Genome-Resolved Secondary Metabolite Mining of a Multi-Species Enterobacteriaceae Consortium

Whole genome sequencing (WGS) of a bacterial isolate initially identified as Staphylococcus epidermidis revealed substantial genomic heterogeneity. Comprehensive metagenomic assembly, binning, and genome-resolved analysis demonstrated the presence of a multi-species Enterobacteriaceae consortium comprising Enterobacter ludwigii, Enterobacter mori, and Kosakonia cowanii. High-quality metagenome-assembled genomes (MAGs) were reconstructed using MetaS-PAdes and MetaBAT2. Species-level assignment was confirmed via Average Nucleotide Identity (ANI) analysis. Genome completeness and contamination were assessed using CheckM.Genome mining using antiSMASH identified diverse biosynthetic gene clusters (BGCs), including non-ribosomal peptide synthetases (NRPS), highly reducing type II polyketide synthases (HR-T2PKS), arylpolyenes, non-ribosomal peptide metallophores, NI-siderophores, terpene precursors, and azole-containing ribosomally synthesized and post-translationally modified peptides (RiPPs). Notably, E. ludwigii harbors a complete azole-containing RiPP cluster with YcaO-dependent cyclodehydratase activity, suggesting bacteriocin-like antimicrobial capability. These findings reveal cryptic secondary metabolite potential and demonstrate the importance of metagenome validation even in presumed monoclonal WGS datasets.