Regulation and Organisation of Glycogen Metabolism in Synechocystis sp. PCC 6803: Insights from in vitro Reconstitutions

Abstract

Cyanobacteria rely on glycogen as their principal carbon and energy reserve to buffer Diel and stress-induced fluctuations, yet the regulatory logic of glycogen metabolism in Synechocystis sp. PCC 6803 has remained incompletely understood. This cumulative work dissects the control architecture of glycogen synthesis and degradation by combining quantitative enzyme kinetics, in vitro pathway reconstitution, kinetic modelling, proteomics, and physiological analysis. First, glucose-1-phosphate adenylyltransferase (GlgC) is characterised as a tightly regulated 3- phosphoglycerate (3-PGA)/Pi ratio-sensing gate that converts changes in photosynthetic output and phosphate availability into ADP-glucose supply, enforcing a strong Diel asymmetry of glycogen synthesis. Second, the two glycogen synthases (GlgA1 and GlgA2) are shown to have comparable intrinsic catalytic efficiencies but distinct operating regimes dictated by primer architecture and branching. Together with the branching enzyme GlgB, they generate glycogen particles with different chain-length distributions and branching patterns, revealing a division of labour between throughput and architecture. Third, glycogen catabolism is resolved into a redox- and stress-responsive module. GlgP2 emerges as the main glycogen phosphorylase under standard and nocturnal conditions, whereas GlgP1 acts as a redox-controlled reserve activated under oxidising, stress-associated states. Deep mobilisation of glycogen during prolonged darkness and resuscitation from chlorosis critically depends on GlgX1-mediated debranching. Pulldown and co-immunoprecipitation experiments further support a glycogen-centred protein neighbourhood linking GlgC–GlgA– GlgB with GlgP/GlgX. Together, these findings establish glycogen metabolism in Synechocystis as a spatially organised, multi-layered control system that integrates light, phosphate, redox and nitrogen signals, providing a mechanistic framework for rational engineering of cyanobacterial carbon storage.