Metabolite-Level Regulation of Carbon Partitioning in Synechocystis sp. PCC 6803

Abstract

Cyanobacteria are promising platforms for sustainable biotechnology, but efficient metabolic engineering requires both improved genetic tools and a detailed understanding of carbon partitioning. Using Synechocystis sp. PCC 6803 as a model organism, this work examined overflow metabolism and metabolite-mediated regulation of central carbon metabolism at key branch points in cyanobacteria. Analyses of the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) and phosphoglucomutase (PGM), which link central carbon metabolism to carbon fixation and carbon storage, revealed tight and partly cyanobacteria-specific regulatory mechanisms. The results presented here show that iPGAM contains two structural features unique to cyanobacteria that integrate its regulation into the PII signaling network via the small regulatory protein PirC, enabling rapid post-translational carbon flux redirection in response to metabolic signals. In Synechocystis, carbon flux between glycolysis and glycogen metabolism is controlled by two specialized enzymes. PGM1, which catalyzes the interconversion of glucose-1-phosphate and glucose-6-phosphate, and PGM2, which generates the PGM1 activator glucose-1,6- bisphosphate from the inhibitor fructose-1,6-bisphosphate. The present work demonstrates that PGM1 activity is further fine-tuned through interplay between its regulatory phosphosite S47 and these bisphosphosugars. In contrast, heterotrophic bacteria use one less strictly regulated, functionally promiscuous enzyme for both the PGM reaction and activator synthesis. Analysis of overflow metabolism in carbon metabolism mutants showed that metabolite excretion primarily occurs when the glycogen sink is impaired. Overflow correlated with the accumulation of central hub metabolites and appeared to be threshold-dependent, thus representing a regulated alternative carbon sink rather than passive diffusion. In addition to providing new insights into Synechocystis metabolism, this work established a protein-based transformation method (BPP Bioportides™) for unicellular cyanobacteria. This method enabled efficient transformation of model and biotechnologically relevant strains, thus expanding the genetic toolbox available for cyanobacterial engineering. Together, the findings presented here demonstrate that central carbon metabolism in Synechocystis is tightly controlled at the metabolite level via cyanobacteria-specific regulatory mechanisms at key branch points, providing both conceptual and methodological foundations for future metabolic and biotechnological applications.