genomic mining on a fermented food metagenome, QC1 and QC2 have potential applications as sanitizers in industrial food facilities where L. monocytogenes contamination is the most prevalent.