Insights into the Polyhydroxybutyrate Biosynthesis in Ralstonia solanacearum Using Parallel 13C Tracers and Comparative Genome Analysis

Bacterial accumulation of poly(3-hydroxybutyrate) [P(3HB)] is a metabolic strategy often adopted to cope with challenging surroundings. Ralstonia solanacearum, a phytopathogen, seems to be an ideal candidate with inherent ability to accumulate this biodegradable polymer of high industrial relevance. This study is focused on investigating the metabolic networks that channel glucose into P(3HB) using comparative genome analysis, ¹³C tracers, microscopy, gas chromatography-mass spectrometry (GC-MS), and proton nuclear magnetic resonance (¹H NMR). Comparative genome annotation of 87 R. solanacearum strains confirmed the presence of a conserved P(3HB) biosynthetic pathway genes in the chromosome. Parallel ¹³C glucose feeding ([1-¹³C], [1,2-¹³C]) analysis mapped the glucose oxidation to 3-hydroxybutyrate (3HB), the metabolic precursor of P(3HB) via the Entner-Doudoroff pathway (ED pathway), potentially to meet the NADPH demands. Fluorescence microscopy, GC-MS, and ¹H NMR analysis further confirmed the ability of R. solanacearum to accumulate P(3HB) granules. In addition, it is demonstrated that the carbon/nitrogen (C/N) ratio influences the P(3HB) yields, thereby highlighting the need to further optimize the bioprocessing parameters. This study provided key insights into the biosynthetic abilities of R. solanacearum as a promising P(3HB) producer.