Multi-omic insights into myo-inositol utilization by anaerobic gut bacteria in laying hens
Myo-inositol (MI) is a key dietary polyol metabolized by gut microbiota, yet its microbial metabolism in the poultry intestine remains poorly characterized. Here, we investigated the temporal dynamics of MI degradation and associated microbial activity in ileal and caecal microbiota from laying hens. Baseline 16S rRNA gene profiling of the inocula revealed distinct community structures between sections, with lower diversity and higher inter-individual variation in ileum compared with caeca. Cultures were incubated in minimal media supplemented with either ¹²C- or ¹³C-labeled MI or the control media having no MI, and metabolite turnover was monitored using ¹³C-NMR spectroscopy. Time-resolved metaproteomics revealed that the identity and functional roles of metabolically active bacteria differed across sections and hens, with caecal Megamonas populations displaying consistently high numbers of labelled proteins spanning multiple COG categories, whereas ileal cultures exhibited more variable taxonomic and functional patterns. Comparative analyses of the full metaproteome and ¹³C-labelled proteomes highlighted section- and time-dependent differentiation of metabolic activity. Integration of proteomic and metabolomic data allowed reconstruction of MI degradation pathways, confirming the enzymatic conversion to downstream fermentation products in the poultry gut. These results provide a comprehensive, isotope-resolved view of MI metabolism and its microbial determinants in laying hens.