Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation

• DOI: 10.1007/s00253-005-1969-3
• Publication Date: 2005-05-27T13:28:13Z
• Authors: Adam G. Lawrence; Joerg Schoenheit; Aimin He; Jiamin Tian; Pinghua Liu; JoAnne Stubbe; Anthony J. Sinskey
• Keywords: 0303 health sciences; Transcription, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Polyesters; Blotting, Western; Hydroxybutyrates; DNA-Binding Proteins; 03 medical and health sciences; Bacterial Proteins; Fermentation; Homeostasis; Cupriavidus necator

Poly-(R)-3-hydroxybutyrate (PHB) homeostasis in Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative Western blotting were carried out on batch cultures of R. eutropha H16 in order to gain insight into how expression of the PHB-related genes phaA, phaB, phaC, phaP, phaR, phaZ1a, phaZ1b, and phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase. phaA, phaB, phaC, phaR, and phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation did not depend on PHB production as in the case of phaP. No evidence of phaZ1c expression was found under the experimental conditions used in this study.