Isotopically Nonstationary 13c-Metabolic Flux Analysis Under Photoautotropic Conditions for a Glycogen Mutant of Synechococcus Sp. PCC 7002

Glycogen is the major carbon storage molecule in cyanobacteria and removal of this native carbon sink could increase the carbon flux towards other value-added products for biotechnological purposes. Comparative analyses of the wild type (WT) and a glycogen deficient mutant could provide insight into the re-routing of carbon through primary intermediates of metabolism and allow assessment of the flux ratios at critical branch points. Specifically, in this study, we have performed ¹³C labeling studies on the model cyanobacterium Synechococcus sp. PCC 7002 using the WT strain and a mutant strain with inactivated glycogen synthesis made by deleting the genes encoding glycogen synthase (A1532/glgA1 and A2125/glgA2). In both WT and mutant strains we observed slow labeling of the TCA cycle relative to glycolysis and Calvin cycle intermediates. In the WT strain, the ¹³C-enrichment of different intermediates at steady state reveals the existence of inactive pools of metabolites that get labelled at reduced rates. Our results also indicate slow labeling of 2-phosphoglycolate (2PG) that is the first committed intermediate in photorespiratory metabolism, thus it is likely that the carboxysome-based carbon concentrating mechanism is fully active in these strains. As might be expected, the transient labeling of the glycogen precursor ADP-glucose was slow in the mutant strain compared to the WT.  Currently, the transient labeling data for the mutant strain is being used to estimate the intracellular fluxes with an isotopically nonstationary metabolic flux analysis approach (INST-MFA). Comparison of flux maps for the WT and mutant strain will provide further insights which will be of value in metabolic engineering efforts in cyanobacteria.

Acknowledgement: Authors acknowledge financial assistance from Reliance Industries Ltd, India.