Analysis of Photomixotrophic Metabolism in Synechocystis Sp. PCC 6803 Via Inverse Tracer-Metabolic Flux Analysis

Analysis of photomixotrophic metabolism in Synechocystis sp. PCC 6803 via Inverse Tracer-Metabolic Flux Analysis

Cyanobacteria have gained considerable attention in recent years for their potential applications in biotechnology. For nearly half a century, extensive research has been undertaken to characterize photosynthetic metabolism in cyanobacteria. However, questions still remain about the presence of a complete TCA cycle or the glyoxylate shunt. We present here a novel method termed Inverse Tracer-Metabolic Flux Analysis to identify and quantify photosynthetic metabolism in Synechocystis sp. PCC 6803. The labeling information of metabolites under the use of ¹²C-tracers (such as glutamate and glyoxylate) in a ¹³C-based background (NaH¹³CO₃ and U-¹³C₆ glucose), were measured by GC-MS to reveal several metabolic insights. First of all, we found that TCA pathway, instead of being a cyclic or an incomplete pathway appeared to be a unique branched structure in Synechocystis, which terminated in the production of succinate. Secondly, the in vivo activity of isocitrate lyase, which involved in the glyoxylate shunt, was not detected under our experimental conditions. The flux analysis revealed that glyoxylate can be synthesized through photo-respiration and can serve as a glycine precursor. Finally, the oxidative pentose phosphate pathway (OPP pathway) showed a measurable flux under glucose-based mixotrophic conditions. This study described an economical labeling approach to precisely characterize functional metabolisms.