In vitro reconstitution of the pantothenic acid catabolic pathway in Geobacillus thermodenitrificans NG80-2

Pantothenic acid (also known as pantothenate or vitamin B5), the obligate precursor of coenzyme A, is universally required in all branches of life. Although its biosynthetic pathways are well characterized, its catabolism remains poorly understood, constraining our insights into its broader biological roles and disease associations. By synergistic analysis of sequence similarity network (SSN) and genome neighborhood network (GNN) of pantothenate permease (UniProt ID: P16256 ) homologues, we identify a compact catabolic gene cluster for pantothenic acid in Geobacillus thermodenitrificans NG80-2. In vitro enzymatic activity assays indicate that pantothenic acid is first hydrolyzed to β-alanine and ( R )-pantoate by pantothenase belonging to the peptidase_M20-M20_dimer family (PF01546-PF07687), structurally distinct from the canonical pantothenase in amidase family (PF01425), and then ( R )-pantoate is sequentially oxidized into aldopantoate, ( R )-3,3-dimethylmalate, and α-ketoisovalerate by three dedicated dehydrogenases. The gene cluster is widespread within Bacillota phylum inhabiting high-temperature, hypersaline or alkaline niches, contrasting with previously described pathway restricted to Pseudomonadota, whose catabolic genes are scattered across the genomes. This work expands our knowledge about the pantothenic acid catabolism and reinforces the value of the transporter-focused genome mining strategy applicable for pathway discovery.