Exploring new approaches to enhance polyhydroxybutyrate accumulation in Paracoccus denitrificans

The bacterium Paracoccus denitrificans was investigated for polyhydroxybutyrate (PHB) production on eight organic carbon substrates. Aerobic cultivation on crotonate resulted in the most significant PHB accumulation. Additionally, PHB production on butyrate increased with an addition of HCO₃⁻ to the mineral medium. Based on the substrate specificity of the cell fraction and the results of the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) for the related gene, it is likely that butyrate and crotonate activations are catalyzed by propionyl-CoA synthetase. Potential FnrP-like binding sequences were identified upstream of certain genes related to PHB metabolism, and RT-qPCR confirmed different expression levels of the concerned genes. FnrP mutant exhibits higher PHB accumulation than the wild-type when grown anaerobically on butyrate and crotonate, transmission electron microscopy revealed an accumulation of PHB granules in the FnrP^- strain. Membrane inlet mass spectrometry demonstrated that the FnrP mutant has an impaired denitrification pathway. The Paracoccus denitrificans citrate synthase was shown to be allosterically regulated by the NAD⁺/NADH ratio and activated by Na⁺, K⁺, and NH₄⁺ ions. K⁺ limitation during aerobic growth led to increased PHB accumulation.