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キーワード:
metabolic flux analysis
sp strain pcc-6803
mass-spectrometry
gas-chromatography
wild-type
cycle
cyanobacteria
microorganisms
purification
arabidopsis
要旨:
Background: Recent studies using transcript and metabolite profiles of wild-type and gene deletion mutants revealed that photorespiratory pathways are essential for the growth of Synechocystis sp. PCC 6803 under atmospheric conditions. Pool size changes of primary metabolites, such as glycine and glycolate, indicated a link to photorespiration.
Methodology/Principal Findings: The C-13 labelling kinetics of primary metabolites were analysed in photoautotrophically grown cultures of Synechocystis sp. PCC 6803 by gas chromatography-mass spectrometry (GC-MS) to demonstrate the link with photorespiration. Cells pre-acclimated to high CO2 (5%, HC) or limited CO2 (0.035%, LC) conditions were pulse-labelled under very high (2% w/w) C-13-NaHCO3 (VHC) conditions followed by treatment with ambient C-12 at HC and LC conditions, respectively. The C-13 enrichment, relative changes in pool size, and C-13 flux of selected metabolites were evaluated. We demonstrate two major paths of CO2 assimilation via Rubisco in Synechocystis, i.e., from 3PGA via PEP to aspartate, malate and citrate or, to a lesser extent, from 3PGA via glucose-6-phosphate to sucrose. The results reveal evidence of carbon channelling from 3PGA to the PEP pool. Furthermore, C-13 labelling of glycolate was observed under conditions thought to suppress photorespiration. Using the glycolate-accumulating Delta glcD1 mutant, we demonstrate enhanced C-13 partitioning into the glycolate pool under conditions favouring photorespiration and enhanced C-13 partitioning into the glycine pool of the glycine-accumulating Delta gcvT mutant. Under LC conditions, the photorespiratory mutants Delta glcD1 and Delta gcvT showed enhanced activity of the additional carbon-fixing PEP carboxylase pathway.
Conclusions/Significance: With our approach of non-steady-state C-13 labelling and analysis of metabolite pool sizes with respective C-13 enrichments, we identify the use and modulation of major pathways of carbon assimilation in Synechocystis in the presence of high and low inorganic carbon supplies.
