Multi-omic stable isotope probing reveals myo-inositol metabolism by anaerobic gut bacteria in laying hens.

Myo-inositol (MI) is a dietary cyclitol and phytate hydrolysis product whose microbial fate in poultry is poorly understood. We combined 13C-labelled MI, metaproteomic stable-isotope probing (SIP), untargeted metaproteomics, and 13C-nuclear magnetic resonance metabolomics to map MI utilization by anaerobic bacterial enrichment cultures established from ileal and caecal digesta of laying hens. This integrative approach enables activity-resolved tracking of carbon flow from substrate to proteins and fermentation end products within a complex anaerobic community. We reconstructed, for the first time, a community-resolved pathway of MI degradation in the chicken gut, linking labelled metabolites to taxon-specific protein synthesis. MI metabolism was spatially structured, with rapid and sustained utilization in the caeca and delayed, host-variable activity in the ileum. Metaproteomic-SIP identified Megamonas as a dominant MI-assimilating genus in the caeca and implicated Gallibacterium as a previously unrecognized contributor in the ileum. Despite minimal shifts in community composition inferred from metaproteomics, protein expression profiles changed markedly, indicating that functional responses to MI occurred independently of taxonomic restructuring. Pathway reconstruction, supported by labelled and unlabelled proteins and labelled metabolite dynamics, revealed conversion of MI into central metabolic intermediates and short-chain fatty acids (SCFAs), providing a mechanistic framework for microbial MI utilization. Together, these findings establish stable isotope probing multi-omics as a powerful strategy for reconstructing metabolic pathways in complex microbiomes and demonstrate the metabolic potential of chicken gut-derived microbial communities to utilize MI under controlled anaerobic enrichment conditions.