Grain-induced rumen fermentation shifts drive ecological enrichment of high-turnover tryptophanase variants for precision microbiome modulation

Indole acts as a critical interkingdom signal, promoting gastrointestinal homeostasis at basal levels while driving cytotoxicity and inflammation when accumulated at supraphysiological concentrations. Current interventions relying on broad-spectrum antibiotics or non-specific inhibitors lack the precision to distinguish pathological overproduction from homeostatic signaling, often disrupting essential commensal networks. Resolving this therapeutic dilemma requires elucidating whether indole hyperproduction arises from proportional expansion of indole-producing bacteria or from ecological enrichment of intrinsically hyperactive tryptophanase (TnaA) variants. Here, using dairy cow rumen as a model ecosystem, we integrated evolutionary genomics, computational structure modeling, and biochemical validation to dissect the structural and kinetic properties of TnaA variants and to determine their contribution to diet-induced indole hyperproduction. By integrating 462 TnaA orthologs from 21,471 reference genomes and 197 rumen metagenomes, we revealed a “conserved-fold, divergent-pocket” architecture: despite extensive sequence divergence (30–100% identity), TnaAs maintained a highly conserved global fold (TM-score 0.89–1.00) while exhibiting distinct active-site geometries and substrate affinities. Phylogenetic reconstruction and genome-scale metabolic modeling are consistent with this architecture reflecting dual, hierarchically operating selective pressures in the bovine rumen. At the macro-evolutionary scale, intense competition for colonization niches in the high-density rumen environment is associated with structural conservation of the TnaA scaffold compatible with a purifying-selection-like constraint across diverse lineages. At the micro-evolutionary scale, diet-induced rumen fermentation shifts were associated with differential enrichment of TnaA variants with distinct kinetic properties. Specifically, high-grain (HG) conditions were associated with enrichment of high-turnover variants consistent with indole-mediated competitive advantages, whereas low-grain (LG) conditions favored attenuated variants compatible with indole’s role as a cooperative signaling molecule. Exploiting these structural distinctions, we identified luteolin as a variant-selective inhibitor that preferentially targets high-turnover TnaA variants (IC 50 = 4.62 µM) through unobstructed active-site penetration, while sparing the sterically gated attenuated commensal forms (IC 50 = 25.27 µM). Our findings reveal that grain-induced fermentation shifts are associated with the enrichment of TnaA variants with distinct kinetic properties within rumen microbial ecosystems, and that exploiting naturally evolved structural vulnerabilities enables precision targeting of dysbiotic metabolic functions while preserving homeostatic microbial networks. These results offer a conceptual framework for transitioning from ecosystem-disrupting antimicrobials to function-selective microbiome modulation. Video Abstract