A FRET-based biosensor for the quantification of glucose in culture supernatants of mL scale microbial cultivations

Microbial Cell Factories

Niklas Tenhaef¹  Roman P. Jansen¹  Johannes Döbber¹  Lisa Jungbluth¹  Wolfgang Wiechert¹  Martina Pohl¹  Stephan Noack¹  Julia Otten¹  Marco Oldiges¹ 

[1] IBG-1: Biotechnology, Forschungszentrum Jülich GmbH;

关键词: Sensor beads;    Online glucose measurement;    Micro cultivation;    Glucose binding protein;    mTurquoise2;    Venus;   

DOI  :  10.1186/s12934-019-1193-y

来源: DOAJ

【 摘 要 】

Abstract Background In 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. Results The 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. Conclusion A 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.

【 授权许可】

Unknown