Glycogen removal enhances ethylene production in Synechocystis sp. PCC 6803 but exposes a 2-oxoglutarate-GS-GOGAT-phosphate constraint
- 1Department of Adaptive Biotechnologies, Global Change Research Institute, Czech Academy of Sciences, Brno, Czechia
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.
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BibTeX
@misc{zavrel2026glycogen,
title = {{Glycogen removal enhances ethylene production in Synechocystis sp. PCC 6803 but exposes a 2-oxoglutarate-GS-GOGAT-phosphate constraint}},
author = {T. Zavrel and J. Sebesta and H. Lee and T. Zlamal and T. Danisova and R. Sandor and M. Cerny and M. Berka and M. Lukes and R. Hoeper and R. Steuer and J. Cerveny and J. Yu},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.25.761650},
url = {https://www.biorxiv.org/content/10.64898/2026.10.25.761650v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Glycogen removal enhances ethylene production in Synechocystis sp. PCC 6803 but exposes a 2-oxoglutarate-GS-GOGAT-phosphate constraint
AU - T. Zavrel
AU - J. Sebesta
AU - H. Lee
AU - T. Zlamal
AU - T. Danisova
AU - R. Sandor
AU - M. Cerny
AU - M. Berka
AU - M. Lukes
AU - R. Hoeper
AU - R. Steuer
AU - J. Cerveny
AU - J. Yu
PY - 2026
DA - 2026/10/25
PB - bioRxiv
DO - 10.64898/2026.10.25.761650
UR - https://www.biorxiv.org/content/10.64898/2026.10.25.761650v1
AB - 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.
ER -