PhaB-independent poly(3-hydroxybutyrate) production in the thermophilic hydrogen-oxidizing bacterium Hydrogenophilus thermoluteolus

Hydrogenophilus thermoluteolus TH-1 is a thermophilic hydrogen-oxidizing bacterium capable of producing poly(3-hydroxybutyrate) (PHB) from CO ₂ . To redirect carbon flux for producing other useful biomaterials, we disrupted the acetoacetyl-CoA reductase genes ( phaB1 and phaB2 ), which are central to the primary PHB synthesis pathway. Unexpectedly, the resulting Δ phaB1B2 mutant still accumulated PHB under autotrophic conditions, reaching approximately 25-35 % of the wild-type level. Furthermore, PHB accumulation in the mutant was significantly restored when fatty acids (butyrate and oleate) were used as carbon sources, whereas acetate and malate resulted in reduced accumulation. These results suggest the existence of a PhaB-independent PHB synthesis pathway. We propose that intermediates from the β-oxidation of fatty acids are converted to ( R )-3-hydroxybutyryl-CoA, bypassing the disrupted PhaB enzymes. Additionally, the basal PHB production from non-fatty acid sources implies the involvement of a reverse β-oxidation pathway. This study highlights the metabolic versatility of strain TH-1 for future metabolic engineering.