TY  - JOUR
AU  - Otten, Julia
AU  - Tenhaef, Niklas
AU  - Jansen, Roman P.
AU  - Döbber, Johannes
AU  - Jungbluth, Lisa
AU  - Noack, Stephan
AU  - Oldiges, Marco
AU  - Wiechert, Wolfgang
AU  - Pohl, Martina
TI  - A FRET-based biosensor for the quantification of glucose in culture supernatants of mL scale microbial cultivations
JO  - Microbial cell factories
VL  - 18
IS  - 1
SN  - 1475-2859
CY  - London
PB  - Biomed Central
M1  - FZJ-2019-04935
SP  - 143
PY  - 2019
AB  - BackgroundIn most microbial cultivations d-glucose is the main carbon and energy source. However, quantification of d-glucose especially in small scale is still challenging. Therefore, we developed a FRET-based glucose biosensor, which can be applied in microbioreactor-based cultivations. This sensor consists of a glucose binding protein sandwiched between two fluorescent proteins, constituting a FRET pair. Upon d-glucose binding the sensor undergoes a conformational change which is translated into a FRET-ratio change.ResultsThe selected sensor shows an apparent Kd below 1.5 mM d-glucose and a very high sensitivity of up to 70% FRET-ratio change between the unbound and the glucose-saturated state. The soluble sensor was successfully applied online to monitor the glucose concentration in an Escherichia coli culture. Additionally, this sensor was utilized in an at-line process for a Corynebacterium glutamicum culture as an example for a process with cell-specific background (e.g. autofluorescence) and medium-induced quenching. Immobilization of the sensor via HaloTag® enabled purification and covalent immobilization in one step and increased the stability during application, significantly.ConclusionA FRET-based glucose sensor was used to quantify d-glucose consumption in microtiter plate based cultivations. To the best of our knowledge, this is the first method reported for online quantification of d-glucose in microtiter plate based cultivations. In comparison to d-glucose analysis via an enzymatic assay and HPLC, the sensor performed equally well, but enabled much faster measurements, which allowed to speed up microbial strain development significantly.
LB  - PUB:(DE-HGF)16
C6  - pmid:31434564
UR  - <Go to ISI:>//WOS:000483292300003
DO  - DOI:10.1186/s12934-019-1193-y
UR  - https://juser.fz-juelich.de/record/865570
ER  -