Revision of the central carbon metabolism in a photoautotrophic organism
The central carbon metabolism of cyanobacteria and other photoautotrophic organisms can be divided generally into assimilation/anabolism (photosynthesis and CO2-fixation) and dissimilation/catabolism (carbohydrate oxidation and respiration). While in photosynthesis CO2 is reduced to carbohydrates, cell respiration describes the opposing reaction in which carbohydrates are oxidized to CO2. When glucose and light are present as sources of energy, photomixotrophic conditions exist. This leads to catabolic (glycolytic pathways) and anabolic (Calvin-Benson-Bassam cycle) reactions taking place at the same time, which can be catalyzed by the same enzymes but in opposing directions in large parts. In contrast to plants (eukaryotes), cyanobacteria (prokaryotes) possess no intracellular compartments to spatially separate opposing processes. How cyanobacteria's metabolism is organized and how futile cycles or competition for intermediates are avoided is still mostly unclear. Particularly interesting is in Synechocystis sp. PCC6803 the glycolytic Entner-Doudoroff (ED) pathway which proceeds exclusively in the catabolic direction and does not overlap with the Calvin-Benson Bassham (CBB) cycle enzymatically. Its function as a catabolic pathway, in Synechocystis, is not yet fully understood and how exactly 6P gluconate for the ED pathway is formed enzymatically is also still ambiguous. Within this work, it could finally be determined that the Enter-Doudoroff (ED) pathway exclusively branches off the oxidative pentose phosphate (OPP) pathway and does not additionally result from a flux via a glucose dehydrogenase /gluconate kinase (GDH/GK) pathway in Synechocystis. Via growth experiments, it could be shown that, in Synechocystis, the Entner-Doudoroff (ED) pathway is essential when the lower glycolysis is interrupted. Synechocystis mutants were created in which an enzyme of lower glycolysis was deleted and examined physiologically under photoautotrophic, photomixotrophic and heterotrophic conditions. Mutants without active lower glycolysis were not viable, which indicated that the ED pathway could not fully compensate for the lower glycolysis. However, the growth of incompletely segregated mutants deteriorated significantly under photomixotrophic and heterotrophic conditions if the ED pathway was unfunctional too. This indicated that the ED pathway might represent an alternative pyruvate source to the lower glycolysis. It is assumed that in Synechocystis, in addition to lower glycolysis, acetyl-CoA can also be produced via the phosphoketolase (PK) pathway. Physiologically and enzymatically, however, the PK pathway has not yet been characterized in Synechocystis in detail. Within this work Synechocystis deletion mutants, in which the putative key enzymes of the PK pathway were deleted, were produced and physiologically examined. Synechocystis cells without a functional ED and PK pathway grew reduced under photoautotrophic, photomixotrophic and heterotrophic conditions in comparison to cells in which the PK pathway was impaired only. We, therefore, hypothesize that the ED pathway might represent an alternative pathway to yield acetyl CoA during these metabolic conditions. It was postulated that the formation of metabolons and enzyme clusters compensates for the lack of cellular compartmentalization and that the formation of metabolons is a way to regulate metabolism, efficiently and rapidly. However, to prove the existence of a protein-protein interaction unequivocally, in vivo methods are required that enable a "look into the cell". Fluorescence microscopy is best suited for this. Various in vitro studies postulated that in Synechocystis, the enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAP2) of the anabolic CBB cycle are regulated in a light-dependent manner. The protein CP12 mediates this regulation by forming a light-dependent trinary complex with PRK and GAP2. In darkness, the PRK-CP12-GAP2 complex is formed stopping the CBB cycle. In light, the complex dissolves and the CBB cycle is reactivated. Within this work, the light- depended organization and dynamics of the PRK-CP12-GAP2 complex formation were demonstrated, for the first time, in vivo. Also, cluster signals of GAP2 under photomixotrophic condition indicated that the assembly of the PRK-CP12-GAP2 complex might also be regulated glucose-dependent.