Glycogen removal enhances ethylene production in Synechocystis sp. PCC 6803 but exposes a 2-oxoglutarate-GS-GOGAT-phosphate constraint

Abstract

Cyanobacteria are promising platforms for converting CO2 directly into value-added chemicals. However, redirecting fixed carbon toward a product requires understanding of how cells balance the energetic and redox supplies and demands - and how efficiently metabolic flux can be channelled into product formation rather than being dissipated or excreted. Glycogen, synthesized via ADP-glucose pyrophosphorylase (GlgC), is the major native carbon and energy sink, and its removal forces cells to redirect excess carbon and reducing power into alternative pathways. As a target product we used ethylene, a major industrial building block currently derived almost entirely from fossil feedstocks, which can be produced photoautotrophically by the ethylene-forming enzyme (EFE) at the expense of 2-oxoglutarate (2-OG), a central node linking carbon and nitrogen metabolism. Here, we compared wild-type Synechocystis sp. PCC 6803 with strains carrying either a complete ({Delta}glgC) or attenuated, light-responsive (LGS; psbA2-driven glgC) glycogen synthesis block, with an EFE-expressing strain (EFE), and with the combined {Delta}glgC+EFE strain. All strains were cultivated across a light gradient (35-300 mol photons m-2 s-1) under nitrogen-replete conditions. Integrating measurements of growth and ethylene production rates, chlorophyll-fluorescence analysis, quantitative proteomics, FTIR profiling, and overflow-metabolite analysis revealed that partial suppression of glycogen synthesis (LGS) enhanced growth under low irradiance but became detrimental above a critical light threshold. EFE expression in the {Delta}glgC background increased ethylene production in a light-dependent manner but further compromised growth. Both strains with EFE increased photosynthetic electron transport consistent with the carbon-sink effect, yet this electron surplus was not channelled proportionally into product or biomass. Instead, continuous 2-OG drain by EFE constrained flux through the GS-GOGAT node and induced a secondary inorganic phosphate limitation in {Delta}glgC+EFE that activated photoprotection and limited productivity. These results point to the 2-OG/GS-GOGAT/Pi axis as a key design constraint determining whether the surplus photosynthetic flux gets converted into product, dissipated through photoprotection, or released as overflow metabolites.