Hyperpolarized ¹³C NMR Reveals Pathway Regulation in Lactococcus Lactis and Metabolic Similarities and Differences 3 Across the Tree of Life

The control of metabolic networks is incompletely understood, even for glycolysis in highly studied model organisms. Direct real-time observations of metabolic pathways can be achieved in cellular systems with ¹³C NMR using dissolution Dynamic Nuclear Polarization (dDNP NMR). The method relies on a short-lived boost of NMR sensitivity using a redistribution of nuclear spin states to increase the alignment of the magnetic moments by more than four orders of magnitude. This temporary boost in sensitivity allows detection of metabolism with sub-second time resolution. Here, we hypothesized that dDNP NMR would be able to provide molecular phenotypes that are not easily accessible with more conventional methods. The use of dDNP NMR allows real- time insight into carbohydrate metabolism in a Gram-positive bacterium (Lactoccocus lactis) and comparison to other bacterial, yeast and mammalian cells shows differences in kinetic barriers of glycolysis across the kingdoms of life. Nevertheless, the accumulation of non-toxic precursors for biomass at kinetic barriers is found to be shared cross the kingdoms of life. We further find that the visualization of glycolysis using dDNP NMR reveals kinetic characteristics in transgenic strains that are not evident when monitoring the overall glycolytic rate only. Finally, dDNP NMR reveals that resting Lactococcus lactis cells use influx of carbohydrate substrate to acetoin rather than lactate during the upstart of glycolysis. This metabolic regime can be emulated using suitably designed substrate mixtures to enhance the formation of the C4 product acetoin more than 400fold. Overall, we find that dDNP NMR provides analytical capabilities that may help clarify the intertwined mechanistic determinants of metabolism and the optimal usage of biotechnologically important bacteria.