Structural elements of cyanobacterial co-factor-independent phosphoglycerate mutase that mediate regulation by PirC

The 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been identified as a crucial regulating key point in the carbon storage metabolism of cyanobacteria. Upon nitrogen starvation, the iPGAM is inhibited by the PII-interacting regulator PirC, released from its interaction partner PII due to elevated 2-oxoglutarate levels. In-silico analysis of 338 different iPGAMs revealed a deep-rooted distinctive evolution of iPGAMs in cyanobacteria. Remarkably, cyanobacterial iPGAMs possess a unique loop structure and an extended C-terminus. Our analysis suggests that iPGAM forms a complex with three individual PirC monomers. Complex affinity is affected by the unique loop and the C-terminal structural elements. A C-terminal truncated enzyme showed loss of control by PirC and two-fold increased enzymatic activity compared to the iPGAM-WT. By contrast, deleting the loop structure drastically reduced the activity of this variant. By replacing the WT iPGAM in Synechocystis with different iPGAM variants, in which these structural elements were deleted, it became apparent that deletion of the C-terminal element showed a similar overproduction of polyhydroxybutyrate as deletion of the iPGAM-regulator PirC. However, in contrast to the latter, these strains showed higher over-all biomass accumulation, making them a better chassis for a production strain for PHB or other valuable substances than the PirC-deficient mutant. These findings significantly contribute to our understanding of the metabolic pathways in cyanobacteria and open up new avenues for further research in this field, inspiring future investigations and discoveries. ImportanceThe primordial cyanobacteria were responsible for developing oxygenic photo-synthesis early in evolution. Through endosymbiosis, they further evolved into the chloroplasts found in the plant kingdom. Many metabolic pathways within chloroplasts originated from cyanobacteria. However, differences emerged during their long separate evolution, providing insights into the endosymbiotic process. In the metabolic pathways involving fixed CO2, the co-factor-independent phosphoglycerate mutase (iPGAM) plays a crucial role by directing the first CO2 fixation product, 3-phosphoglycerate, towards critical anabolic path-ways. Our findings reveal a distinct evolution of iPGAM within oxygenic photo-synthetic organisms. We have identified two specific segments in cyanobacterial iPGAMs that tightly control the cellular carbon/nitrogen state through a specific protein interactor (PiC). This understanding of iPGAM has allowed us to engineer cyanobacterial strains with altered carbon fluxes. Since cyanobacteria can directly convert CO2 into valuable products, our results demonstrate a novel approach for developing a chassis for biotechnical use.