Investigation of the Polyhydroxybutyrate Cycle Regulation and the Redox Homeostasis in Rhodospirillum rubrum during Dark Chemoheterotrophic Growth

As the world transitions away from fossil-based resources, there is a growing need for innovation in the development of alternative biomolecules and efficient bioprocesses based on renewable feedstocks. Among the promising biobased materials are polyhydroxyalkanoates (PHAs), a family of polyesters naturally produced by microorganisms. PHAs, especially poly(3-hydroxybutyrate) (PHB), offer viable alternatives to conventional plastics in specific applications due to their biocompatibility, biodegradability, and comparable physicochemical properties. However, despite this potential, microbial PHB production remains economically uncompetitive compared to petroleum-based plastics. One promising way to address this challenge is to unravel how PHB synthesis is regulated and integrated into cellular metabolism, in order to improve productivity. Rhodospirillum rubrum, a purple non-sulfur bacterium (PNSB) with a versatile metabolism, can produce PHB under a wide range of growth conditions. Intracellularly, PHB is both synthesized and degraded simultaneously, forming a dynamic cycle under tight regulation. Yet, the regulation of this cycle and its relationship to the hostâ s metabolism remains poorly understood. This thesis aims to advance our understanding of the PHB cycle regulation in R. rubrum during chemoheterotrophic growth in particular on acetate. Specifically, we sought to identify environmental factors influencing PHB content and to explore the regulatory mechanisms controlling the PHB cycle under different growing conditions. We performed transcriptomic profiling across carbon sources and oxygen levels and found that PHB biosynthetic genes are constitutively expressed, while genes responsible for PHB mobilization exhibit condition-specific regulation. This suggests that transcriptional regulation plays a minor role in PHB synthesis, but a more significant role in PHB degradation. Our data also support the idea that the transcriptional regulator PhaR controls carbon partitioning between the ethylmalonyl-CoA (EMC) pathway for biomass formation and the PHB cycle. To assess whether PHB accumulation functions as an intracellular redox buffer, we used the genetically encoded Peredox biosensor to monitor NADH/NADâ º ratios in vivo. Combined with experiments manipulating dissolved oxygen and adding reductants, our results indicate that PHB can act as an electron sink under certain conditions, likely as part of a broader hierarchy of electron sink mechanisms. This feature could be exploited as a driving force to enhance PHB yields under continuous cultivation. However, culture redox potential and NADH/NADâ º levels did not always correlate with PHB content, suggesting that other redox couples, such as NADPH/NADPâ º, may be more directly involved in the PHB cycle. Altogether, this work offers new insights into the regulation of PHB metabolism in R. rubrum and provides a foundation for metabolic engineering and bioprocess optimization aimed at reducing the