A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis.
Background:The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth.Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively.Results:The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producingCorynebacterium glutamicum(C. glutamicum) strain DM1933 in a BioLector®microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.
IBG-1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. vi.steffen@fz-juelich.de.
Victoria
V
Steffen
IBG-1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. j.otten@fz-juelich.de.
Julia
J
Otten
IBG-1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. susann.engelmann@rwth-aachen.de.
Susann
S
Engelmann
IBG-1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. a.radek@fz-juelich.de.
Andreas
A
Radek
IBG-1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. m.limberg@fz-juelich.de.
Michael
M
Limberg
ICS-6, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. b.koenig@fz-juelich.de.
Bernd W
BW
Koenig
IBG-1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. s.noack@fz-juelich.de.
Stephan
S
Noack
IBG-1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. w.wiechert@fz-juelich.de.
Wolfgang
W
Wiechert
IBG-1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany. ma.pohl@fz-juelich.de.
Martina
M
Pohl
">l-lysine detection; FRET-based biosensors; metabolite analysis; microbial strain development; online monitoring
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