Highly expressed ACSF2 may determine the butyrate metabolic preference of the rumen epithelium

Ruminant epithelia preferentially catabolize butyrate to fuel ketogenesis, yet the mechanism by which the rumen epithelium establishes this preference remains unclear. Here, we identify ACSF2 as a mitochondrial acyl-CoA synthetase (ACS) that catalyzes the activation of butyrate to butyryl-CoA, thereby enabling ketogenesis and accounting for this bias. We found that ACSF2 is markedly enriched in the forestomachs across ovine organs, with expression far exceeding other ACSs in the rumen epithelium, and it is rising during postnatal establishment of fermentative function. Single-cell transcriptomics and immunostaining localize ACSF2 to the mitochondria-rich layers, where it is co-expressed in mitochondria with ketogenesis genes, notably the rate-limiting enzyme HMGCS2. Further gain- and loss-of-function experiments show that ACSF2 activates butyrate to butyryl-CoA, enhances butyrate-supported growth, and is required for efficient butyrate consumption and cell fitness under butyrate-dependent conditions. These findings define ACSF2 as a key mitochondrial gatekeeper for butyrate utilization and ketogenesis in the rumen epithelium, providing a molecular mechanism for butyrate-biased energy metabolism during rumen maturation.