gutSMASH 2.0: Extended Identification of Primary Metabolic Gene Clusters From the Human Gut Microbiota

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• gutSMASH provides an integrated platform to analyze the metabolic potential of gut microbiomes. • gutSMASH 2.0 incorporates 14 new detection rules that covering 12 additional types of metabolic gene clusters (MGCs) • Integration of transcription factor binding site predictions. • gutSMASH 2.0 is freely available at https://gutsmash.bioinformatics.nl . Microbiota-derived metabolites serve as key messengers mediating host–microbe and microbe–microbe interactions, often through specialized primary metabolic pathways. gutSMASH was initially developed to systematically identify the metabolic gene clusters (MGCs) that encode these pathways in anaerobic gut microbial genomes. Here, we present gutSMASH 2.0, a major update that significantly expands its functionality. This version introduces 14 new detection rules covering 12 additional types of MGCs. The comparative genomics framework was enhanced with 26 experimentally validated MGCs and 15,024 gene clusters from the Cultivated Genome Reference 2 (CGR2) collection. Furthermore, gutSMASH 2.0 integrates transcription factor binding site prediction using LogoMotif’s methodology, enabling investigation of MGC regulatory elements. Together, these improvements make gutSMASH a more powerful tool for automated discovery and analysis of niche-determining metabolic pathways in the gut microbiome. gutSMASH 2.0 is freely available at https://gutsmash.bioinformatics.nl/ .