Abstract
Megasphaera hexanoica KCCM 43214T, isolated from cow rumen, is capable of producing medium-chain carboxylic acids such as hexanoate and octanoate. In this study, we present a high-quality genome assembly, along with intracellular metabolomic profiling and pangenomic analysis. Illumina sequencing generated 2.3 Gbp from 15,293,634 reads with a GC content of 49.5%, while PacBio HiFi sequencing produced 331.5 Mbp across 45,266 reads, with an average read length of 7,323 bp and a HiFi read N50 of 8,214 bp. Hybrid assembly of short and long reads resulted in a single 2.88 Mbp contig, containing 2,835 protein-coding genes. Genome-scale metabolic models were constructed to evaluate its metabolic capabilities under specific growth conditions. Intracellular metabolomic analysis of cells grown in medium containing fructose and lactate revealed key metabolic activities associated with chain elongation. Pangenomic analysis across nine annotated genomes identified 6,721 orthologous genes using OrthoMCL, emphasizing the genetic and functional diversity within the Megasphaera genus. This dataset offers valuable insights into the metabolism and biotechnological potential of M. hexanoica KCCM 43214T.