​S-5. Visualizing fermentation in living yeast cells

​EBC Symposium: Resources for the Future Session

Sebastian Meier, Carlsberg Laboratory, Copenhagen, Denmark Co-authors: Magnus Karlsson, Pernille Jensen, and Mathilde Lerche, Albeda Research, Copenhagen, Denmark; Jens Duus, Carlsberg Laboratory, Copenhagen, Denmark

ABSTRACT: The visualization of biotechnologically relevant metabolic pathways in living microbial cells provides direct insight into cellular biochemistry and facilitates the optimization and control of cells in production. Special emphasis and interest is placed on the optimization of yeast fermentation with assays on intact cells with the hope of obtaining improved cell factories for fermentation. Classical studies of intracellular enzymes in isolation do not reconstruct the complex composition and macromolecular crowding of the intracellular milieu. In addition, enzymes do not necessarily operate in isolation inside the cell, but are often subpartitioned into functional complexes. The complexity of functional and structural organization inside the cell therefore makes the direct detection of cellular processes in their natural surroundings desirable for advancing the biochemical understanding and biotechnological control of metabolism. Direct observations of cellular reaction chemistry in living organisms call for noninvasive methods that resolve reactant signals and ideally detect natural substrates rather than chemically introduced reporter groups. The combined need for chemical detail on molecular transformations and sufficient time resolution to detect transient reaction intermediates is not met by conventional spectroscopic methods. Instead, the time course of cellular reactions in vivo is commonly approximated using isotope labeling patterns of metabolic products extracted from cells grown on defined substrates. We describe a novel methodology termed dynamic nuclear polarization, which yields substrate solutions with an approximately millionfold enhanced nuclear magnetic resonance (NMR) spectral signal relative to the complex cellular background to make real-time observations of fast metabolic reactions and short-lived pathway intermediates in vivo realistic. We show that the enhancement of nuclear spin polarization allows us to directly follow the flux of the glucose signal through rather extended reaction networks of central carbon metabolism in living fermentations. Experiments are conducted as real time assays of a few minutes duration that detect metabolic bottlenecks, pathway use, reversibility of reactions, and reaction mechanisms in vivo with subsecond time resolution.
Sebastian Meier received his diploma in biochemistry from the University of Regensburg (Germany) in 2000 and his Ph.D. degree in biophysics from the University of Basel (Switzerland) in 2004. He began his employment with the Carlsberg Laboratory (Copenhagen, Denmark) in 2007 as a staff scientist and was named senior scientist in 2010, serving as an expert on spectroscopy of complex systems.



Brewing Resources

Ask the Brewmasters Technical Quarterly MBAA Webinars MBAA Podcasts Food Safety Brewery Safety Vendor Search