Fructose metabolism in Entner-Doudoroff pathway-deficient Cupriavidus necator H16 depends on the Calvin shunt.

As a facultative chemolithoautotrophic bacterium, Cupriavidus necator H16 uses the Entner-Doudoroff (ED) pathway for heterotrophic growth on carbohydrates such as fructose and the Calvin cycle for lithoautotrophic carbon dioxide fixation. In a previous study, we found that an ED pathway-deficient C. necator strain can survive on fructose, but the underlying metabolic pathway remained unclear. This study aimed to elucidate the metabolic mechanism of fructose metabolism in this ED pathway-deficient C. necator strain. First, the metabolic characteristics of fructose catabolism in the deficient strain were examined. Then, the roles of glycolysis/gluconeogenesis, the Calvin shunt, and the non-oxidative pentose phosphate pathway (non-OxPPP) in the metabolism of fructose were identified through comparative transcriptomic analysis combined with 13C tracer experiments. Further growth experiments using knockout strains of key genes involved in these pathways confirmed that the non-OxPPP compensates for the blocked ED pathway to metabolize fructose and provide a precursor for the Calvin shunt, thereby driving subsequent carbon fluxes. Additionally, phosphoglycolate salvage pathways, particularly the malate cycle, are crucial for recycling glycolate-2-phosphate produced during RuBisCO-catalyzed oxidation. This study revealed a novel fructose metabolism mechanism in C. necator and highlighted its metabolic flexibility, thereby deepening our understanding of its carbon utilization strategies and providing a theoretical basis for further metabolic engineering research.