Journal Article

FZJ-2019-04935

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png A FRET-based biosensor for the quantification of glucose in culture supernatants of mL scale microbial cultivations

Otten, J.FZJ* ; Tenhaef, N.FZJ* ; Jansen, R. P.FZJ* ; Döbber, J. ; Jungbluth, L.FZJ* ; Noack, S.FZJ* ; Oldiges, M.FZJ* ; Wiechert, W.FZJ* ; Pohl, M. (Corresponding author)FZJ*

2019
Biomed Central London

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Please use a persistent id in citations: http://hdl.handle.net/2128/23068  doi:10.1186/s12934-019-1193-y

Abstract: 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.

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Contributing Institute(s):

1. Biotechnologie (IBG-1)
2. Strukturbiochemie (ICS-6)

Research Program(s):

1. 581 - Biotechnology (POF3-581) (POF3-581)

Appears in the scientific report 2019

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Database coverage:
; ; ; ; BIOSIS Previews ; Clarivate Analytics Master Journal List ; DOAJ Seal ; Ebsco Academic Search ; IF < 5 ; JCR ; NCBI Molecular Biology Database ; PubMed Central ; SCOPUS ; Science Citation Index Expanded ; Web of Science Core Collection

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