Stefan Jakovljević1, Jelena Đokić1, Emilija Brdarić1, Aleksandar Bisenić1*, Sergej Tomić2, Nataša Golić1, Dušan Radojević1, Miroslav Dinić1 and Hristina Mitrović1
1Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia
2Institute for the Application of Nuclear Energy, University in Belgrade
aleksandar.bisenic [at] imgge.bg.ac.rs
Abstract
Although most intestinal butyrate is produced through the pyruvate pathway, alternative butyrate synthesis pathways, including the glutarate pathway, are consistently detected across human gut metagenomes. However, the taxonomic basis of this highly prevalent functional capacity remains poorly resolved, as currently recognized glutarate pathway-utilizing gut butyrogens do not adequately explain its near-universal occurrence. Here, we present Pilosibacter species as highly prevalent gut commensals that help explain the taxonomic basis of glutarate pathway-mediated butyrate synthesis. Fecal isolate NGB245 was identified as a novel Pilosibacter species and characterized as a glutamate-fueled butyrate-producing gut commensal with in vitro anti-inflammatory and intestinal barrier-preserving properties. To define its metabolic specialization, we combined hybrid genome sequencing and genome-resolved comparative functional analysis, integrating KEGG module completeness, COG category enrichment, GO term enrichment, and targeted annotation searches across Pilosibacter genomes and dominant saccharolytic butyrate producers. This revealed a coherent genomic signature centered on glutamate acquisition and fermentation via the glutarate pathway, including expanded capacities for the acquisition and salvage of diverse glutamate equivalents. These predictions were supported experimentally, as glutamate supplementation enhanced growth and butyrate production in Pilosibacter sp. NGB245. Finally, genome-resolved metagenome tracking across 142 healthy adult fecal metagenomes from five geographically distinct cohorts identified Pilosibacter sp. NGB245 and Pilosibacter fragilis as highly prevalent human gut taxa, while relative abundance positioned Pilosibacter species as the leading identified contributors to glutarate pathway-mediated butyrate synthesis within the analyzed reference panel. This study provides the first reported functional genomics, metagenomics, and in vitro probiotic characterization of Pilosibacter. Our findings define Pilosibacter as a low-abundance but highly prevalent glutamate-specialized butyrogenic lineage, provide a strong taxonomic basis for the widespread glutarate pathway signal in human gut metagenomes, and expand the current view of prevalent gut butyrate producers beyond saccharolytic species.
Keywords: Pilosibacter, butyrate, glutamate, comparative genomics
Acknowledgement: This work was supported by the Science Fund of the Republic of Serbia under Grant IDEAS (7744507, NextGenBiotics); the Ministry of Science, Technological Development and Innovations of the Republic of Serbia under Grant (451-03-66/2024-03/200042).