Abstract
Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: . The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D = 0.1 h−1) conditions, to be 22.0 ± 2.6 and 15.6 ± 0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05 ± 0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.
Highlights
In S. cerevisiae, the NADPH producing reactions, e.g. glucose-6-P dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) of the Pentose Phosphate Pathway (PPP), are reactions www.nature.com/scientificreports/
In contrast NADPH consuming reactions typically operate close to the equilibrium[4], the NADPH/ NADP concentration ratio has a significant contribution to the driving force of the synthesis reactions, e.g. fatty acids, amino acids and products from the mevalonic acid pathway
The cytosolic free NADPH/NADP ratio was found to correlate with increasing extracellular glucose concentrations
Summary
In S. cerevisiae, the NADPH producing reactions, e.g. glucose-6-P dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) of the Pentose Phosphate Pathway (PPP), are reactions www.nature.com/scientificreports/. In contrast NADPH consuming reactions typically operate close to the equilibrium[4], the NADPH/ NADP concentration ratio has a significant contribution to the driving force of the synthesis reactions, e.g. fatty acids, amino acids and products from the mevalonic acid pathway. It was found that by switching the NADH-dependent dehydrogenase DhaT to the NADPH-dependent dehydrogenase YqhD for the conversion of the toxic aldehyde 3-HPA to PDO, a higher PDO titer was achieved and the differences in the cofactor reduced/oxidised ratios contributed to the high PDO titer[8,9]. Metabolite sensor reactions have been proposed to monitor compartment specific concentrations (or ratios), especially for cofactor couples like NAD/ NADH, NADPH/NADP, and ATP/ADP12–15. The reaction can be a dead-end or within a pathway, as long as the enzyme capacity is sufficient to reach pseudo equilibrium
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