Combined In-Cell NMR and Simulation Approach to Probe Redox-Dependent Pathway Control

Dynamic response
of intracellular reaction cascades to changing
environments is a hallmark of living systems. As metabolism is complex,
mechanistic models have gained popularity for describing the dynamic
response of cellular metabolism and for identifying target genes for
engineering. At the same time, the detailed tracking of transient
metabolism in living cells on the subminute time scale has become
amenable using dynamic nuclear polarization-enhanced 13C NMR. Here, we suggest an approach combining in-cell NMR spectroscopy
with perturbation experiments and modeling to obtain evidence that
the bottlenecks of yeast glycolysis depend on intracellular redox
state. In pre-steady-state glycolysis, pathway bottlenecks shift from
downstream to upstream reactions within a few seconds, consistent
with a rapid decline in the NAD+/NADH ratio. Simulations
using mechanistic models reproduce the experimentally observed response
and help identify unforeseen biochemical events. Remaining inaccuracies
in the computational models can be identified experimentally. The
combined use of rapid injection NMR spectroscopy and in silico simulations
provides a promising method for characterizing cellular reactions
with increasing mechanistic detail.