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.
VIEW PRESENTATION S-5
