Positional <scp>¹³C</scp> enrichment analysis of aspartate determines <scp>PEPC</scp> activity <i>in vivo</i>

Photoautotrophic organisms fix inorganic carbon (Ci) by RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (RUBISCO) and PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC). Monitoring Ci assimilation rates in vivo is a major challenge in analyzing photoautotrophic metabolism and engineering improved photosynthesis, as conventional methods do not distinguish between these two fluxes. We explored widely applied gas chromatography mass spectrometry (GC-MS) metabolite profiling for C-positional fractional ¹³C enrichment (E¹³C) analyses of aspartate to differentiate within one molecule between PEPC, RUBISCO, and CBB cycle activities by ¹³C pulse-labeling. We validated this method using two GC-MS instruments and two prevailing chemical derivatization methods. We selectively determined E¹³C at each carbon position of aspartate with accuracy < 1% and precision < 2.5%. In combination with dynamic ¹³CO₂ labeling of Synechocystis cultures, we determined PEPC activity in vivo alongside assessments of RUBISCO and CBB cycle activities. We demonstrate that RUBISCO is inactive in the dark, whereas PEPC remains active but at a lower rate than during the day. Accurate quantifications of aspartate concentrations and positional E¹³Cs provide molar Ci assimilation rates of photoautotrophic Synechocystis cultures. This technology can be combined with C-positional analyses of other metabolites, for example 3-phosphoglycerate, and may be adapted to characterize natural and biosynthetically engineered Ci-assimilation.