Characterization of the antipathogenic molecules of lactobacilli in vegetable fermentations Tom Eilers , Wannes Van Beeck, Jelle Dillen, CarolineDricot, Peter Bron, Sander Wuyts, Dieter Vandenheuvel, Sarah Lebeer 1 (ENdEMIC), Uantwerpen, Belgium Vegetable fermentation-dominating microorganisms produce various antimicrobial molecules to combat competitors. In the Belgian citizen science project “Ferme Pekes”, we have previously gathered more than 600 novel microbial strains from fermented carrot juices. These were mainly Lactic Acid Bacteria (LAB), primarily belonging to the genus Lactiplantibacillus , which clearly dominated the final product 1 . When we spiked carrot juice fermentations with Salmonella Typhimurium NTCT 13347, Escherichia coli O157:H7 or Listeria monocytogenes MB2022, these pathogens were initially abundant but not able to persist as fermentation progressed, showing that the LAB were able to produce antipathogenic molecules and outcompete them 2 . Lactic acid is known to be a key antimicrobial in most vegetable fermentations. However, the presence and activity of other antimicrobial molecules, such as bacteriocins and non-ribosomal peptides (NRPs), is not well studied. In this project, we combined a computational analysis of the genomes of the obtained isolates with phenotyping of the antimicrobial activities . At the genome level, we screened for the presence of several different putative biosynthetic gene clusters and identified 70 novel clusters (51 bacteriocins, 16 RiPPs, and 3 NRPS) in 82 strains. To validate the function of these clusters and molecules, and to facilitate the structural analysis, dedicated gene disruption mutants are required. However, natural LAB-isolates are typically recalcitrant to transformation and quite strain specific. We propose a CRISPR-Cas9 prime editing approach to make precision gene disruption mutants, which is feasible in strains with very low transformation efficiencies. This technique allows for site specific mutagenesis without leaving an unwanted resistance marker in the final construct. Structural information on the antimicrobial components combined with head-to-head comparison of wild-type and gene deletion mutants in vegetable fermentation experiments will provide in depth information on the microbial community in this man- made ecosystem. References 1. Wuyts, S. et al. Carrot juice fermentations as man-made microbial ecosystems dominated by lactic acid bacteria. Appl. Environ. Microbiol. 84 , (2018). 2. Van Beeck, W. et al. Robustness of fermented carrot juice against Listeria monocytogenes , Salmonella Typhimurium and Escherichia coli O157:H7. Int. J. Food Microbiol. 335 , 108854 (2020).
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