| Systems biology requires comprehensive insight into the molecular physiology of an organism, but important components and regulatory mechanisms of the network of central metabolic pathways in cyanobacteria remain unclear. To determine components of a potential alternative tricarboxylic acid (TCA) cycle in the cyanobacterium Synechocystis sp. PCC 6803, the role of N-acetylornithine transaminase (S1r1022) in transamination of gamma-aminobutyrate was probed. The results are consistent with Slr1022 being part of an alternative route for converting 2-oxoglutarate to succinate via glutamate and gamma-aminobutyrate (a traditional 2-oxoglutarate dehydrogenase is missing in this organism). Another new enzyme component identified in the TCA cycle, S1r0201, was the C subunit of succinate:quinone reductase; this subunit was analyzed regarding its function in the succinate dehydrogenase complex and in the overall respiratory pathway. Metabolite analysis performed by gas chromatography-mass spectrometry on a series of genetically engineered strains with altered redox state, central metabolic pathways or carbon fixation efficiency showed that levels of intermediates in the TCA cycle remained relatively stable in Synechocystis sp. PCC 6803, indicating that even when specific electron transport pathways have been deleted, cells retain homeostasis in critical central metabolism, and it helps to maintain cellular redox poise. However, mutations and conditions that disturbed the redox state greatly affected carbon storage mechanisms of the cell. A high NADPH/NADP ratio led to increased biosynthesis of poly-3-hydroxybutyrate, a carbon storage compound that does not accumulate in Synechocystis sp. PCC 6803 under normal growth conditions but upon its formation leads to NADPH conversion to NADP. The results of the project highlight the physiological flexibility and biochemical diversity of cyanobacteria such as Synechocystis sp. PCC 6803, and emphasize the presence of homeostasis in central metabolites. |
