Controlled Carbon Loss: Threshold-Dependent Overflow Metabolism in Synechocystis sp. PCC 6803

Cyanobacteria such as <i>Synechocystis</i> sp. PCC 6803 are promising chassis for sustainable bioproduction. During nitrogen starvation, <i>Synechocystis</i> redirects fixed carbon from biomass growth toward glycogen accumulation as a carbon and energy reserve. Inhibiting glycogen synthesis results in the excretion of excess carbon as organic acids, predominantly pyruvate and 2-oxoglutarate. Efficiently rerouting this carbon toward the formation of value-added products such as the plastic alternative polyhydroxybutyrate requires a deeper understanding of carbon partitioning and overflow metabolism. To investigate this, we quantified intra- and extracellular metabolites in <i>Synechocystis</i> wild-type and mutant strains with altered glycogen metabolism (Δ<i>pgm</i>, Δ<i>glgC</i>, Δ<i>glgA1</i>, Δ<i>glgA2</i>), nitrogen signaling (Δ<i>glnB</i>), and carbon allocation (Δ<i>pirC</i>), including the double mutant Δ<i>glgC</i>Δ<i>pirC</i>. Metabolites were analyzed after two days of nitrogen-replete or -depleted growth using enzymatic glycogen quantification and liquid chromatography-mass spectrometry. Excretion was primarily triggered by inhibition of glycogen synthesis but modulated by other changes in carbon flow, such as <i>pirC</i> deletion. Besides pyruvate and 2-oxoglutarate, small amounts of glutamate, succinate, and malate were excreted. Our findings suggest that, rather than a passive consequence of metabolite accumulation, excretion is a selective, threshold-dependent process that limits intracellular metabolite buildup, revealing an additional layer of metabolic control relevant to cyanobacterial bioengineering.